Cell surface glycosyltransferases

Biochimica et Biophysica Acta, 415 (1975) 473-512 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA85155

C E L L S U R F A C E G L Y C O S Y L T R A N S F E R A S E S

B A R R Y D. S H U R and ST E PH E N R O T H

Department of Biology, The Johns Hopkins University, Baltimore, Md. 21218 (U.S.A.)

(Received August 8th, 1975)

C O N T E N T S

I. Introduct ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474

A. Purpose of the review . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474

B. The glycosyltransferases . . . . . . . . . . . . . . . . . . . . . . . . . . . 475

C. The glycosyltransferase assays . . . . . . . . . . . . . . . . . . . . . . . . . 477

II. Surface glycosyltransferases in physiological systems . . . . . . . . . . . . . . . . 478

A. Hemostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 478

B. Intestinal cell differentiation . . . . . . . . . . . . . . . . . . . . . . . . . 480

C. Asialoglycoprotein clearance . . . . . . . . . . . . . . . . . . . . . . . . . 481

D. Glycoprotein reabsorpt ion in kidney tubules . . . . . . . . . . . . . . . . . . 483

E. Concanavalin A binding sites . . . . . . . . . . . . . . . . . . . . . . . . . 483

III. Surface glycosyltransferases in development . . . . . . . . . . . . . . . . . . . . 485

A. Embryonic neural retina . . . . . . . . . . . . . . . . . . . . . . . . . . . 485

B. Amoeba aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487

C. Embryonic liver . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488

D. Gamete recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489

E. Gastrulating chick embryos . . . . . . . . . . . . . . . . . . . . . . . . . 490

IV. Surface glycosyltransferases on cultured cells . . . . . . . . . . . . . . . . . . . 492

A. Activities on normal and malignant cells . . . . . . . . . . . . . . . . . . . . 492

B. Adhesion and repair of cell surfaces . . . . . . . . . . . . . . . . . . . . . . 496

C. Cell-contact- and culture-density-dependent surface glycosyltransferase activity . . . 499

D. Cell-cycle-dependent surface glycosyltransferase activity . . . . . . . . . . . . . 504

E. Potential role in growth control . . . . . . . . . . . . . . . . . . . . . . . . 505

F. Potential role of lipid intermediates . . . . . . . . . . . . . . . . . . . . . . 506

G. Inability to demonstrate surface glycosyltransferases . . . . . . . . . . . . . . . 507

V. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510

Abbreviation: EGTA, ethyleneglycol bis(a-aminoethylether)-N,N'-tetraacetic acid.

I. INTRODUCTION

1,4. Purpose of the review During the past few years, over forty publications have appeared reporting

glycosyltransferase activities on intact cells. The conclusion in all of these reports is that some transferases are bound to the outer surface of the cell plasma membrane, where these enzymes retain their catalytic activities (Fig. 1) or, at least, their binding properties.

If it is true that glycosyltransferases exist on the outsides of some cells as well as on their Golgi [1,2], endoplasmic reticulum [3] and mitochondrial [4,5] membranes, then the importance of this fact would be greater than just another subcellular location for an additional enzyme. Functionally, these surface proteins would be logical candidates for a role in many cell interactions known to occur via protein and carbohydrate moieties on the cell surface.

In a sense, the surface glycosyltransferases could be acting as a class of endogenous lectins capable of interacting with surface glycosides. Lectins, proteins and glycoproteins that bind specifically to monosaccharide residues have been extensively utilized to probe the carbohydrate components of cell surfaces (for review, see ref. 6). Exogenously applied lectins alter contact-mediated phenomena as diverse as growth control and fertilization, as well as acting as mitogens. Lectins agglutinate malignant cells and normal mitotic cells more readily than normal interphase cells, although the reason for this remains unclear [6]. The interaction of these potential in situ lectins, the surface glycosyltransferases, with their surface glycoside acceptors might be carefully controlled by plasma membrane architecture. Their catalytic activities offer an additional regulatory mechanism via the cells' ability to control the supply of requisite sugar donors to the surface enzymes.

In this manner, one can easily imagine surface transferases playing a role in specific intercellular recognition and association, cell migration on cellular and acel-

-sugar acceptor

UDP N ~(~

sugar donor

enzyme

- react ion product

free nuc leo t ide

G L Y C O S Y L T R A N S F E R A S E REACTION

Fig. 1. Glycosyltransferase reaction. Various monosaccharides are represented by circles, triangles, squares and diamonds. A trisaccharide sugar acceptor is depicted, linked at its reducing end to a protein or lipid moiety represented by the wavy line. The enzyme, usually in the presence of divalent metal cations, catalyzes the transfer of a monosaccharide from its uridine diphosphate donor to the nonreducing terminus of the sugar acceptor. The reaction products are a tetrasaccharide, represented here with a terminal "diamond" moiety, and the free nucleotide.

: = ~ Initial adhesive ( ~ ~ recognition as

i > o result of transferase- 13 Reaction completion substrate complex Iland consequent cell

U DU DP../P -X"~ ~" modification

Separation Fig. 2. Model for generalized cell-cell recognition. Each cell is represented with a cell surface trisaccharide sugar acceptor and surface glycosyltransferase specific for that sugar acceptor. Enzyme- substrate interaction occurs only between transferases and acceptors on adjacent cells. Adhesion is stable so long as the requisite sugar nucleotide is not made available. In the presence of this sugar nucleotide, catalysis occurs, forcing the cells to separate, producing cell surfaces with the added monosaccharide, X. In an analogous manner recognition is postulated to occur between surface glycosyltransferases and sugar acceptors, bound not only on other cell surfaces but also within extracellular matrices. Catalysis in this instance would produce a modified substrate through the addition of monosaccharide X residues. From Roth et al. [21].

lular substrates, covalent modifications by surface enzymes of neighboring cells

(Fig. 2) or substrates and specific binding of circulating glycoproteins or glycolipids. These functions would make surface transferases crucial to morphogenesis, malignant

invasiveness, fertilization, hemostasis, liver physiology and other contact-mediated cell interactions. Indeed, there are data consistent with each of these possibilities and with others. The overall purpose o f this review will be to evaluate the data in support o f a functional role for the surface glycosyltransferases and, more importantly, to

judge the overall evidence for the existence o f surface transferases in each of the biological systems that has been pursued.

In some cases, the evidence for a surface localization for transferases is quite

good and in some cases the evidence is virtually nonexistent. For many reasons, functional roles are often more difficult to establish than enzyme localization and, as a result, the quality o f the data suggestive o f a particular function is less rigid than that o f the data for surface locale. This review will take a reasonably critical, and hopefully objective, approach to the work accomplished to date. We will be especially alert to reports unable to rule out equally plausible hypotheses concerning both the existence and function o f surface transferases. These studies appear to be an exciting new development in our eventual understanding, in molecular terms, o f how cells interact with other cells and substances in their immediate environment.

IB. The glycosyltransferases These enzymes catalyze the transfer o f a monosacchar ide residue f rom a sugar

nucleotide (sugar donor) to the non-reducing terminus of a specific sugar acceptor that may vary in size from less than 200 daltons to greater than a million [7-9]. Fig. 1 illustrates the reaction which often requires divalent metal cations for optimum activity. The transferases are named according to the sugar donors they utilize. That is, all galactosyltransferases transfer galactose from uridine diphosphate galactose (UDPgal) to their specific acceptors, while neuraminyltransferases transfer neuraminic acid from its cytidine monophosphate derivative (CMPNeu) to the required acceptor. As far as is known [7-9], no transferase can utilize more than one type of sugar donor. Acceptor specificities, however, can be less stringent. For example, the galactosyltransferase that transfers galactose to a non-reducing N-acetylglucosamine on some glycoproteins will also transfer galactose to free N-acetylglucosamine. The Km of the monosaccharide, however, appears to be much higher than that for the larger molecular weight compound [10].

The transferases may be classified according to other criteria, namely their solubilities. Most cell-associated glycosyltransferases are attached to membranes and, unless the broken tissues are treated specifically, the transferase activities will be pelleted during a high speed centrifugation (100000 × g, 1 h) [7-9]. Most of the membrane-bound transferases are thought to reside on the Golgi and smooth endoplasmic reticulum membranes [1,2], although virtually all of the evidence for this comes from two approaches; subcellular fractionation data [1,2] and autoradiography after incubation with labeled sugars [11,12]. Although there are no real reasons to doubt this conclusion, subcellular fractionation schemes and autoradiography can not ordinarily be considered unequivocal data in favor of either an internal or a surface locale for the transferases. Membrane fractionation studies usually cannot rule out small contaminations of highly active membrane fragments. For example, Golgi preparations may possess a small plasma membrane contaminant and vice versa. Autoradiography localizes the transferase product, a 3H-labeled glycoside, not the transferase.

Studies attempting to demonstrate surface glycosyltransferase activities have increasingly utilized whole-cell preparations. Whether internal enzymes contribute to enzyme assay products in these preparations needs to be carefully controlled for in each study. Nevertheless, studies that have compared surface and internal transferases conclude that the bulk of the enzymes are internal and probably Golgi- associated. If surface membrane arises, even in part, by the evagination of Golgi vesicles [11-13], then it is possible that the Golgi transferases are carried to the surface by these same vesicles. To date, however, there are no data showing the specific succession of transferases from Golgi membranes to the cell surface. In those cases, therefore, where there are reasonable data for surface transferases, the mechanism of appearance on the plasma membrane must remain total speculation.

Soluble transferases have been found in colostrum [ 14], milk [ 15,16], serum [ 17], chick embryo cerebrospinal fluid [18], vitreous humor [18] and amniotic fluid [18]. The colostrum and milk galactosyltransferases synthesize lactose by transferring galactose to glucose in the presence of the modifier protein a-lactalbumin [19]. In

the absence of this protein, the same enzyme will transfer galactose to N-acetylglucosamine, preferentially synthesizing N-acetyllactosamine. Except for this enzyme, it is not clear what functions, if any, are performed by the other soluble transferases. For a thorough review of the chemistry of both soluble and membrane-bound glycosyltransferases, see refs 7-9.

IC. The glycosyltransferase assays Assays for the glycosyltransferases are usually simple and are based on the

separation of low molecular weight compounds (unused sugar donors) from high molecular weight compounds (glycosylated acceptors). The most common procedure is acid precipitation of the heavy molecular weight product using either trichloroacetic acid or phosphotungstic acid. Of these two, phosphotungstic acid is the recommended precipitant, since some heavily glycosylated products are either not precipitated by trichloroacetic acid or precipitated poorly [20]. A slightly more complex but much more rigorous assay is high-voltage electrophoresis of the reaction mixture on borate- impregnated paper [21]. Under these conditions, unreactedsugarnucleotidemigrates rapidly leaving the glycosylated product(s) at the origin. Additionally, any labeled sugar 1-phosphate or free sugar formed during the incubation as a result of pyro- phosphorylase or phosphatase activity, respectively, will also migrate as a saccharide- borate complex. When assaying N-acetylglucosaminyl- or N-acetylgalactosaminyltransferases, free acetylated hexosamines do not leave the origin to a significant extent during borate electrophoresis. They will, however, migrate away from the origin when the electrophoretogram is subjected to ascending chromatography in 70 ~ ethanol.

The advantage of this borate electrophoresis assay is that the formation of sugar donor breakdown products may be monitored along with the formation of the transferase product. Since the labeled sugar donors are expensive, and since the Km values for these substrates are usually high in the case of the membrane-bound transferases, many of the experiments reported in the literature are carried out under subsaturating conditions with respect to this substrate. When this is true, any difference in transferase levels between two samples might just as well be the result of increased phosphatase activities or product hydrolysis as of decreased transferase activities. The most direct way to test for this is to measure the degree to which the sugar donor is broken down or free sugar liberated. The borate electrophoresis assay allows this to be done easily and routinely.

The data in the literature regarding cell surface glycosyltransferases are grouped, here, according to the potential functions implicated for these enzymes. Reports are dealt with first that ascribe a physiological significance to surface transferases, followed by systems that are developmentally oriented. Finally, work on cultured normal and malignant cells is evaluated.

