MOLECULAR AND CELLULAR BIOLOGY, May 1985, p. 923-929 Vol. 5, No. 50270-7306/85/050923-07$02.00/0Copyright C 1985, American Society for Microbiology

Membrane Mutants of Animal Cells: Rapid Identification of Thosewith a Primary Defect in Glycosylation

PAMELA STANLEYDepartment of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 10461

Received 26 November 1984/Accepted 6 February 1985

Membrane mutants of animal cells have been isolated by several laboratories, using a variety of selectionprotocols. The majority are lectin receptor mutants arising from altered glycosylation of membrane molecules.They have been obtained by selection for resistance to cytotoxic plant lectins or by alternative protocolsdesigned, in many cases, to isolate different classes of receptor mutants. The identification of most membranemutants expressing altered surface carbohydrates is rapidly achieved by determining their resistance to severallectins of different carbohydrate-binding specificities. For Chinese hamster ovary mutants, genetic novelty maysubsequently be determined by complementation analysis with selected members of 10 recessive, glycosyla-tion-defective complementation groups defined by this laboratory. In an attempt to identify new complemen-tation groups, 11 Chinese hamster ovary membrane mutants independently isolated in different laboratorieshave been investigated for their lectin resistance and complementation properties. Only one new complemen-tation group was defined by these studies. The remaining 10 mutants fell into complementation group 1, 2, 3,or 8. Although no evidence for intragenic complementation was observed, indirect evidence for differentmutations within some genes was obtained. Seven of the independent isolates fell into complementation group1, reflecting the high probability of isolating the Lecl phenotype from Chinese hamster ovary populations. Theresults emphasize the importance of performing a genetic analysis before biochemical characterization ofputative new membrane mutants.

Almost any selection for an animal cell mutant mightinclude among the survivors those with a primary defect inglycoconjugate formation. This is because carbohydratebiosynthesis and metabolism involve lipid, protein, andnucleic acid precursor molecules (22, 23). In addition,mature glycoconjugates (glycoproteins and glycolipids) per-form many functions within a membrane environment in-cluding transport, receptor, enzymatic, and structural func-tions. Changes in glycosylation may therefore alter thebiosynthesis, degradation, compartmentalization, or confor-mation of one (or several) membrane molecules. Con-versely, a change in the function of a glycosylated moleculemight reflect an alteration in the structure of its carbohydratemoieties. For example, Thy-1- mouse lymphoma cells se-lected for the ability to survive cytotoxic anti-Thy-1 antibod-ies have been shown to arise, in several cases, from differentdefects in N-linked carbohydrate biosynthesis (37). As aresult of certain (though by no means all) carbohydratestructural changes, the Thy-1 molecule seems to be de-graded more rapidly intracellularly and therefore does notlocalize correctly to the plasma membrane (38, 39).Over the last 10 years, this laboratory has identified 18

different glycosylation mutations in Chinese hamster ovary(CHO) cells (28; J. Ripka and P. Stanley, unpublished data).All are lectin receptor membrane mutants and were obtainedby selection for resistance to cytotoxic plant lectins. Ten ofthe mutant phenotypes (Lecl through Lec9 and Leci3)exhibit unique lectin-resistant (LecR) properties and belongto separate, recessive complementation groups. Four of themutants (LEC10, LEC11, LEC12, and LEC14) behave dom-inantly in hybrids but exhibit unique LecR and biochemicalphenotypes, suggesting that they arise from mutations indifferent genes. The remaining four mutant types (LeclA,Lec2A, Lec2B, and Lec13A) appear to represent alternativemutant alleles within complementation groups 1, 2, and 13,respectively. They fall into the latter groups by comple-

mentation analysis, although they exhibit unique LecR phe-notypes.With the aim of identifying new glycosylation mutants,