II. SURFACE GLYCOSYLTRANSFERASES IN PHYSIOLOGICAL SYSTEMS

IIA. Hemostasis

Wounding of vascular endothelium exposes collagen fibrils that serve as adhesion sites for circulating platelets, thus initiating hemostasis. Certain platelet constituents, such as ADP, are then released causing the aggregation of additional platelets onto those already adhering to collagen [22]. Two separate laboratories have reported the existence of plasma membrane glucosyltransferases on the surface of blood platelets. One particular enzyme, collagen:glucosyltransferase, has been implicated in the adhesion of these cells to endothelial collagen, thus initiating hemostasis [22-25]. Surface sialyltransferases have been suggested to mediate the consequent platelet aggregation [26].

Jamieson and coworkers [22-24] have shown that soluble collagen, from which glucose has been removed (thereby exposing galactose residues) inhibited the binding of platelets to connective tissue. Also, intact platelets were able to transfer glucose to this same soluble collagen. 55 % of this intact platelet glucosyltransferase activity could be recovered on isolated plasma membranes prepared by the glycerol lysis technique [27]. The resultant optimized enzyme activity was purified 19-fold. Plasma membrane markers, i.e. ATPases, phosphodiesterases and acid phosphatases, followed the glucosyltransferase activity, but did not exceed 8-fold enrichments.

Product characterization was carried out utilizing highly purified collagenase to digest the glucose-collagen product. Elution from a Sephadex column produced coincident peaks of radioactivity and ninhydrin-positive material. Hydrolysis of the product under conditions specific for glucose residues [28] removed the radioactivity from the ninhydrin-positive peak. This subsequently cochromatographed with known glucose in two paper chromatographic solvent systems. The incorporated glucose moiety was recovered as glucosylgalactosylhydroxylysine residues.

Acceptor specificity was demonstrated utilizing various potential glycoprotein acceptors. The most active glycoprotein assayed was only 7.6 % as active as soluble collagen. A small endogenous acceptor component was detectable in these preparations and characterized as a glycogen synthetase system strongly bound to these membranes. The activity represented less than 1.0 % of that obtained using exogenous collagen acceptors. Various sugar nucleotides were assayed as well, with only UDP- glucose serving as the appropriate sugar donor.

Less than 10 % free glucose was generated during the incubation from UDPglu- cose, and incubation mixtures utilizing [14C]glucose rather than UDPglucose resulted in no incorporation of radioactivity. These data indicated UDPglucose, rather than free glucose, as the authentic sugar donor.

Isolated platelet plasma membranes also possess a collagen:galactosyltransferase although intact platelets did not demonstrate this enzyme activity [29]. Plasma membrane exhibiting appropriate enzyme markers express this activity with a four-fold purification over the cell lysate. Unlike glucosyltransferase activity, which shows no detergent stimulation, galactosyltransferase activity was stimulated two-fold

with 0.1% Triton X-100. The observed detergent stimulation of enzyme activity and the inability to demonstrate activity with intact platelets suggests [29] that galactosyltransferase may be buried within the plasma membrane, or exposed only to the cytoplasmic side. Glucosyltransferase, on the other hand, is likely to be exposed to the outside of the plasma membrane and to be able to interact with collagen fibrils.

To determine whether platelet plasma membrane collagen :glucosyltransferase plays any role in platelet-collagen adhesion, studies [22-24,29] were undertaken to show parallel behavior transferase activity and adhesion. Collagen digests rich in carbohydrate side-chains inhibited glucose transfer to soluble collagen by 40 % of control values and inhibited adhesion by 84 %. Carbohydrate-poor portions of collagenase digests inhibited enzyme activity and adhesion only 10% and 17%, respectively. Inhibitors of platelet-collagen adhesion, such as D-glucosamine, aspirin, chlorpromazine and sulfhydryl blocking reagents, produced a parallel inhibition of collagen :glucosyltransferase activity. Aspirin and glucosamine had no inhibitory effect on collagen:galactosyltransferase. Sulfhydryl blocking reagents produced partial inhibition of galactosyltransferase.

Similarly, Bosmann's laboratory [25] has reported the existence of collagen: glucosyl- and collagen:galactosyltransferases on platelet plasma membranes. Ad- ditionally, polypeptide:N-acetylgalactosaminyl- and glycoprotein:galactosyltransferases were also characterized. A crude plasma membrane purification demonstrated close to 100 % of all enzyme activity, but did not clearly rule out contamination from internal membranes. Partial enzyme characterizations were conducted, and unlike previous reports [22-24], these studies could not detect any endogenous activity. Bosmann [25] suggested that the glycoprotein:galactosyl- and polypeptide:N-acetylgalactosaminyltransferases in adult platelets are residual enzymes used by immature ceils to actively synthesize glycoprotein components. The collagen:glycosyltransferases, however, may function as mediators of platelet-collagen adhesion similar to those proposed by Jamieson [22-24].

Taken together, these studies strongly indicate that platelet plasma membrane collagen :glucosyltransferase exists. Evidence for its role in collagen-platelet adhesion is based upon correlations of enzyme activity and adhesion. A clear demonstration of this enzyme's role in hemostasis awaits more direct tests.

Subsequent to platelet-collagen adhesion, certain cellular components are released, initiating the aggregation of additional platelets onto those already adhering to collagen. While platelet plasma membrane glucosyltransferase has been implicated in platelet-coUagen interactions, a surface sialyltransferase has been implicated [26] in platelet-platelet aggregation. 52 % of platelet homogenate sialyltransferase activity could be recovered on isolated plasma membranes with a purification of over 50-fold. However, the purity of the plasma membrane preparation, relative to internal membrane contamination, was not tested.

Optimized enzyme assays revealed a Km of 9.0" 10 -5 M for CMPNeu and 67.5 • 10 -5 M for desialyzed fetuin as substrate, and as in most studies, sugar nucleotide was utilized at subsaturating amounts.

Two observations suggest that endogenous sialyl acceptors are present on platelet plasma membranes. (1) Endogenous acceptor activity remained on the isolated plasma membranes, and (2) intact platelets maintained higher endogenous activity than homogenates. Exogenous activity increased in homogenates. Exogenous product characterization revealed authentic sialic acid incorporation onto desialyzed fetuin. Acceptor specificity studies showed that most glycoproteins tested were not nearly as active as desialyzed fetuin although prothrombin, from which sialic acid was removed, was six times more effective as an exogenous acceptor than desialyzed fetuin.

Components that inhibit platelet-platelet and platelet-collagen adhesions were tested for their inhibitory ability on sialyltransferase activity. UDP, ADP and aspirin were most effective (approximately 96 ~) at inhibiting endogenous and exogenous activities, o-Glucosamine was less effective, inhibiting activities about 80 ~o. As above, these studies show parallel behavior of adhesion and enzyme activity but not causation.

Human blood platelets also possess a neuraminidase activity. The ability of this neuraminidase fraction to remove sialic acid residues from intact platelets was shown by a decrease in platelet electrophoretic mobility of approximately 40 ~o after enzyme treatements.

Plasma membrane sialyltransferases, their surface endogenous acceptors, and the neuraminidase activity capable of cleaving sialic acid residues from platelet surfaces were compiled into a model for cell-cell adhesion. Bosmann [26] suggested that surface acceptors (sialic acid acceptors) bind surface sialyltransferases on adjoining cells forming an adhesive bridge between them. After catalysis (CMPNeu, cation), the adhering cells are forced to separate but are able to regenerate new surface acceptors by neuraminidase removal of newly incorporated surface glycosides. In this way adhesive sites can be regenerated for further aggregation.

Considerable evidence does exist, therefore, for platelet plasma membrane glycosyltransferases. Most notable was the ability of intact platelets to transfer glucose to soluble collagen and the enrichment of transferase activity on isolated plasma membranes. Furthermore, it is likely that platelet adhesion to collagen galactosides is mediated by surface glucosyltransferases. Surface sialyltransferases have been implicated in platelet-platelet aggregation, but not very strongly. The ability of platelets to adhere to collagen that has had its carbohydrate components destroyed [30] suggests that additional mechanisms are likely.

liB. Intestinal cell differentiation In the intestinal wall, epithelial cells begin as mitotically active, undifferentiated

cells at the base of intestinal crypts. As differentiation proceeds, these cells migrate up the crypt wall and reach maturity at the upper third of the villus [31 ]. Cell surface glycosyltransferases have been investigated on these cells as a function of their differentiation. Enzyme activity was concentrated on the mitotically active crypt cells.

Weiser [31] has employed dissociation procedures that isolate selective

populations of intact intestinal epithelial cells. The initial ethylenediamine tetraacetic acid (EDTA) treatment dissociated cells predominantly of villus tip origin. Through sequential EDTA dissociations, populations of cells were recovered that lie further and further within the intestinal crypt. In this way, a graded distribution of cells, from villus tip to crypt base, could be isolated. Sucrase and alkaline phosphatase activities were used as markers of mature villus cells, while thymidine kinase was indicative of mitotically active crypt cells.

Utilizing these preparations, intact undifferentiated crypt cells expressed up to a ten-fold higher endogenous glycosyltransferase activity than mature villus cells with GDPfucose, GDPmannose, UDP-N-acetylglucosamine, UDPgalactose and UDPglucose. In contrast, CMPsialic acid:sialyltransferase activity was prefentially localized on villus cells. Use of lactose as an exogenous acceptor for sialyltransferase and N-acetylglucosamine as an exogenous acceptor for galactosyltransferase suggested that differential endogenous enzyme activities were a result of increased enzyme as well as acceptor levels on the particular cell types [32].

Surface localization of enzyme activities was suggested by the following observations. (l) Neither glycosyltransferase nor endogenous acceptor activity was released into the medium. Only exogenous acceptor products were found in the cell-free supernatant. (2) Internal epimerization was ruled out after identification of 3 labeled products showed them to be the same monosaccharides as the original sugar donors, utilizing three chromatographic systems. (3)Homogenization of villus and crypt cells revealed a comparable increase in both cell types of N-acetylglucosaminyltransferase activity rather than an eight-fold selective increase in crypt cells. (4) Villus exogenous sialyltransferase activity could be localized on purified villus cell brush borders. (5) Free, labeled glucosamine was incorporated predominantly into villus cells [31], in contrast to the high N-acetylglucosaminyltransferase activity on crypt cell surfaces.

While these studies probably represent real cell surface transferase differences between villus and crypt cells, it is not clear that each class of transferases was being assayed under optimal conditions. More importantly, hydrolysis of the sugar nucleotides was not ruled out, allowing for the possibility that the low villus cell transferase activity may be the result of high levels of phosphatases degrading the necessary sugar nucleotide substrates. Indeed, villus cells are known to be rich sources of these degrading enzymes. Experiments in which equal aliquots of villus and crypt cells were mixed produced much lower activities than the expected intermediate values. Although the author concluded [32] that villus ceils sterically interfered with crypt cell glycosylation of neighboring cells, it is equally plausible that villus phosphatases destroyed the substates in the mixed incubations. The functions that these surface transferases participate in on differentiating intestinal epithelial cells is entirely unknown.

IIC. Asialoglycoprotein clearance Cell surface sialyltransferases have been suggested to mediate the specific liver

clearance of desialyzed plasma proteins. Circulating glycoproteins from which some terminal sialic acid residues have been removed, thereby exposing galactose moieties, are cleared by the liver ten times more rapidly than when these glycoproteins maintain a more complete carbohydrate complement [33,34]. Isolated liver plasma membranes retain this specific binding capacity for disialyzed glycoproteins [35,36]. A glycoprotein:sialyltransferase activity was demonstrable on these isolated plasma membranes and was a likely candidate for the membrane binding sites for the desialyzed plasma glycoproteins since sialyltransferases recognize terminal galactosides [35]. Consistent with this was the observation that neuraminidase treatment of liver membranes prevented the subsequent binding of asialoglycoprotein, possibly by creating additional endogenous binding sites for sialyltransferase. Incubation with CMPNeu reversed the inhibition produced by neuraminidase treatment.

More recently, Aronson et al. [37] suggested that plasma membrane galactosyltransferase, rather than sialyltransferase, is a binding receptor for asialoglyco- proteins. Two of their observations critically speak to this conclusion. First, agalactofetuin inhibited the binding of asialofetuin to liver plasma membranes by 80~. Contaminating asialofetuin, which may have been the true inhibitor, is supposedly ruled out by the inability to demonstrate exogenous sialyltransferase activity utilizing an agalactofetuin preparation as acceptor. However, membrane sialyltransferase transferred sialic acid to asialofetuin and extensive galactose transfer occurred to agalactofetuin. Therefore, agalactofetuin, an inhibitor of asialofetuin binding, was suspected of binding to plasma membranes via a galactosyltransferase. While in these studies agalactofetuin was 80 ~ as effective as asialofetuin in inhibiting labeled asialofetuin binding to liver membranes, a previous report [36] indicated that agalacto derivatives of orosomucoid and ceruloplasmin needed to be present at 970 and 1800 times, respectively, the molar concentration of the asialo derivatives to get 50 ~ binding inhibition.

Second, a-lactalbumin, a protein known to modify the acceptor specificity of galactosyltransferase from N-acetylglucosamine to glucose [19], inhibited the binding of asialofetuin to liver plasma membranes. These authors concluded [37] that the naturally occurring liver plasma membrane receptor for asialofetuin, prior to its phagocytosis and degradation, is a plasma membrane galactosyltransferase. Since asialofetuin has a terminal galactose residue, end product binding to galactosyltransferase was the suggested mechanism for asialofetuin binding.

In a responding communication, Hudgin and Ashwell [38] found no sialyl-, galactosyl- or N-acetylglucosaminyltransferase activity associated with the purified plasma membrane receptor protein. Considerable glycosyltransferase activity did exist in the original Triton X-100 extract under optimal conditions. While the asialoglycoprotein binding protein was purified almost 200 times utilizing an asialoorosomucoid affinity column, most transferase activity, over 90~ of the original extract activity, could be recovered in the initial effluent.

Hudgin and Ashwell [38] confirmed the inhibitory activity of a-lactalbumin on the binding of asialoorosomucoid to purified binding protein, but failed to demon-

strate any inhibitory action on crude extract galactosyltransferase activity utilizing asialo, agalactoorosomucoid as acceptor. However, the crucial enzymatic activity, that of glucose:galactosyltransferase was not assayed in the presence of a-lactalbumin using purified binding protein. The bulk of the data suggests that glycosyltransferases are not directly involved in glycoprotein binding to liver plasma membranes. The fact that a-lactalbumin inhibits this binding could mean that the binding protein and galactosyltransferase are related, but this is pure speculation at present.

HD. Glycoprotein reabsorption in kidney tubules Specific absorption of soluble glycoproteins is characteristic of kidney tubules,

as well as liver parenchymal cells. Here, however, unlike the liver, absorption is of low molecular weight glycoproteins that have passed through the glomerular filtration barrier. The potential role of plasma membrane, or renal tubule, glycosyltransferases in this specific glycoprotein reabsorption, as well as in the biosynthesis of glomerular and tubule basement membrane, is being investigated [119]. Data indicate that intact glomeruli and isolated kidney plasma membranes demonstrated considerable glycosyltransferase activity.

Traditional plasma membrane markers were enriched to the following degrees, on isolated kidney plasma membranes, relative to the tissue homogenate; alkaline phosphatase, 1.11; UDPase, 2.60; 5'-nucleotidase, 3.72; (Na ÷ + K÷)-ATPase, 11.4. Collagen: glucosyl-, fetuin: galactosyl- and fetuin: sialyltransferases were also enriched in these membrane preparations 1.89-, 5.62- and 4.28-fold, respectively. These transferase activities were demonstrable on intact isolated glomeruli, with specific activities of 0.2--0.5 those in homogenized preparations. Since glomeruli are essentially cellular aggregates, their decreased specific activity relative to the homogenate may simply be due to an inaccessibility of the cell surface during the transferase assay.

These authors have previously shown [120] that folic acid is able to stimulate apparent glycosyltransferase activity, although the precise mechanism of folate transferase stimulation is not yet clear [121]. In both isolated kidney plasma membranes and glomeruli, 1.4 mM folic acid stimulated glucosyl- and galactosyltransferase activities ~2-fold, and inhibited sialyltransferase activity to about the same degree.

Unfortunately, no controls were included in these studies to show that sugar nucleotides were the authentic sugar donors or that enzyme activity was surface localized on intact glomeruli. Nevertheless, the enrichment of transferase activity on isolated kidney plasma membranes suggests their presence on cell surfaces. The role of these transferases in normal renal physiology, whether in glycoprotein reabsorption or basement membrane biosynthesis, has yet to be determined.

liE. Concanavalin A binding sites Concanavalin A, a lectin isolated from jack beans, is thought to bind cell

surface a-mannoside residues, thereby mediating diverse biological effects [6]. Malignant cells are preferentially agglutinated by polyvalent concanavalin A, unlike

their normal counterparts which are less readily agglutinated unless treated with protease. Binding of monovalent concanavalin A to malignant cell surfaces appears to slow their growth and migration in vitro [6,39]. Similarly, migration of primordial germ cells can be altered in vivo after monovalent concanavalin A treatment [40]. The receptor at the cell surface to which concanavalin A binds is being investigated in Isselbacher's laboratory [41,42] among others. They have indicated that at least one of the cell surface residues to which concanavalin A binds on agglutinating cells appears to be a galactosyltransferase.

Cells that were readily agglutinated by concanavalin A (rabbit erythrocyte ghosts, intestinal crypt cells) showed high galactosyltransferase activities. Similar cell types that were not concanavalin A agglutinable (human erythrocyte ghosts, intestinal villus cells) showed negligible enzyme activity. When concanavalin A agglutinable cells were pretreated with concanavalin A, galactose transfer decreased by 5 0 ~ but other membrane-bound glycosyltransferase activities (N-acetylglucosaminyl- and N-acetylgalactosaminyltransferase) were unaffected. A partially purified galactosyltransferase from rabbit erythrocytes was precipitable with concanavalin A as determined by Ouchterlony immunodiffusion and by direct precipitation in poly- acrylamide gels. Furthermore, a concanavalin A-galactosyltransferase interaction was demonstrated by a ten-fold purification after elution from a concanavalin A/ Sepharose affinity column.

The ability to confer concanavalin A agglutinability to normally non-agglutinable cells was shown after purified rabbit erythrocyte galactosyltransferase was absorbed onto human type 0 erythrocytes [41]. Evidence for enzyme absorption onto these cells arises from (1) previously galactosyltransferase-negative erythrocytes which exhibit active galactosyltransferase after treatment and (2) enzyme activity that disappears from the supernatant after incubation with the cells. Enzyme absorption alone did not initiate agglutination, but when concanavalin A was added to these treated cells, they were agglutinated. Other glycoproteins, extracted from the rabbit erythrocyte ghost, were unable to initiate concanavalin A agglutinability when incubated with type 0 human erythrocytes.

The observed correlation between concanavalin A agglutinability and galactosyltransferase activity was further studied using thymus and spleen lymphocytes [42]. Thymus lymphocytes were agglutinated by much lower concentrations of concanavalin A than were necessary to agglutinate spleen cells. Conversely, wheat germ agglutinin agglutinated spleen lymphocytes more readily than thymus cells. However, both thymus and spleen cells possessed active cell surface galactosyltransferases.

Enzyme activity expressed per cell or per mg protein suggested that spleen lymphocytes possessed higher enzyme levels, although activity expressed per cm 2 of cell surface indicated that the concanavalin A-agglutinable thymus cells exhibited slightly higher specific activity.

Three lines of evidence suggested that enzyme activities were cell surface localized. (1) Enzyme activities were expressed on intact lymphocytes that were able to exclude trypan blue and undergo blast transformation. (2) The heavy mole-

cular weight exogenous acceptor utilized became glycosylated and accumulated in the medium. (3) During the course of a 2-h preincubation, detectable enzyme activity was not released into the surrounding medium. Partial kinetic characterizations were conducted and labeled product identification demonstrated the product radioactivity to have been galactose.

Concanavalin A blast transformation of thymus cells leads to a subsequent elevation in surface galactosyltransferase activity. These studies agree well with other observations [32] from the same laboratory that indicated higher surface transferase activities on rapidly dividing intestinal crypt cells than on nondividing mature villus cells.

Podolsky and Weiser [43] have been able to define further the role of surface galactosyltransferases in concanavalin A agglutination of erythrocytes. Purification of the galactosyltransferase was based upon extensive sonication and chromatography on concanavalin A/Sepharose followed by UDPSepharose. The resultant enzyme was purified 620-fold relative to the membrane-bound preparation. Complete kinetic characterization indicated an Mn 2+ requirement for activity and a Michaelis- Menten constant of 7.5 ' l0 -6 M for UDPgalactose as substrate.

Carbohydrate analysis of the purified enzyme revealed a mannoside constituent, among other sugars. Consequently, it was suspected [43] that concanavalin A binding to galactosyltransferase may be mediated by the mannoside residue. Con- sistent with this were studies utilizing heat- and p-chloromercuribenzoate-inactivated transferases. These treated enzymes were still capable of conferring concanavalin A agglutinability onto human erythrocytes. Treatment with a-mannosidase, an exoglycosidase cleaving terminal a-mannoside residues, released carbohydrate identified as mannose. Demannosized galactosyltransferase was unable to confer concanavalin A agglutinability to human erythrocytes. Transferase treated with heat-inactivated a-mannosidase still possessed the ability to bind concanavalin A.

These data indicated that at least one of the cell surface concanavalin A receptors was a galactosyltransferase. Concanavalin A-enzyme binding appeared to involve a-mannoside residues and was not dependent upon enzymatic activity. While not all cell surface galactosyltransferase-positive cells were concanavalin A agglutinable (example, PyNIL) all concanavalin A-agglutinable cells tested expressed surface galactosyltransferase activity [41].

The degree to which the diverse biological effects of concanavalin A binding to cell surfaces is caused by binding to surface galactosyltransferases has yet to be determined.

III. SURFACE GLYCOSYLTRANSFERASES IN DEVELOPMENT

IIIA. Embryonic neural retina

The mechanisms that govern cellular interactions during morphogenesis are still to be determined. Much attention, however, has been given to adhesive specificity

between embryonic cells. Numerous in vitro systems have been developed to measure the degree of this adhesive specificity between various cell types (for review, see ref. 44). Evidence suggests that, in some cases, surface glycosyltransferase-acceptor binding mediates the observed recognition.

Cell surface galactosyltransferase activity was reported on embryonic neural retina cells by Roth et al. [21] and McGuire [45]. It was suggested that these surface enzymes might underlie the adhesive specificity typical of these cells by binding to their naturally occurring substrates on the neighboring cell surface. These enzymes and substrates represented specific components for the "lock and key" models of cell recognition postulated much earlier by both Weiss [46] and Tyler [47].

Neural retina galactosyltransferase activity [21,45] was optimized and the labeled endogenous product was identified as authentic galactose transferred to primarily glycoprotein acceptors.

Five observations suggest that the observed galactosyltransferase activity was cell surface localized.

(l) All enzyme assays were conducted in 10 mM NaN3 and utilized intact neural retina cells. Although 10-20 ~ of the cells tested took up trypan blue after 4 h of incubation, there was no detectable drop in cell number.

(2) Large molecular weight mucin and glycoprotein acceptors were galactosylated by intact cells and could be recovered in the extracellular medium after incubation.

(3) After preincubating intact cells in incubation medium without added UDP[14C]galactose and removing all cells from the medium by centrifugation, negligible enzyme activity could be found in the supernatant, using both large molecular weight mucin and glycoprotein acceptors, as well as N-acetylglucosamine.

(4) Homogenized cell activity represented 109-125 ~ of intact cell endogenous and exogenous activity. However, Triton X-100 was able to stimulate homogenate activity so that it represented 256 ~ of endogenous, and 714 ~o of exogenous, acceptor activity in whole cells. Therefore, under usual incubation conditions, i.e. without detergent, almost all of the cells would have to be lysed to account for the observed "intact cell" activity. Autoradiographic examination of a neural retina suspension incubated with UDp[aH]galactose indicated that greater than 90~ of the cells was galactosylated.