particularly those in which different mutations might havegiven rise to the same membrane phenotype as well as thoseexhibiting intragenic complementation and those selectedfrom protocols designed to obtain alternative types of mem-brane mutant, this laboratory has developed a combinedaproach involving a phenotypic test for lectin resistance(LecR P-test) and complementation analysis (26). If a cellline exhibits altered resistance to at least two lectins ofdifferent carbohydrate-binding specificity, it is highly likelyto be a glycosylation mutant (27, 29). Complementationanalysis with selected members of the 10 recessive LecRCHO complementation groups (25, 27, 31) will subsequentlydetermine whether the glycosylation mutation is novel. Thisapproach allows the identification of a probable glycosyla-tion mutant within 4 days and its assignment to a comple-mentation group within 1 month. The advantages of perform-ing a genetic analysis before biochemical characterizationare revealed in this paper in which 11 independently isolatedCHO membrane mutants are classified into comple-mentation groups. Only one of the mutants was found todefine a new complementation group. The remainder fellinto complementation group 1, 2, 3, or 8, even though theywere selected by a variety of protocols and, in some cases,exhibited significant variations in LecR phenotype comparedwith other mutants in the same group.

MATERIALS AND METHODS

Cell lines. CHO membrane mutants isolated by five dif-ferent laboratories were kindly donated for these studies:Carol Jones (Eleanor Roosevelt Institute for Cancer Re-search, Inc., Denver, Colo.) provided clone Ji, a hybridCHO-Ki line, which carries human chromosome 11, fromwhich were selected the cell lines termed 17B, 19A, and 21B

923

924 STANLEY

(14); Stuart Kornfeld (Washington University, St. Louis,Mo.) provided the cell lines 15B and clone 13 which wereselected from a CHO-Kl population originally obtained fromPotter Stewart (2, 9); Ernest Chu (University of Michigan,Ann Arbor) provided two clones (D4-lb and D4-5) selectedfrom CHO-Kl cells (18); April Robbins (National Institutesof Health, Bethesda, Md.) provided clone 34-2-1 which wasselected from a ,-galactosidase-deficient CHO cell mutant,N211-1-8 (21); Carlos Hirschberg (St. Louis University, St.Louis, Mo.) provided mutants 687, 699, and Cl which wereselected from the CHO line 51-liB, an auxotroph for glycineand proline (10; N. Nuwayhid and C. Hirschberg, un-published data). The cell lines from this laboratory usedin forming somatic cell hybrids include the parental auxo-troph Gat-2 and the mutants Gat-Lecl.lN, Gat-Lec2.4C,Gat-Lec3.6F, Gat-Lec8.2B, Gat-Lec5.D11A,Gat-Lecl.Lec6.3B, and Pro+Lec9.Lec2.1A. Pro-5 parentalcells and the mutants Pro-Lecl.3C, Pro-Lec2.6A,Pro-Lec3.4B, Pro-Lec8.3D, and Pro-LeclA.3B were usedin addition to the Gat- lines as controls in P-tests for thedetermination of lectin resistance.

Cells were cultured in monolayer or suspension at 37 or34°C in complete alpha medium containing 10% fetal calfserum as described previously (26). Media and sera wereobtained from GIBCO Laboratories, Grand Island, N.Y. Allcell lines were tested for mycoplasma by the Hoeschst 33258staining method (4). None were positive except clone 15Bwhich was also shown to carry mycoplasma in tests per-formed by G. J. McGarrity (Institute for Medical Research,Camden, N.J.). The mycoplasma contamination did notappear to alter the glycosylation phenotype of this mutant,which was cultured in isolation.

Lectin resistance properties. Cell lines were tested for theirresistance to the five lectins L-phytohemagglutinin (L-PHA)(from Proteus vulgaris; Burrough's Wellcome, Poole, En-gland), wheat germ agglutinin (WGA) (from T. vulgaris;Sigma Chemical Co., St. Louis Mo.), concanavalin A (CONA) (Pharmacia, Uppsala, Sweden), and RIC (toxin from R.communis) and LCA (agglutinins from L. culinaris preparedas described previously (26). The lectin resistance propertiesof mutants and hybrids were determined either by P-test orby D1o analysis (26). The P-test is performed in 96-wellmicrotiter dishes in which each well is seeded with 2,000cells in medium containing 10% fetal calf serum and differentamounts of lectin (26). The endpoint is taken as the lectinconcentration that gives approximately 10% survival whenwells containing no lectin have reached confluency (about 4days at 37°C in a CO2 incubator). D1o values are moreaccurate, being calculated from survival curves of relativeplating efficiency at increasing lectin concentrations (26).