(5) Incubations in the presence of 5-fold excess unlabeled galactose-l-P or free galactose failed to lower the radioactivity associated with the product. If sugar nucleotide could be transported across the plasma membrane intact, which is not believed to be the case [48], internal galactosyltransferase could utilize the transported UDP-[t4C]galactose. The substrate, UDPglucose, should equilibrate with internal pools of UDP[14C]galactose via UDPglucose-4'-epimerase. However, a 5-fold excess of unlabeled UDPglucose did not affect product radioactivity.

Consistent with the possibility that these cell surface glycosyltransferases are involved in the adhesive specificity expressed by neural retina cells, were observations utilizing low molecular weight exogenous acceptors. A collecting aggregate assay for

adhesive specificity [49] was employed, .in which unlabeled neural retina aggregates were incubated with 32p-labeled dissociated neural retina cells. When low molecular weight galactosyltransferase exogenous acceptors were present in the collecting aggregate medium, the adhesion of labeled neural retina cells to neural retina aggregates was inhibited. The degree of inhibition was not great (20-50 ~) but was consistently observed with N-acetylglucosamine, N-acetylgalactosamine and fl-linked phenyl N-acetylglucosaminide. No inhibitory effects were observed with a-linked phenyl N-acetylglucosaminide, N-acetylmannosamine, lactose or N-acetyllactosamine, none of which were galactosyl acceptors.

A neural-retina-specific cell-aggregating factor has been isolated by Moscona and colleagues (see ref. 50 for references). This macromolecular glycoprotein complex, released from the surfaces of dissociated ceils, has been postulated to be similar, if not identical, to the naturally occuring surface adhesive ligands. In the light of the evidence [21,45] implicating surface galactosyltransferases in neural retina adhesive recognition, Garfield et al. [50] assayed partially purified neural-retina-aggregating factor for galactosyltransferase and acceptor activity.

Exogenous galactosyltransferase activity, present in the crude factor preparation, subsequently failed to copurify with the aggregating factor. A particulate retina galactosyltransferase preparation was added to the purified factor to test for acceptor activity, but no endogenous acceptor was demonstrable. The presence of the aggregating factor also failed to influence endogenous or exogenous galactosyltransferase activity in neural retina cell suspensions. These data indicate that the neural retina aggregation factor is probably not an asialo-ovine submaxillary mucin: galactosyltransferase or a galactose acceptor. Of course, the experiments [50] are not addressed to the possibility that the factor may be a galactosyltransferase with specificity for a different acceptor, e.g. glycoprotein [8,9] or, for that matter, any other transferase or acceptor.

Cell surface glycosyltransferase activities have been described in other neural tissue besides embryonic neural retina. An age-dependent glycoprotein:galactosyltransferase has been indicated on rat neuronal cell bodies [51 ]. Also, synaptosomal preparations of embryonic chick brain were reported [52-54] to have high glycosyltransferase activities and it was hypothesized that these enzymes may partially mediate neuronal specificity [55]. This area has been recently reviewed [56].

IIIB. Amoeba aggregation The possibility that surface glycosyltransferases may mediate adhesive specificity

in amoebae was investigated by Hoover [57]. In addition to microelectrophoresis, phytohemagglutinin and glycosidase studies, glucosyl-and galactosyltransferase activities were measured in putative plasma membrane preparations from two amoeba strains.

Surface galactosyl- and glucosyltransferases varied according to the adhesiveness of these strains. That is, more galactosyltransferase activity was present on the more adhesive amoeba type. Glucosyltransferase activity was predominantly located on

the less adhesive strain. Unfortunately, enzyme assay conditions were not shown to be optimal, and UDPgalactose hydrolysis and glucosyltransferase specific activities were not described in sufficient detail to make these data interpretable.

IIIC. Embryonic fiver Arnold et al. [58] investigated the correlation of age-dependent changes in liver

cell surface glycosyltransferases with the progressive decrease in reaggregation competence characteristic of dissociated liver cells. Five different endogenous transferase activities were expressed using intact cells. Utilizing preliminary assay conditions, mannosyltransferase was more than twice as active as galactosyltransferase. N-Acetyl- glucosaminyl-, sialyl- and fucosyltransferases showed activities decreasing in that order. However, these transferase activities may be an underestimate since the liver cells were assayed immediately after trypsinization.

Mannosyltransferase activities were optimized and, as with other systems, substrate (GDPmannose) levels were routinely subsaturating because of the high concentration (8 • 10 -4 M) required for saturation. Besides utilizing intact cells during the incubations, enzyme activities were thought to be surface-associated by the following two criteria. (1) Under conditions that facilitated GDPmannose degradation (i.e. no divalent cation) to mannose-l-P and free mannose, no endogenous product formation could be detected. (2) Incubations substituting [~H]mannose for GDP- mannose also failed to produce any endogenous product. Mannosyltransferase activity was decreased only 40 ~ in the presence of 100-fold molar excess of unlabeled mannose. These results speak against the hydrolysis, internal resynthesis and subsequent utilization of GDPmannose.

Of the product radioactivity, 70-85~ was soluble in organic solvents. In addition to GDPmannose, mannose-l-P and mannose, three unidentified mannose derivatives were detectable in their thin-layer chromatographic system. The least of the three, product C, was present at all ages tested and remains unidentified. Products A and B were most demonstrable between ages 11 and 16 embryonic days. Mild acid hydrolysis of product B revealed mannose-l-P and mannose constituents. Product A was tightly bound to the cells, could not be extracted with lipid solvents from aqueous incubation mixtures, could be chloroform/methanol/water-extracted from lyophilized reactions, and could act as a sugar donor substrate in the mannose incorporation assay. These authors considered the possibility that they were dealing with a mannosyl lipid intermediate identified in similar systems (see below).

Surface mannosyltransferase activity in intact liver cells of varying ages revealed an age-dependent decrease in endogenous activity. These studies agree with those [59] on isolated rat liver plasma membrane transferase activities. Glycoprotein: galactosyltransferase specific activity decreased 5-fold from 17 embryonic days to 35 post-natal days. Whether this decreased enzymatic activity is at all associated with the decreased reaggregation competence of these cells is not known. However, the evidence presented supports the existence of surface transferases on embryonic liver

cells and shows that some of these vary with age as does adhesive competence in this

tissue.

IIID. Gamete recognition Sperm and egg surface carbohydrates have been implicated in the specific

adhesion manifested by these cells [60]. It seems both plausible and testable that Lillie's classical anti-fertilizin-fertilizin model for sperm-egg adhesion may involve surface transferases and carbohydrate acceptors. Consistent with this is a report of enzyme-substrate complimentarity in the union of Chlamydomonas gametes [61].

Gamete recognition in this unicellular green algae occurs initially as a result of flagellar membrane adhesion [61]. The adhering flagella break their connections after a cytoplasmic bridge forms between the two cell bodies. These newly separated flagella are no longer adhesive to other gametes.

Ftagellar membrane vesicles, which are naturally found in the cell medium, are capable of agglutinating with the opposite mating type. Membrane vesicles from two opposite mating types of Chlamydomonas moewussi, as well as intact gamete cells, were investigated for the presence of surface glycosyltransferase activities [61]. Six glycosyltransferase activities toward endogenous acceptors were indicated on these membrane vesicles and gametes from both ÷ and - - mating types. All assays utilized a common set of incubation conditions in salt concentrations that were definitely not physiological for the algae.

Nevertheless, mixing equal aliquots of both mating type vesicles resulted in a four- to eight-fold stimulation in endogenous transferase activity over the expected intermediate value. Solubilization of membrane vesicles with Triton X-100 resulted in not more than a two-fold stimulation of endogenous activity. Upon mixing these solubilized membranes, less stimulation occurred than when mixing intact membrane vesicles.

Whole gamete cells were also assayed for surface transferase activity and, as with the membrane vesicles, all six transferase activities were detected but at a lower specific activity. Mixing + and - - gametes produced a two- to three-fold increase in all transferase activities above the expected intermediate value, except for fucosyltransferase activity, which remained constant.

Vegetative, nonsexual cells that do not adhere to vegetative cells of the opposite mating type showed some transferase activities but were not stimulated when opposite mating types were mixed. Likewise, Triton-treated gametes and vegetative cells did not show stimulation upon mixing + and - - cells.

The stimulated glycosyltransferase activities observed upon mixing + and - - mating types suggest enzyme-substrate complimentarity between mating gametes. However, no controls for substrate hydrolysis were included which poses a potential problem in interpreting these data. The observed stimulation upon mixing ÷ and - - gametes may have been due to a particular segregation of active phosphatases on one mating type and phosphatase inhibitors on the opposite mating type. Upon mixing, the inhibitor from one type may have inactivated the other's phosphatase,

permitting much greater apparent transferase activities on the mixed cell types. Similar situations have been observed in the case of some slime mold species [62].

However, the ability to assay transferases on isolated membrane vesicles and the marked stimulation of activity upon mixing either gametes or membrane vesicles indicate that surface transferases do occur and may be involved in gamete recognition. The authors point out [61] that catalysis of the enzyme-substrate complex between two adjoining flagella could separate the flagellae and yield membranes that are no longer able to agglutinate with other gametes.

IIIE. Gastrulating chick embryos Gastrulation is the single most complex example of cellular interactions. The

chick embryo, however, is amenable to autoradiography and invites an investigation of glycosyltransferase-acceptor complexes in the gastrula. Incubation of early chick embryos with 3H-labeled sugar nucleotides indicated in a preliminary investigation [63] that enzyme-substrate complexes were associated with migrating and inductive cell types. Autoradiographic examination of whole mount chick embryos, after 3H-labeled sugar incorporation, suggested that endogenous glycosyltransferase activities showed both temporal and spatial specificity within the embryo. Galactosyl-, N-acetylglucosaminyl- and glucosyltransferase activities were reported in the notochord-somite-lateral plate mesoderm vicinity, within the optic cup-skin ectoderm junction and in the area vasculosa.

More recently (Shur, B. D., unpublished data), these studies have been extended to include enzymological controls that suggest sugar nucleotides were the substrates directly involved, that enzyme activities were surface associated, and more directly implicate particular cell types utilizing a wider variety of sugar nucleotides. Serial section autoradiographs, rather than whole mount preparations, indicated as before that migrating and inductive cell types expressed the most active surface transferase activities.

The observed patterns did not result from sugar nucleotide binding to the cell surfaces, since (1) the sugar nucleotide patterns were highly specific, (2) some sugar nucleotides, including UDPglucose, produced no detectable incorporation, (3) a 1000-fold molar excess of "chase" unlabeled sugar nucleotide failed to alter grain patterns, and (4) these patterns show time, temperature and cation dependency.

That the substrates directly employed by these embryos were the sugar nucleotides and not the sugar-l-P or free sugars comes from four observations. (1) When incubations were conducted substituting 3H-labeled sugars for the sugar nucleotides, at similar concentration and specific activity, less specific grain patterns resulted than from the parent sugar nucleotide. Additionally, [3H]glucose produced very heavy incorporation whereas UDp[3H]glucose, at the same concentration and specific activity as [3H]glucose, produced only a barely detectable grain activity. (2) Embryo- incubations that were directly assayed for hydrolysis of sugar nucleotides to free sugars indicated that 8 0 ~ or more of the original sugar nucleotide was still intact after a 4 h incubation. When incubations were designed to mimic the potential

contribution of this 10-20 ~o 3H-free sugar component (20 700 all-free sugar and 80 ~o unlabeled sugar nucleotide for the entire 4 h) only negligible grain patterns resulted. (3) An unlabeled excess of phosphatase substrate, such as a different sugar nucleotide, which inhibited the 20 70 free sugar production, did not interfere with the observed sugar nucleotide patterns. (4) Phloridzin, a sugar transport inhibitor, blocked free sugar utilization without decreasing sugar nucleotide activity significantly.

As mentioned above, UDP[aH]glucose failed to produce significant incorporation. This observation also speaks to the extracellular utilization of the sugar donors. Since sugar nucleotide synthetases and epimerases are internally located (for review, see ref. 64) then similar activities should have been observed with both [all]glucose and internalized sugar nucleotide, but this was not the case. The final glycosylated products of the various transferases were also externally located, since grains were clearly peripheral to cell surfaces, or in the extracellular spaces.

Of those glycosyltransferases assayed, galactosyl-, N-acetylglucosaminyl- and fucosyltransferases were most active. Glucuronyltransferase showed some activity, while glucosyl-, N-acetylgalactosaminyl- and sialyltransferases produced negligible grains. Examples of the endogenous product locations and the transferases giving heavy labeling are (a) primitive streak primary mesenchyme cells (galactosyl and fucosyl), (b) newly aggregating somites (galactosyl), (c) mature somites (N-acetylglucosaminyl), (d) precardiac mesoderm (N-acetylglucosaminyl), (e) neural crest (galactosyl, N-acetylglucosaminyl, and fucosyl) and (f) primordial germ cells (galactosyl and N-acetylglucosaminyl).

Taken together, these temporally and spatially specific patterns are suggestive of cell surface glycosyltransferase activities that are localized primarily on migrating cells as well as on cells undergoing inductive interactions. It is possible that these cell types utilize their cell surface enzymes to accomplish, at least in part, these phenomena.

Migrating cells may use a carbohydrate matrix as a substrate. For example alcian blue staining of glycosaminoglycans was demonstrated in the vicinity of migrating neural crest cells (Weston, J., unpublished data) and autoradiographic detection of [aH]glucosamine incorporation into chick embryos demonstrated a carbohydrate matrix deposited within the cranial extracellular space prior to neural crest migration into this area [65]. Primordial germ cells, mesenchymal cells and neural crest cells may bind these substrate oligosaccharides with their surface glycosyltransferases allowing the cell to advance in the direction of increasing enzyme- substrate formation. Complexes at the trailing end of the cell may be broken through catalysis.

A mechanism of this type could also explain some inductive interactions in embryos. Lash and coworkers [66] have indicated that notochord and ventral neural tube proteoglycans appear to be potent inducers of somite chondrogenesis. Auto- radiographic analysis of chick embryo glycosyltransferase activity showed that mature somites, suspected of undergoing induction, showed high levels of N-acetylglucosaminyltransferase activity [63]. These monosaccharides are known residues in

proteoglycans [67]. Incomplete side chains of these glycosaminoglycans may be acceptors for cell surface transferases (receptors) on inducible tissues.

Hay and coworkers [68] have investigated the distribution of collagen in chick embryo inductive systems. They have hypothesized that collagen fibrils may act as an inducer in some of these interactions. Since investigators [22-25] have suggested that platelet binding to incomplete collagen side chains is initiated by surface glucosyltransferases, Hay [68] has likewise suggested that these same surface glucosyltransferases could act as receptors on inducible tissues for collagen binding.

IV. SURFACE GLYCOSYLTRANSFERASES ON CULTURED CELLS

IVA. Activities on normal and malignant cells

The metastasizing ability of a malignant cell is ultimately manifested at the cell surface. Since surface carbohydrates are likely to be involved in cell social phenomena, the glycosyltransferase composition of transformed cells has received much attention. In order to define the localization of some glycosyltransferases in subcellular fractions, Hagopian et al. [69] assayed seven HeLa cell membrane fractions from a discontinuous sucrose gradient. The composition of all fractions was determined by enzyme markers, chemical analysis including cholesterol:phospholipid ratios and electron microscopy.

Three glycosyltransferase activities, with endogenous and exogenous acceptors, were investigated. Smooth membrane fractions exhibited polypeptide:N-acetylgalactosaminyl- and glycoprotein:galactosyltransferase activities, purified 47-fold and 28-fold, respectively, compared to the homogenate. Plasma membranes, devoid of these two transferase activities, exhibited collagen:glucosyltransferase activity, purified 145-fold, which compared well with the 120-fold purification of 5'-nucleotidase. Further evidence for a differential localization for these transferases comes from experiments with detergents. Both N-acetylgalactosaminyl- and galactosyltransferase activities were stimulable by Triton X-100. Glucosyltransferase activity toward collagen remained unaffected.

Soon afterwards, Bosmann [70] reported similar data indicating a plasma membrane localization for over 5 8 ~ of HeLa cell collagen:galactosyltransferase activity. Final enrichment was over 160-fold. Comparable studies by Molnar et al. [71] cited evidence for the existence of plasma membrane galactosyl- and N-acetyl- hexosaminyltransferases on Ehrlich ascites tumor cells.

When glycosyltransferases were measured on intact cells [21] rather than in plasma membrane preparations, Roseman [8,9] proposed an enzyme-substrate model for recognition phenomena (Fig. 2), and considerable interest developed concerning malignant cell surface transferase activities. Indeed, more literature has appeared concerning transferase activities on cultured normal and malignant cell surfaces than in any of the above systems. As in cellular homogenate studies [72-87], these investigations fail to show any clear correlation between surface activity and trans-

formation. Intact cell transferase activities have been shown to increase after viral transformation of chick embryo fibroblasts [79,80], decrease after transformation of hamster [81] and murine [82] fibroblasts, and in two other reports [83,84] on mouse cells to show an increase, decrease or no change at all.

Bosmann et al. [79] and Morgan and Bosmann [80] studied cell surface transferase activities on chick embryo fibroblasts and their viral transformants. Seven cell lines were employed and all cells were harvested from confluent cultures. Initially [79] four of these cell lines were screened for intact cell enzyme activity. Under common incubation conditions, endogenous galactosyl- and mannosyltransferase activities were most active. Glucosyl-, N-acetylglucosaminyl-, N-acetylgalactosaminyl- and sialyltransferase activities were less active, but demonstrable. Other sugar nucleotides showed negligible incorporation.

Galactosyl and mannosyl enzyme activities were partially characterized and suggested to be surface localized from two observations. First, either pre- or post- treatment with 0 .25~ trypsin for 15 min inhibited activity significantly. Second, heavy molecular weight exogenous acceptors were able to specifically interfere with endogenous incorporation.

Endogenous galactosyl and mannosyl activities on transformed cells were approximately ten times higher than on either normal chick embryo fibroblasts or on fibroblasts infected with a nontransforming, Rous-associated virus. Likewise, cells infected with a temperature-sensitive virus exhibited enzyme levels comparable to the transformed cell line at the permissive temperature. These values fell to normal levels at the nonpermissive temperature of 41 °C. Enzyme stability at 41 °C was demonstrated by assays on the other cell lines at this temperature.

Consequently, these authors suggested [79] that the increased endogenous glycosyltransferase activity on virus-infected cells resulted from transformation rather than from viral infection.

Morgan and Bosmann [80] employed four strains of Rous sarcoma virus to vitally transform chick embryo fibroblasts. As above [79] all resulting cell lines showed a four- to 14-fold increase in endogenous galactosyl- and mannosyltransferase activity over normal cells. These authors suggested that the degree of loss of contact inhibition in these four virally transformed cell lines did not correlate well with their surface glycosyltransferase activities.

The two most transformed strains exhibited an increase in cell surface transferase activity similar to those of the cell lines which still maintained some degree of contact inhibition. These conclusions must be considered preliminary, however, since no effort was made to opt imize for enzymatic conditions or to determine whether differential sugar nucleotide hydrolysis could account for the observed transferase activities.

Sasaki and Robbins [81] investigated cell surface sialyltransferase activities on normal and polyoma virus-transformed hamster cells. Chromatographic analysis of 5-h reaction components showed 79 ~o of the original substrate, CMPNeu, remained, 19~o was hydrolyzed to neuraminic acid and 0 . 5 ~ was heavy molecular weight

product. It appears that CMPNeu was the substrate for the observed incorporation, rather than free sialic acid, since incubations with [3H]neuraminic acid, rather than CMP[aH]neuraminic acid, resulted in no product radioactivity and since an 8.8-fold molar excess of unlabeled neuraminic acid did not interfere with CMp[3H]neuraminic acid activity.

Further evidence that surface sialyltransferase activity was being assayed comes from the observation that trypsin-harvested cells had 37 ~ less activity than ethyleneglycol bis (a-aminoethylether)-N,N'-tetraacetic acid (EGTA)-harvested cells. Neura- minidase pretreatment of cells resulted in a three-fold increase in neuraminic acid incorporation, but did not affect other assayed glycosyltransferase activities. Finally, activity was not increased upon homogenizing the cells, and was inhibited by adding 0.1 ~ Triton to the homogenate.

Throughout all sialyltransferase assays, normal and polyoma-transformed hamster fibroblasts displayed similar kinetics. However, transformed cells continually showed approximately half the endogenous product activity of normal cells.

After the incorporated radioactivity was identified as authentic sialic acid, pro- nase digests of the material were chromatographed on Sephadex G-50. Glycopeptides from both transformed and sparsely grown normal cells were slightly enriched in high molecular wight components, relative to digests from densely grown normal cells.

CMPNeu labeling of cell surfaces was examined by Datta [82] on normal and temperature-sensitive transformed cell lines. Well characterized sialyltransferase assays on monolayer cultures seemed to be measuring surface activities since a 1000- fold molar excess of unlabeled neuraminic acid did not affect product radioactivity and free [14C]neuraminic acid incubations resulted in only 10-15~ of the product obtained with CMPNeu. Also, neuraminidase pretreatment stimulated activity two to three times, and post-treatment with neuraminidase removed most incorporated radioactivity. The initial rate of uptake of CMPNeu into acid-soluble pools was only 25 ~ that incorporated into acid-insoluble products. Finally, various inhibitors such as phlorizin, sodium cyanide and iodoacetate did not interfere with CMPNeu product activity. Enzymatic and acid hydrolysis of the product indicated sialic acid incorporation into glycoprotein and glycolipid acceptors.

The temperature-sensitive transformed BHK cells, when grown at the permissive temperature of 32 °C, incorporated half the sialic acid that these cells incorporated when grown at 38.5 °C, the non-permissive temperature. The ability of transformed phenotype cells to demonstrate half the surface sialyltransferase activity of normal phenotype cells was found when cells were assayed at either 32 or 38 °C. Contrary to temperature-sensitive polyoma transformed BHK cells, normal BHK and 3T3 cells showed a two-fold stimulation of activity when grown at 32 °C rather than at 38.5 °C. These results suggest [82] that the transformed phenotype was associated with decreased endogenous sialyltransferase activity.

Patt and Grimes [83] determined the optimal glycosyltransferase activities present on surfaces of normal and virally transformed 3T3 mouse fibroblasts. They were careful to demonstrate that their activities were due to cell surface enzyme

activities rather than uptake and utilization of sugar nucleotides or their degradation products. Adding unlabeled exogenous free sugars or sugar phosphates had no effect on incorporation from labeled sugar nucleotides. Detergent disruption of whole cells did not significantly increase activity, suggesting that a few broken cells could not account for the observed whole cell activity. Consistent with surface activity were results from experiments using free, 3H-labeled sugars. Free galactose appeared immediately in the soluble fractions of the cell. Incorporation into heavy molecular weight acceptors occurred only after long incubation times and appreciable label uptake. UDPgalactose activity, however, immediately appeared in acid-precipitable extracts and was not present in soluble form for a considerable time. In double label experiments with [3H]galactose and UDP[14C]galactose, aH could be detected in glucose whereas no epimerization of [14C]galactose could be detected. Therefore, UDPgalactose incorporation probably occurred extracellularly. Hydrolysis of other radioactive products indicated no epimerized monosaccharides. Additionally, no breakdown of sugar nucleotides could be demonstrated, and no labeled sugar nucleotides could be found in internal pools after incubation.

The data indicate [83] that normal (3T3) and transformed (Py3T3, SV3T3) cells possess a wide spectrum of cell surface transferases but no clear quantitative variation correlates with transformation.

Patt and Grimes also tested the ability of 2.5 mg/ml concanavalin A to inhibit surface enzyme activity, as reported by Isselbacher and coworkers [41], as well as the ability of neuraminidase pretreatment to stimulate sialyltransferase activity. They found no effect with either agent.

Patt et al. [84] compared surface transferase activities on normal cells with two transformed cell types: cells that produce tumors which regress in immuno- competent hosts, and transformed cells that produce malignant tumors.

While maximal activities were generally lower in transformed cell lines, some enzyme activities showed no relationship to transformation or malignancy. The authors [84] suggested that simple measurement of surface activity does not allow one to predict the normal or malignant potential of cell lines.