Complementation analyses. All cell lines obtained fromother laboratories carried the Pro- auxotrophic marker.Therefore, complementation analyses were performed withthe Gat-LecR CHO lines isolated by this laboratory asdescribed previously (26). Briefly, LecR cells carrying thePro- and Gat- auxotrophic markers were cultured in mixedmonolayers and treated with polyethyleneglycol, and viablehybrids were selected in medium lacking proline, glycine,adenosine, and thymidine and containing 10% dialyzed fetalcalf serum. The hybrid cell selection was performed in thepresence and absence of increasing concentrations of alectin to which both parents were resistant. In this way, theLecR properties of the freshly fused hybrid population weredetermined. In addition, some hybrid colonies were pickedfrom plates which contained no lectin, cultured, and subse-quently tested for their lectin resistance properties by P-test

or D1o analysis. In all crosses, auxotrophic reversion fre-quencies and spontaneous fusion frequencies were shown tobe s-si0.

Karyotype analysis. Hybrid cell cultures in exponentialgrowth were incubated with 0.25 ,ug of colcemid (GIBCOLaboratories) per ml for 2 h, and the number of chromo-somes per mitotic cell nucleus was determined for 25 intactchromosome spreads. Hybrids examined by P-test or D1oanalysis (see Tables 2 to 5) were shown to be pseudotetra-ploid before testing.

kESULTSLeCR phenotypes of the independent mutants. The methods

by which the independently isolated membrane mutantswere obtained are summarized in Table 1. The first threegroups of mutants were selected for lectin resistance insingle-step protocols, B4-2-1 survived a selection for amannose-6-P receptor deficiency, and the remaining mutantswere survivors of 3H-labeled sugar suicide protocols. All ofthe mutants had been previously tested for their lectinresistance or lectin binding properties or both with one ormore lectins and, in many cases, exhibited phenotypessimilar to those of mutants from our repertoire: 15B cells(isolated about the same time as Lecl CHO cells; 29)possessed a typical Lecl phenotype (1, 9); clone 13 (a novelphenotype when isolated; 2) and the abrin-resistant CHO-Kimutants (18) appeared similar to each other and to Lec8cells; the Jl LecR mutants isolated recently (14) exhibitedphenotypes typical of Lecl (17B), Lec2 or Lec3 (19A), andLec8 (21B) CHO cells; 1B4-2-1 was initially shown to be ricinresistant (21) and, subsequently, to be deficient in dolichol-P-mannose synthetase, resulting in the synthesis of alteredN-linked carbohydrates (33); and the LecR properties ofmutants 687 and 699 were consistent (for five differentlectins) with those of a Lecl rtutant (699) and a relatedphenotype (10). The Cl mutant is a recent isolate which hasnot been extensively characterized.Although several of the mutants had already been tested

for lectin resistance, it was important to compare themdirectly with our CHO mutants under uniform experimentalconditions (Table 1). Most of the independent isolates ex-hibited a LecR phenotype similar to that of one of the mutantgroups previously described by this laboratory (26, 28). Asexpected, 15B, 17B, and 699 expressed typical Lecl pheno-types, whereas 13 and 19A expressed typical Lec8 pheno-types. Surprisingly, D4-lb and D4-5 were not similar to Lec8cells as predicted but exhibited characteristic Lecl pheno-types. Mutant 687 was sensitive to CON A like Lecl cellsbut its degree of resistance to WGA and RIC was moretypical of the LeclA phenotype (27). Mutant 21B wasqualitatively similar to both Lec2 and Lec3 CHO cells butwas less resistant to WGA than Lec2 cells and markedlymore sensitive to RIC than Lec3 cells (26). Finally, the twomutants B4-2-1 and Cl exhibited LecR phenotypes whichwere novel. All of the RicR CHO mutants previously de-scribed by this laboratory are affected in their interactionswith more than one of the five P-test lectins (26, 28), yetB4-2-1 cells were not significantly altered in sensitivity toany but RIC. In contrast, Cl cells were resistant or hyper-sensitive to all five lectins. The novelty of the Cl phenotypelies in its concomitant sensitivity to CON A and RIC whichhas not previously been observed among the CHO glycosyla-tion mutants (26, 28). Therefore, both the B4-2-1 and Cl celllines exhibited the hallmarks of putative new membranemutants.Complementation analyses of the independent mnutants.

MOL. CELL. BIOL.

IDENTIFICATION OF NEW CHO GLYCOSYLATION MUTANTS

TABLE 1. Origins and LecR properties of CHO membrane mutants obtained from other laboratories

Parental cell line and LecR phenotype'Mutant Most similarselection protocol cell line' L-PHA WGA CON A RIC LCA phenotype"(plg/mI)"' o(3r (2) (18) (0.005) (20)

Jl (CHO-Kl)PHA (200) 17B' RR> S12 R3o R,10 LeclWGA (10) 19A R1 RI,, (S) S3 S4 Lec8WGA (10) 21B" _ R SIM- Lec2/Lec3

CHO-KlRIC (0.1) 15B R>65 R30 SI R--.( R>,,, LeclWGA (4) 13 RIO VI( (S) S4 Lec8

CHO-KlAbrin (1) D4-lb R30 R30 SI R-40 R-, LeclAbrin (1) D4-5 RIO R30 S5 R ,40 R-.2 Lecl

CHO (N211-1-8)Mannose-6-P-ricin B4-2-1 - - R4 New

conjugate (0.43 nM)

CHO (51-llB)[3H]fucose suicide 687 R-65 R6 S3 RI( R>, LeclA[3H]fucose suicide 699 R-65 R30 S5 R(oR~>,( Lecl[3H]N-acetylmannosamine suicide C1 R,65 R-.,, S5 S1 R->,, New

"Numbers in parentheses are the concentration of selective lectin. Abrin is the toxin from Abrius precatoritts. The parental line JI (from which 17B. 19A, and21B were selected) was slightly more resistant to L-PHA (R) and WGA (R) and more sensitive to LCA (S3). whereas the parental line N211.1.8 (from which B4-2-1was selected) was slightly more sensitive to both CON A (S) and LCA (S) compared with parental CHO cells.

" The references describing the isolation of each mutant are given in the text.'Cells were tested for their abilities to grow in the presence of five cytotoxic plant lectins by P-test or D,, analysis (see text). The values represent fold

resistance (R) or fold sensitivity (S) compared with parental cells. No difference from parental cells is denoted by-." The LecR phenotype(s) from our CHO mutant collection which most closely resembles the phenotype of the line tested (see reference 26).Numbers in parentheses are D,,, values in micrograms per milliliter. These values are for the parental CHO lines Pro-5 and Gat-2 used in this laboratory.The LecR values for 17B and 21B were obtained by C. Jones, using lectins from different commercial sources which gave slightly different DI,, values for

parental Jl cells (14). However, with the exception of PHA, the differences were -twofold.

Although the LecR phenotype is extremely helpful in iden-tifying potential glycosylation mutants and in tentativelyassigning them to mutant groups, it can be misleading forseveral reasons (see Discussion). Therefore, it is necessaryto perform complementation analyses to distinguish newrecessive mutations. Only by complementation analysis canit be known when mutants with identical phenotypes arisefrom mutations in different genes or when mutants withdifferent phenotypes are the result of mutations in a singlegene (e.g., Lecl and LeclA).

Ideally, in performing complementation studies, all reces-sive mutants should be crossed with each other in anexhaustive, pairwise analysis. However, because of theenormous amount of time required to fuse and select somaticcell hybrids and to subsequently analyze their properties,only mutants of related phenotype were crossed in thesestudies. To define a new recessive mutation, all LecRmutants resistant to the selective lectin used in isolating thatmutant were crossed. In assigning a new isolate to a pre-viously defined complementation group, it was consideredsufficient to show non-complementation with a mutant inthat group and complementation with a mutant of relatedLecR phenotype from another group.