An attempt has been made [85] to clarify the present state of affairs by stressing the need to fully characterize enzyme assays on cultured cells. Spataro et al. [85] showed that surface sialyltransferases on Rous sarcoma virus-transformed fibroblasts required enzyme conditions different from those required by normal chick embryo ceils. Depending upon the assay conditions employed, normal and malignant fibroblast surface transferase activity could be shown to change relative to one another. They suggested that a different surface environment distinguishes transformed cells from normals. Consequently, it is clear that assay conditions should be separately optimized before any conclusions can be drawn concerning total surface enzyme activity and transformation.

As was the case with glycosyltransferases in cellular homogenates of normal and transformed cells [72-78], cell surface glycosyltransferase activities show no clear correlation to transformation. As best exemplified by the data of Grimes and

coworkers [83,84], transformed cell surface glycosyltransferase activity can be increased, decreased, or equal to normal surface activity. Surface transferase assays that have been carried out in relation to particular cellular functions have fared better, however. In terms of surface repair mechanisms, cell adhesion and growth control, a number of correlations between function and transferase activity are becoming discernible.

IVB. Adhesion and repair of cell surfaces Surface glycosyltransferases and their endogenous acceptors on neural retina

cells [21], blood platelets [22-26,29] and algal gametes [61] have been implicated in cellular adhesion. Likewise, transferase activities on cultured cells have been linked to some adhesive phenomena. A partially purified galactosyltransferase has been shown [86] to markedly stimulate fibroblast-substrate adhesion. Cell surface sialyltransferases have been implicated in the repair of neuraminidase treated cell surfaces [81,82,85,87,88].

Pierce et al. [86] have identified a soluble galactosyltransferase activity associated with bovine fetuin. The purified enzyme displayed kinetics similar to a previously purified [14] soluble transferase, requiring Mn z+ and having a Km of 6.0' 10 -5 M with respect to UDPgalactose. Mouse 3T12 fibroblasts rapidly adhered to, and flattened on, a plastic substrate in the presence of 15/~g/ml of this purified enzyme (Fig. 3). An equivalent weight of fetuin, from which galactosyltransferase had been removed, did not stimulate 3T12 cell adhesions. These authors [86] suggested that the previously reported [89] cell flattening factor identified in fetuin is a contaminating serum galactosyltransferase that binds to fetuin via an enzyme-substrate interaction.

Neuraminidase treatment of rat dermal fibroblasts produced an increased adhesiveness between these cells. Lloyd and Cook [87] utilized this phenomenon to explore the possibility that neuraminidase stimulated adhesion by creating desialyzed cell surface components which acted as acceptors for adjacent cell surface sialyltransferases. Lectin agglutination studies on pre- and post-neuraminidase treated cells showed that fl-galactosyl (Ricinus eommunis agglutinin) and /%N-acetylgalactosaminyl (soybean agglutinin) residues were uncovered by neuraminidase treatment. These exposed sites may have been responsible for the observed adhesivity. Accordingly, glycoproteins with terminal fl-N-acetylgalactosamine residues (asialo- bovine submaxillary mucin) were shown to reverse the effect of neuraminidase treatment, presumably by competing for receptor sites with endogenous acetylated galactosamine residues. Ovalbumin (terminal fl-N-acetylglucosamine) and desialyzed fetuin (terminal/3 galactose) were ineffective. Asialo-bovine submaxillary mucin was shown not to exert its action by directly inhibiting neuraminidase.

Cell surface sialyltransferases bound the asialo-bovine submaxillary mucin since, in the presence of CMP [14C]sialic acid, whole cells catalyzed the sialylation of bovine submaxillary mucin. No enzyme leakage into the supernatant could be detected when incubations were conducted in phosphate-buffered saline. To test

Fig. 3. Mouse fibroblasts (3T12) 30 min after seeding. These cultures contain 15 ~zg/ml of either (A) fetuin: galactosyltransferase purified from commercial fetuin or (B) fetuin from which galactosyltransferase activity has been removed. In the presence of the active galactosyltransferase, 3T12 fibroblasts can be seen adhering to the plastic substratum long before such flattening is seen with enzyme-free fetuin, or in the absence of additions. Both cultures contain 1 mg/ml bovine serum albumin.

further the surface localization of this sialyltransferase, isolated plasma membranes were prepared and examined by electron microscopy, enzyme markers and lipid analysis [90]. These membranes exhibited sialyltransferase activity toward asialo- bovine submaxillary mucin. Plasma membrane galactosyltransferase could also be detected and utilized asialo-agalacto-fetuin as an acceptor. While all enzyme activities were appreciable, no effort was made to measure optimal activity. These data strongly indicate that cell surface glycosyltransferases and acceptors exist on these cells and suggest further that adhesion between the cells occurs via enzyme-substrate complexes.

Neuraminidase pretreatment of transformed cells was utilized by four groups [81,82,85,88] to study the resialylation of desialyzed cell surfaces. Bernacki [88] has examined intact cell sialyltransferases on leukemic cells before and after neuraminidase treatment. Enzyme pretreatment, shown to release sialic acid residues from intact cells, increased sialyltransferase activity six-fold. 7 5 ~ of the newly incorporated sialic acid was removed with a second neuraminidase treatment.

Excess unlabeled sialic acid did not inhibit [14C]sialic acid incorporation from CMP[14C]sialic acid, suggesting that the sugar nucleotide, rather than the free sugar, was the actual substrate. Consistent with this was the inability to demonstrate extracellular hydrolysis of CMPNeu to sialic acid. Chromatographic analysis of ethanol- soluble radioactivity (approximately 10 ~o of insoluble) extracted from cells indicated, on the other hand, that internal pools of free sialic acid increased during the incubation. This could be resulting from internal hydrolysis of CMPNeu that had leaked into the cell. However, the degree of this hydrolysis in intact neuraminidase treated cells, with a six-fold higher sialyltransferase activity, was half that of controls.

Cell surface sialyltransferases were suggested by the neuraminidase increase in sialyltransferase activity but, as with most intact cell studies, the possibility of sialylation from internal pools of sialic acid could not be definitely ruled out. The most likely interpretation, however, is that CMPsialic acid was utilized externally.

To demonstrate more clearly a plasma membrane localization tbr sialyltransferase, Porter and Bernacki [122] prepared electron microscope autoradiographs of cells after CMp[3H]Neu or [3H]galactose incubation. To make this feasible, leukemic L-1210 cells were neuraminidase pretreated to increase endogenous sialyltransferase acceptor activity 6-fold. Grain distributions over various cellular compartments were compared in cells incubated for 15 min in either 10 #M sugar nucleotide or free sugar. Over 8 3 ~ of the grains examined (780 grains counted) in CMP[ZH]Neu incubations was localized over the plasma membrane, while < 10~ was associated with the Golgi apparatus. A reciprocal distribution was found in [3H]galactose autoradiographs (850 grains counted): 64 ~ of the grains was over the Golgi membranes and only 10 ~ was associated with the cell surface.

Autoradiography necessarily localizes the transferase product, not the enzyme itself. However, controls have been included to insure that the observed plasma membrane grains were representative of the complete transferase system, enzyme as well as acceptor. 100-fold molar excess of unlabeled N-acetylneuraminic acid,

N-acetylmannosamine, mannosamine or mannose did not interfere with CMp[3H]Neu product radioactivity. < 1 ~ of the total radioactivity in the incubation medium could be identified as free [3H]sialic acid. Over 90~o of the incorporated radioactivity was neuraminidase sensitive, and was subsequently identified as sialic acid. Finally, the cell surface grains in CMp[aH]Neu autoradiographs were shown not to result from soluble enzyme leakage into the supernatant.

Taken together, the differential grain distributions in CMp[aH]Neu and [3H]galactose incubations, with the reported biochemical controls, clearly indicate CMPNeu to be the authentic sugar donor in these assays and that the transferase- acceptor complex exists on the cell surface.

In similar studies, neuraminidase pretreatment of hamster [81] and chick embryo [85] fibroblasts has stimulated surface sialyltransferase activities two- to six-fold. Consequently, these studies, as well as those discussed above, strongly indicate sialyltransferases on these cell surfaces that seem to be able to function as repair enzymes and as receptors for adhesive purposes.

IV(?. Cell-contact- and culture-density-dependent surface glycosyltransferase activity In order to understand more fully the biochemical basis of malignancy, in-

vestigators are increasingly turning to tissue culture systems for their studies. Trans- formed cells in vitro at least partially mimic their behavior in vivo. Normal fibroblasts tend to respond to contact by ceasing to divide. Under identical conditions, transformed cells fail to respond to these growth controls, and continue to multiply forming numerous cell layers. An understanding of the biochemical basis of these normal growth controls, and how transformed cells escape this contact-dependent signal is crucial to our understanding of cell interactions.

Evidence available indicates that this contact-mediated growth control may, in part, be initiated by glycosylation of surface acceptors via adjacent, cell surface glycosyltransferases (Fig. 4). A number of reports [91-97,123] have suggested that normal fibroblasts in culture preferentially glycosylated one another's surfaces on contact, utilizing endogenous sugar nucleotides. Transformed cells, on the other hand, were able to glycosylate acceptors on their own surfaces, and were more dependent on exogenously added sugar donors. As a result, endogenous activity decreases with increasing cell contact in normal cultures, while some transformed cells maintained high endogenous activity.

Roth and colleagues [91-94,123] have presented data indicating cell surface galactosyltransferase activity on normal 3T3 mouse fibroblasts and spontaneously transformed 3T12 cells. Surface endogenous activity on normal mouse fibroblasts was dependent upon the extent of cell contact in culture [91 ]. Cells taken from sparse cultures exhibited high endogenous activity, while confluent cultures showed reduced endogenous surface galactosylation. Transformed fibroblasts maintained a constant level of activity per cell independent of culture density. Normal cells showed negligible glycosylation of endogenous acceptors when incubated in suspension as compared to incubations in which the cells were allowed to pellet. Endogenous surface glyco-

N on-transformed cell recognition

J Transformation or normal cell in mitosis

UDPu -DpX~ ~ ' ~

Normal cells after contact in culture

Fig. 4. Model for cell-cell interactions between normal and between transformed cells. Recognition between non-transformed cells is on left, as in Fig. 2. Normal cells in mitosis, or transformed ceils at all stages of their cell cycle, are able to bind their surface acceptors with their own surface transferases, rather than bind acceptors on adjacent cells. At lower right, non-transformed cells in culture have glycosylated each other's surface, upon contact, through the addition of monosaccharide X.

sylation on malignant cells was unchanged in both suspension and pellet assays.

Autoradiographs were prepared on normal and transformed cultures after incubation with UDP[3H]galactose. Transformed fibroblasts expressed active incorporation over their surfaces while sparse normal cells produced negligible activity. However, pellets prepared of normal cells exhibited many grains on the cell periphery. These observations were extended [92] to include other surface glycosyltransferases

on these cells. After partial optimization for enzyme activity, N-acetylglucosaminyl- and sialyltransferase activities displayed similar contact requirements.

These results indicate that surface galactosyltransferases on normal cells were able

to glycosylate neighboring cell surfaces and did so. As a result, endogenous activity decreased with increasing cell density and sparsely grown cells required contact for

optimal glycosylation. Transformed fibroblasts, on the other hand, seemed to glycosylate themselves only when UDPgalactose was present, thereby producing a contact- and growth-independent transferase activity. Further studies (Webb, G. C. and Roth, S., unpublished data) indicated that contact-dependent glycosylation between cells diminished as a function of the saturation densities shown by these cells. Three different clones of 3T3 cells displayed saturation densities of 4.5, 8.6 and 18.0" 104 cells/cm 2. The ratio of contact-dependent glycosylation to contact- independent glycosylation for these clones was 2.2, 1.4 and 0.92, respectively. Malignant 3TI 2 cells, wich yielded the highest number of cells per dish, also exhibited contact independence for maximal glycosylation.

Controls for sugar nucleotide utilization and for contact-dependent glyco-

sylation between normal cells have been reported [91,93]. Four-fold molar excess of galactose-l-P, galactose or UDPglucose did not interfere with UDPgalactose activity. Transferase activity was considered to be surface localized, since enzyme activity towards exogenous acceptors resided with intact cells while the glycosylated product could be recovered in the cell-free supernatant. Additionally, leakage of transferases into the supernatant and hydrolysis of sugar nucleotides was minimal and equal in suspension and pellet incubations. Nor could differential cell viability resulting from two different incubation conditions explain the observed difference in glycosylation.