Since all independent mutants from other laboratoriescarried the Pro- auxotrophic marker, they were crossedwith the Gat-LecR cells from our collection. Hybrid popu-lations were selected in deficient medium in the presenceand absence of a lectin to which both parents were resistant.In this way, the lectin sensitivity of the hybrid cell popula-tion was determined immediately after hybrid formation. Inseveral cases, hybrid colonies were also picked from plates

which contained no lectin, cultured, and subsequently testedfor lectin resistance and karyotype.The mutants isolated by Jones' laboratory fell into three

different complementation groups (Table 2). Clone 17Bexhibited non-complementation with Lecl CHO cells butfull complementation with Lec8 cells and thus belongs tocomplementation group 1; clone 19A exhibited the oppositebehavior and therefore belongs to complementation group 8;and clone 21B exhibited complementation in hybrids formed

TABLE 2. Complementation analyses of independent mutants17B, 19A, and 21B"No. of colonies in WGA

Cell lines (pg/mI) Comple-crossed mentation

0 3

17Bx parental -800 4 +x Lecl -400 -400

19Ax Lecl 213 3 +x Lec8 168 120

21Bx Lec2 302 1 +x Lec3 439 54

" Cell lines 17B. 19A. and 21B were fused with Gat-2 (parental) orGat-LecR cell lines from complementation group 1. 2. 3. or 8. Hybrids wereselected from 3 x 1Wi cells plated in deficient medium containing 0. 1. 2. or 3pLg of WGA per ml. After 8 to 10 days. the plates were stained and colonieswere counted.

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926 STANLEY

TABLE 3. Complementation analyses of independent mutants15B, 13, D4-lb, and D4-5"

No. of colonies in ResistanceCell lines WGA (,tglml) of hybrids to Comple-crossed WGA mentation

0 3

15Bx Lecl 294 ND 100 (9)x Lec8 416 ND 3-5 (5) +

13x Lecl 289 1 3-5 (3) +x Lec8 293 253 200 (5)

D4-lbx Lecl -1,800 -1,800 NDx Lec8 -2,000 70 ND +

D4-5x Lecl -2,000 --2,000 NDx Lec8 -2,000 100 ND +

" Cell lines 15B, 13, D4-lb, and D4-5 were fused with Gat-LecR cell linesfrom complementation groups 1 and 8. Hybrids were selected in deficientmedium containing 0, 1, 2, or 3 ,ug of WGA per ml from 3 x 105 (15B and 13crosses) or 4 x 105 (D4-lb and D4-5 crosses) cells plated. After 8 to 10 days,several colonies were picked from plates containing no WGA. All plates weresubsequently stained and the colonies were counted.

" Selected colonies were cultured, shown to be pseudotetraploid by karyo-type analysis, and P-tested for resistance to WGA. Hybrids from crossesexhibiting complementation (+) displayed wild-type sensitivity to WGA.whereas hybrids from crosses exhibiting non-complementation (-) wereappropriately WGA resistant. The numbers in parentheses represent thenumber of hybrids tested from each cross. ND, Not determined.

with Lec2 CHO cells but non-complementation in hybridsformned with Lec3 CHO cells and therefore belongs tocomplementation group 3. The partial sensitivity of Lec3 xLec3 hybrids to WGA is observed when hybrid populationsare tested for WGA resistance immediately after fusion (25,31). However, when Lec3 x Lec3 hybrids are culturedbefore testing, their resistance is typical of pseudodiploidLec3 mutants (31). The LecR phenotypes of these mutantsare consistent with their respective complementation assign-ments (Table 1).The two mutants provided by Kornfeld's laboratory were

found to belong to two different complementation groups(Table 3). Each mutant type behaved recessively, exhibitingfull complemeniation with one of the recessive mutantstested. The 15B mutant exhibited non-complementation withLecl cells and therefore belongs to complementation group1, consistent with its lectin resistance phenotype (Table 1),whereas clone 13 exhibited non-complementation with Lec8cells and a typical Lec8 lectin resistance phenotype (Table1). It therefore belongs to complementation group 8.Mutants D4-lb and D4-5 both exhibited non-cotmplemen-

tation with Lecl CHO cells and full complementation withLec8 cells (Table 3). They therefore belong to complemen-tation group 1 as predicted by the LecR phenotypes pre-sented in Table 1. In contrast, mutant B4-2-1 appears tobelong to a new complementation group (Table 4). In twocrosses, the mass population of hybrids was tested for RICsensitivity immeliately after fusion. The freshly formedhybrids exhibited essentially parental sensitivity to RIC,indicating that the B4-2-1 mutation behaves recessively aswould be predicted by the fact that these cells lack dolichol-P-mannose synthetase activity (33). The sensitivity to RIC oftwo hybrids from the cross of B4-2-1 with Gat- parentalCHO cells was confirmed by D1o analysis. However, when