Another demonstration of 3T12 cell surface galactosyltransferase activity has come from Cebula and Roth [123]. Plasma membranes from malignant 3T12 fibroblasts have been isolated by homogenization and sucrose gradient centrifugation. Approx. 10 ~ of total cell homogenate glycoprotein: galactosyltransferase sedimented with 5'-nucleotidase and (Na ÷ q- K÷)-ATPase, both traditional plasma membrane markers. 100~ of galactosyltransferase activity towards desialyzed ovine submaxillary mucin, an activity not detectable with intact cells, was clearly separable from the plasma membrane markers at the other end of the sucrose gradient. A comparison of this mucin: transferase activity profile, thought to represent Golgi membranes, with the glycoprotein:galactosyltransferase profile suggests a plasma membrane localization for approx. 10~ of total glycoprotein:galactosyltransferase activity.

Bosmann and his colleagues have also analyzed cell surface glycosyltransferases on normal and transformed mouse fibroblasts [95] and melanoma cells [96] as a function of their culture density. Their studies, consistent with those above, indicate that on normal and some melanoma cells endogenous transferase activity decreased with increasing cell density, and that confluent transformed fibroblasts displayed higher endogenous activity than confluent normal cultures.

Initially, 3T3 mouse fibroblasts and lines transformed by murine sarcoma virus, Rous sarcoma virus and polyoma virus were assayed [95]. All cell types displayed surface glycosyltransferase activity when incubated with labeled nucleotide sugars. Partially characterized enzyme reactions suggested that 3T3 cells and their viral transformants possessed many surface transferase activities. Glucosyl-, mannosyl-, sialyl- and galactosyltransferase activities were higher than N-acetylglucosaminyl- and N-acetylgalactosaminyltransferases. Xylosyl- and arabinosyltransferases were not detectable.

Trypsin pretreatment of whole cells inhibited subsequent enzyme activity by 85-100~, and trypsin treatment after sugar nucleotide incubation removed 95-100~o of the incorporated radioactivity. Cell-associated soluble label was less than 10 ~ of the trichloroacetic acid-precipitable product indicating the lack of large cellular pools of label. Again, however, this observation does not rule out more rapid utilization of the internalized substrate than can be detected.

The data indicate that transformed fibroblasts displayed high endogenous transferase activity independent of culture density. Normal ceils from sparse cultures expressed similar specific activity to some transformed fibroblasts. 3T3 cells from

confluent cultures, on the other hand, exhibited less than half the activity of sparse cells.

Utilizing confluent cultures, Bosmann suggested [95] that enzymes as well as acceptors were increased on transformed cells. Surface glycosylation of exogenously added acceptors was somewhat more active with viral transformants than with normal fibroblasts, indicating higher sialyl-, N-acetylglucosaminyl- and galactosyltransferase levels on malignant cells. However, not all transformed cell lines produced an equivalent increase in exogenous enzyme activity. Collagen:glucosyltransferase was decreased on transformed cell surfaces.

While these results probably represent actual glycosyltransferase differences between sparse and confluent cultures of normal and transformed cells, they would have been more compelling if controls were included to rule out differential hydrolysis of sugar nucleotides. A decreased availability of substrate could conceivably explain the decreased surface transferase activity on densely grown normal cells.

Two different cell lines of melanoma origin, which showed different degrees of metastasizing ability, were assayed for various surface parameters [96]. The low- metastasis line produced fewer pulmonary tumors than those produced when cells from the high-metastasis line were injected. Endogenous glycosyltransferase activities were assayed on these two cell lines, as a function of their density in culture. The high-metastasis line, when taken from sparse cultures, contained 2-4 times the glucosyl-, galactosyl-, mannosyl- and sialyltransferase activities of either high-metastasis cells from confluent cultures or low-metastasis cells from either sparse or confluent conditions. That is, the highest metastasis cell line showed the highest endogenous glycosyltransferase activity. As extensive cell contact occurred, the endogenous transferase activity decreased to a level equal to that of the low metastasis cell. N-Acetylglucosaminyl- and N-acetylgalactosaminyltransferase activities were unaffected by culture density.

Internal utilization of the sugar nucleotide or utilization of its breakdown products have not been definitively ruled out. However, the data appear to be consistent with previously described results [91-95,123].

Similar to surface transferase activities were results[96] for total cell homogenate glycosidase, glycosyltransferase and proteolytic activity. Sparse high-metastasizing cells exhibited more active enzymes than sparse low-metastasizing cells or confluent cultures of either cell line.

The ability of normal cells, and the inability of malignant cells, to show contact- dependent glycosylation has been further investigated by Yogeeswaran et al. [97]. Glycosphingolipids were covalently linked to fine glass beads 2-10 ~m in diameter or onto cover slips. Normal BHK and NIL cells glycosylated these glass-bound carbohydrate residues upon contact. Cells utilized either endogenous or exogenous sugar donors to complete the reaction. Glycosylated glass beads and cover slips were extensively washed before counting, to remove any non-covalently attached material. Incubations with glass cover slips, or with glass powder, were shown not to be toxic. No glycosylation occurred to glass particles without attached corbohydrate.

A number of glycosyltransferase activities was indicated by the use of three different sugar nucleotides as well as with cells prelabeled with [14C]galactose. In all cases, polyoma virus-transformed BHK cells glycosylated the carbohydrate-glass complex less (20-50 ~o) than did normal cells. The ability to glycosylate glycolipid- glass particles upon contact under physiological conditions, employing endogenous sugar nucleotides synthesized from exogenously added [~4C]galactose, is consistent with the concept of contact-dependent glycosylation in normal cells.

Patt and Grimes [83] also measured surface glycosylation on normal cells in shaking and pellet incubations. Some glycosyltransferase activities (glycoprotein: sialyltransferase and glycoprotein: and glycolipid: galactosyltransferases) were equal in pellet and suspension incubations of growing 3T3 cells. On the other hand, two transferases (glycoprotein :N-acetylglucosaminyl- and glycoprotein:N-acetylgalactosaminyltransferases) were 50 ~ more active in pellet incubations. One activity (glycolipid :sialyltransferase) was 70 ~ higher in suspensions. However, in these experiments it is not clear that the shaking conditions employed actually minimized cell contact.

A functional significance for cell surface glycosyltransferases is strongly suggested by the demonstration of contact-dependent glycosylation between cells. While not all observations are consistent with this possibility [83,84], several well controlled studies have argued for the ability of normal cells to glycosylate neighboring cell surfaces, and the relative inability for these cells to glycosylate their own surfaces. On the other hand, some transformed cells appear to be able to glycosylate their own surfaces, and only when sugar nucleotides are added exogenously.

The ability of normal cells to glycosylate one another on contact agrees with studies by Hakomori [98] and Robbins and Macpherson [99] in which complex carbohydrates increased in complexity as cultured normal cells reached confluency. Transformed ceils showed less complex carbohydrates, similar to those of sparse normal cells, at all stages of culture density.

Strongly consistent with these studies are data presented by Nicolson and Lacorbierre [100]. They measured the number of receptor sites for the Ricinus

communis lectin (specific for fl-galactosides) on various cell lines. Of those cells examined, only normal 3T3 cells showed an increase in cell surface receptor sites as the cells became more confluent. The transformants, 3T12 cells among others, showed no such change in these exposed galactosides.

The ability of some transformed fibroblasts to glycosylate themselves, and the relative inability of normal cells to do so, is further clarified by lectin agglutination [101,102] and fluorescein-labeled antibody studies [103]. Agglutination of transformed cells with polyvalent lectins occurs at much lower concentrations than required to agglutinate normal cells, unless the normal cells are protease treated [6]. However, similar quantities of lectins bind to both cell types [101]. Subsequent studies [102] on pre- and post-fixed lectin-treated cells demonstrated that lectin binding sites were more clusterable on transformed cells than on normal cell surfaces. This suggested that lectin agglutination of transformed cells is made possible by clustering of binding sites allowing a greater number of adhesive sites to interact with one another. The

observed difficulty in lectin agglutination of normal cells may be the result of a relative inability of lectin sites to cluster on normal surfaces.

These studies, along with demonstrations of differences in membrane antigen mobility [103], suggested that the clustering of glycosyltransferases and acceptors on malignant cells may be analogous to clustering of lectin binding sites. In fact, Weiser and coworkers [41-43] suggested that one of the concanavalin A cell surface receptors is a galactosyltransferase. The inability of sites to cluster on normal cells may prevent surface glycosyltransferases from interacting with their acceptors, leaving them accessible for contact-dependent glycosylation.

IVD. Cell-cycle-dependent surface glycosyltransJbrase activity Lectin studies [6] have shown a similar ease of agglutination of normal mitotic

and all malignant cells. Normal interphase cells are relatively poorly agglutinated. These studies suggested a re-expression of a more mobile membrane during normal mitosis that malignant cells continually display. Two reports [93,104] have suggested variations of cell surface glycosyltransferase activities as the cell cycle proceeds. Since the ability of transformed cells to glycosylate their own surfaces correlated well with an increased mobility of malignant cell plasma membranes, normal mitotic cells were suspected [93] of being able to glycosylate their own acceptors, unlike their interphase counterparts. Malignant 3T12 cells did not show a requirement for intercellular contact for optimal enzyme activity when assayed at various stages of their cell cycle [93]. Normal interphase cells required cell contact for maximum glycosylation. Normal cells in mitosis, however, showed the same ability to glycosylate themselves in suspension incubations as did all stages of malignant cells (Fig. 4).

Autoradiographs of cultures incubated with UDp[3H]galactose were consistent with these observations. Mitotic 3T3 cells showed heavy grain deposits over their surfaces. This was in contrast to interphase normal 3T3 cells, elsewhere in the culture, which showed low levels of silver grains. All cells in 3T12 cultures displayed active enzyme activity, independent of cell contact [91,93].

Controls indicated that normal mitotic contact-independent glycosylation was not explicable by release of transferase into incubation supernatants, differential hydrolysis of sugar nucleotides, or differential viability of cells assayed as resulting from the methods of synchronization or collection.

Utilizing lymphoma suspension cells, Bosmann [104] has suggested that cell surface glycosyltransferases showed peak activity during the S phase of the cell cycle. Surface endogenous and exogenous sialyl-,galactosyl- and N-acetylglucosaminyltransferase activities all behaved similarly, i.e. a rise at S phase and virtually no activity during mitosis. Previously, internal glycosyltransferase activity in these cells has also been shown to peak at the S phase [105]. Unfortunately, sugar nucleotide hydrolytic activity as a function of the cell cycle was not presented, nor was any evidence presented that enzyme activities were optimized. Bosmann's results represented total cell surface activity and not contact-dependent activity, so comparisons are difficult to make between these two reports. Any differences existing between

these studies may be due, at least in part, from the use of two vastly different cell types (substrate grown [93] vs suspension [104]). Indeed, Bosmann suggested [104] that, since lymphoma suspension cells grow ascitically in vivo, their surface enzymes may function in other capacities than those on substrate bound cells.

IVE. Potential role in growth control Various approaches have enabled investigators to retard transformed cell

growth in vitro. Treatment with monovalent concanavalin A [39], with adenosine 3',5'-monophosphate (cyclic AMP) [106] and, in some cases, challenge with normal fibroblasts [107] have made malignant cells slow their growth rate. Each of these three treatments has been tested for its effect on cell surface glycosyltransferases. As previously discussed (see Section IIE) a surface galactosyltransferase has been implicated as one of the cell's surface receptors for concanavalin A [41-43].

Sudo and Onodera [108] have studied the effect of dibutyryl cyclic AMP on cell surface glycosyltransferases on normal C3H-2K and SV40-transformed C3H-2K mouse cells. Endogenous galactosyl-, glucosyl-, fucosyl- and mannosyltransferase activities were similar in both cell suspension types. Normal cells demonstrated decreased endogenous sialyltransferase activity and increased exogenous enzyme activity. Isolated C3H-2K plasma membranes retained this increased exogenous sialyltransferase activity.

Endogenous and exogenous galactosyltransferase activities were unaffected by dibutyryl cyclic AMP when added to cell suspension assays of normal C3H-2K cells. However, dibutyryl cyclic AMP markedly reduced both activities on SV40 transformants. Endogenous transferase activity was inhibited 40~o by 1 mM dibutyryl cyclic AMP, while exogenous activity decreased by 60 ~ . Exogenous sialyltransferase activity was also inhibited on transformed cell surfaces in the presence of dibutyryl cyclic AMP but, similarly to galactosyltransferase activities, normal enzyme levels remained unaffected. Microsomal and isolated plasma membrane sialyl- and galactosyltransferase activities were also examined in the presence of dibutyryl cyclic AMP. Its effect was partially stimulatory in these membrane assays.

Whether the preferential inhibitory action ofdibutyryl cyclic AM P on transformed cell surface glycosyltransferases is at all related to cyclic AMP's growth regulatory ability, is, of course, not yet known. However, before the inhibitory action of dibutyryl cyclic AMP can be thought of as specific for transformed cell surface transferases in this system, any potential detergent effects from butyric acid must be ruled out.

The possibility that surface transferases are involved in growth control has received support from experiments indicating that contact-inhibited cells also exhibited contact-dependent glycosylation of surface components [91-97,123]. Similarly, the endogenous activities of many [91,92,95] but not all [80], normal cells decreased with increasing cell contact. These observations agree well with those of Hakomori [98] and Robbins and Macpherson [99] in which the length of surface carbohydrates increased on normal cells as they reached confluency.

Transformed cells, on the other hand, are not as sensitive to contact inhibition

of growth, did not exhibit contact-dependent glycosylation, did not show reduced endogenous activity with increasing cell contact and did not display an increase in length of surface carbohydrates as confluency was reached. Similarly, in at least one case there is a decreasing dependence upon cell contact for glycosylation as cultured cells lose their contact inhibition growth controls (Webb, G. C. and Roth, S., unpublished data). Why transformed cells fail to complete their endogenous glycosyltransferase reactions in culture is not known. Presumably the lack of some required cation or the inability to transport sugar nucleotides from within the cell could explain this phenomenon. This prompted the prediction [109] that, if some transformed cells could be made to glycosylate themselves in culture, their growth would be retarded. Preliminary experiments support such a prediction [94].

Sparse cultures of highly malignant 3TI 2 fibroblasts were treated [94] in serum- free tissue culture medium supplemented with 100 #M amounts of UDPgalactose, UDP-N-acetylglucosamine, and UDP-N-acetylgalactosamine, 1 mM MnCI2 and 10 mM NAN3. After 2 h, the cells were washed and plated under standard conditions. 24 h later, the cultures were trypsinized and the cell number determined. Untouched controls and controls treated with just NaN3 and MnCI 2 showed one population doubling during this time period. Cultures treated with the complete reaction mixture showed little increase in cell number compared to control cultures.

Interestingly, 3T12 fibroblasts have been shown [110] to be contact-inhibited by normal 3T3 cells. This suggested that, while 3T12 cells are not able to convey growth controlling signals to one another, they are able to respond to a normal growth-inhibiting command.

IVF. Potential role of lipid intermediates Since it is generally accepted that sugar nucleotides do not permeate cell mem-

branes, what mechanism is used to transport the sugar donor outside the cell so that extracellular glycosylation can occur? Indeed, what mechanism is used to transport the sugar donor into the Golgi apparatus from the surrounding cytoplasm?

It has now been firmly established that some membrane-bound glycoproteins are synthesized, at least partially, not directly via sugar nucleotides but rather through lipid-linked sugar intermediates (for review, see ref. 111). Sugar nucleotides donate their monosaccharides to phosphorylated derivatives of polyprenols, such as dolichol and retinol (vitamin A). Oligosaccharide chains are synthesized on these lipids, which then transfer sugar moieties to membrane-bound protein or lipid acceptors. Additionally, polyprenol intermediates may play a role in individual, monosaccharide transfer. In this way, activated sugar donors may penetrate cell membranes.

Although most polyprenol intermediates have been associated with internal membrane transferases, a number of investigators [58,83,97, 112-114] have suggested their importance in cell surface glycosyltransferase assays as well. Sus- pensions of intact oviduct cells were shown [112] to synthesize both mannosyl- phosphoryl-dolichol and oligosaccharide-lipid from GDPmannose. Arnold et al. [58] identified a mannose derivative in liver cell mannosyltransferase assays. This man-

nose product was tightly bound to the cells and could not be extracted with lipid solvents from aqueous incubation mixtures, but could be extracted from lyophilized incubations with lipid solvents. Most important, this compound acted as a substrate in the mannose incorporation assay.

Patt and Grimes [83] observed that fibroblast cell surface mannosyltransferase activity was associated with a lipid component having many of the characteristics of mannosylphosphorylisoprenol. A retinol intermediate in collagen:platelet glucosyltransferase has been identified [113]. Preliminary results from Hakomori's [97] laboratory demonstrate that glycosylation of derivatized glass particles by intact cells was greatly enhanced in a medium enriched with retinol.

Another report [114] assessed the effect of polyprenols on,cell surface galactosyltransferase activity on normal and transformed fibroblasts. Normal 3T3 mouse- fibroblasts showed twice the endogenous galactosyltransferase activity when assayed in the presence of 0.05 mM ficaprenol. Retinol and dihydrophytol were less active at the same concentrations. Malignant 3T12 fibroblasts showed no stimulation of activity when assayed in the presence of these polyprenols. Exogenous N-acetylglucosamine:galactosyltransferase activities were slightly elevated in both cell types utilizing any of these three lipids. Addition of retinyl acetate, retinoic acid or dolichol had no effect on endogenous activity in either 3T3 or 3T12 cells.

Some of the diverse biological effects of retinol (vitamin A) could be explained by either altered mucopolysaccharide synthesis or abnormal cell recognition proces- ses. Vitamin A increases mucopolysaccharide synthesis in cultured cells [115], acts as a teratogen in mice causing cleft palate among other abnormalities [116] and affects metastasis of murine breast cancers [117]. The potential role of vitamin A as a necessary lipid intermediate in surface transferase reactions is particularly inviting considering its effects on recognition phenomena that may also utilize glycosultransferases.

IVG. Inability to demonstrate surface glycosyltransferases Deppert et al. [48] examined BHK cells for cell surface galactosyltransferase

activity and suggested that galactose incorporation resulted from the external hydrolysis of UDPgalactose, followed by the subsequent transport and internal utilization of free galactose. In incubations in which UDP[3H]galactose was applied to monolayer cultures, the sugar nucleotide was first degraded to galactose-l-P, and subsequently to galactose. 1 mM MnC12 markedly stimulated the degradation of sugar nucleotide and, subsequently, all glycosyltransferase assays contained this level of Mn 2÷.

A large excess of unlabeled free galactose, phloridizin, a competitive inhibitor of galactose transport [118], and sodium azide all produced parallel inhibitions of galactose incorporation whether [~4C]galactose or UDP[3H]galactose was the sugar precursor. These compounds did not interfere with homogenate galactosyltransferase. [3H]Galactose incorporation into product radioactivity, from either free galactose or UDPgalactose, occurred with a lag time of 10 min. Supposedly, the lag

represented galactose equilibration with internal monosaccharide pools. Homogenate galactosyltransferase activity occurred without a lag period.

While this report does indicate extracellular hydrolysis of sugar nucleotides, the data are unrelated to surface transferases. First was the routine use of monolayer cultures for their assays. A number of studies [91-96] showed that normal cells in culture express negligible, endogenous galactosyltransferase activity unless assayed well before confluency and under conditions allowing cell contact. Therefore, cells from subconfluent cultures may have exhibited low endogenous activity because they were separate, while cells from confluent cultures may have already glycosylated their acceptors. A more convincing demonstration by these authors would have been to assay sparse cultures in pellet incubations.

Second, the only exogenous acceptor tested was an "antifreeze glycoprotein" from the serum of an artic fish. It is quite likely that BHK cells do not possess a surface transferase for this compound even though they may possess such an enzyme internally. None of the large or small molecular weight, glycoprotein or glycolipid acceptors that have been shown to be active by others was tested.

Third, utilizing the same cell type, BHK, Yogeeswaran et al. [97] showed a monosaccharide transfer to glycolipids linked to glass particles. Similarly, surface sialyltransferases have been strongly indicated on BHK cells by Datta [82].

While cell surface galactosyltransferases may not, of course, be present on the cells used by Deppert et al. [48], if such enzymes were present the assay conditions described would not have detected them.

Recently, Keenan and Morr6 [124] have evaluated some of the evidence implicating glucosyltransferases on the surfaces of eucaryotic cells. Their contention was that no clear evidence yet exists to support a plasma membrane localization for glycosyltransferases. However, they appear to be familiar with only a limited part of the relevant literature. Their first criticism centered on the potential extracellular breakdown of sugar nucleotides and subsequent internalization and reutilization of free monosaccharide. While some investigations have failed to control carefully for this important possibility, most reports [21-24,29,45,58,63,81-83,88,91,93,122] have dealt with this problem. Secondly, they suggested [124] that glycosyltransferases liberated into the cell supernatant from undetected lysed cells could account for the observed endogenous and exogenous transferase activities. Contrary to their state- ment that this possibility has never been tested, investigators have repeatedly assayed cell supernatants for "leaked" glycosyltransferase activity. Never has the leakage of enzyme into the medium [21,32,41,42,45,87,91,93,122] or homogenized cell activity [21,32,45,61,81,83,91,93] been able to account for the observed intact cell incorporation.

Keenan and Morr6 [124] have also suggested that glycosyltransferases, if they exist on the plasma membranes of eucaryotic cells, may simply be responsible for the elaboration of cell surface coats, rather than participating in cell recognition phenomena. While it would be premature to rule out this possibility, considerable evidence

suggests that transferases are involved in diverse recognition phenomena [21-26, 29, 41--43,45,52-55,57,58,61,63,86,87,91-94,95-97].

V. CONCLUSIONS

A variety of criteria attest to a surface localization for some glycosyltransferase activities. The earliest reports relied upon membrane fractionation procedures, the strength of which depended upon the purity of the putative plasma membrane fraction. Soon afterwards, the use of intact cells alleviated the drawbacks of fractionation studies but introduced new problems. Intact cell transferase assays must include controls for extracellular hydrolysis of the sugar nucleotide and internal utilization of the breakdown products. The use of excess cold sugars and sugar phosphates, incubations that assayed the rate of free, labeled sugar incorporation, the use of metabolic poisons, and the inability to detect sugar nucleotide hydrolysis in many systems, are all indications that sugar nucleotides are the authentic sugar donors.

Other recommendations would include product characterization, cell viability tests, tests for enzyme leakage into the extracellular medium, and the trypsin and glycosidase sensitivity of the intact cell reaction. Many of the papers discussed in this review have included such precautions, although not one has included them all.

Furthermore, the ability to assay surface transferase activities does not necessarily ensure valid comparisons between cell types. It is essential that enzyme activities be relatively well characterized so that maximal activity is being assayed. While the existence of surface transferases may, at times, be clear, it is equally clear that negligible enzyme optimization can lead to faulty comparisons between cells.

Although it is true that no single paper yet published on the topic of cell surface glycosyltransferases contains all of the appropriate controls, this is also true in the area of Golgi transferases. What is more, it will probably always be true in most developing fields of biology. Nevertheless, in the short time that surface transferases have been a subject for investigation, the data in their favor are equal to those showing a Golgi location for many glycosyltransferases in the rat liver cell. That is, plasma membranes of high purity and autoradiographic data are as supportive of surface enzymes as Golgi membranes and Golgi autoradiographs are supportive of Golgi enzymes. In fact, if one omits consideration of the rat liver cell, there is more evidence for surface transferases in more systems than for Golgi enzymes. Most of the transferases in most systems examined do seem to be inside the cell but pre- cisely where remains unknown except for a remarkably limited number of well studied cell types. In sum, the available data make surface transferases a most reasonable conclusion in a number of cases and a very exciting possibility in a wealth of others. Additionally, the bulk of the data indicate that transferases and their acceptors may mediate recognition and social phenomena. This latter possibility awaits more rigorous testing which, even if totally unsuccessful, cannot help but tell us much about cell surface carbohydrates and the proteins that make them.

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