examined by P-test, these hybrids were significantly moreresistant to RIC. The latter result may be caused by celldensity effects since many more cells are initially plated inthis type of test. In general, the levels of lectin resistancedetermined by P-test are slightly higher than those obtainedby D1o analysis (see reference 26). The combined resultssuggest that B4-2-1 cells carry a recessive mutation to RICresistance which behaves as partially dominant under cer-tain culture conditions. A similar result is observed with thetemperature sensitivity of B4-2-1 cells. The reduced abilityof B4-2-1 cells to grow at 39.5°C is largely overcome whenthe cells are plated at high cell density (S. Krag, personalcommunication).The complementation data in Table 4 and the LecR

phenotypic data in Table 1 show that the B4-2-1 mutation isnot similar to any of the previously described RIC-resistantCHO mutants Lecl, LeclA, Lec5, Lecl.Lec6, Lec8, Lec9,or LEC10 (26). It therefore belongs to a new complementa-tion group (number 15) and will subsequently be referred toas the Lecl5 CHO phenotype.The mutants isolated by Hirschberg and colleagues, using

3H-labeled sugar suicide protocols, exhibited LecR pheno-types typical of Lecl (699) and LeclA (687) CHO mutants ora novel phenotype (Cl) not represented among previousisolates (Table 1). The complementation analyses in Table 5confirmed that 699 and 687 are recessive mutants and thatboth belong to complementation group 1. In addition, thedata show that Cl is a recessive double mutant carrying aminimum of two genetic markers, Lecl and Lec2. There-fore, the novel LecR phenotype of Cl cells appears to be dueto the combined effects of at least two glycosylation lesionsand is not representative of a single new mutation.

DISCUSSIONMembrane mutants are easily selected from animal cell

populations, using lectins (which interact with moleculescarrying the appropriate carbohydrates) or specifically tar-geted agents (such as antibodies and ligand-toxin conjugates)or radiolabeled precursors capable of incorporation intomacromolecules leading to "suicide." The problem lies not

TABLE 4. Complementation analyses of the independent mutantB4-2-1"

Cell line No. of colo- Resistance of hybrids to RICCrsedl with nies in RIC (gm)Comple-crossedwigtmI)mentationB4-2-1

0 0.01 P-testh DI(Parental -800 15 0.025 (4) 0.006 (2) +

LecS --1000 ND >0.02 < 0.05 (3) ND +'Lecl.Lec6 -1,200 ND >0.02 < 0.05 (3) ND +-Lec9.Lec2 310 2 ND ND +

Cell line B4-2-1 was fused with Gat-2 (parental) CHO cells and theGat-RicR lines belonging to complementation groups 1. 5. 6. and 9. TheLec9.Lec2 line is reverted from Pro- to Pro'. It may be selected against bythe inclusion of RIC at 0.5 ng/ml in the deficient medium. This is toxic to theLec2 phenotype but not to hybrids in which the recessive Lec2 phenotype is

suppressed (Ripka and Stanley. unpublished data). Hybrids were selectedfrom 105 cells plated in deficient medium containing 0 (or 0.0005 in theLec9.Lec2 cross). 0.005. 0.01. or 0.025 ,pg of RIC per ml. Plates were stainedafter 8 to 10 days at 34°C. Three to six hybrids from plates containing no RICwere picked, cultured, and tested for resistance to RIC by P-test or DI,,analysis.

6 In these P-tests, the endpoint for B4-2-1 was t).t)5 ,g of RIC per ml. andfor parental CHO cells it was 0.01 ,ug of RIC per ml. The hybrids exhibited anintermediate level of resistance. ND. Not determined.

Complementation is concluded on the basis that non-complementaryhybrids would be significantly more RIC resistant and that hybrids from theB4-2-1 x parental cross were completely sensitive to RIC by DI( analysis.

MOL. CELL. BIOL.

IDENTIFICATION OF NEW CHO GLYCOSYLATION MUTANTS

in obtaining such mutants but in their subsequent biochem-ical characterization. Three different selections designedspecifically to isolate mutations in the Thy-1 molecule (12),the mannose-6-P-receptor (21), or the low-density lipopro-tein receptor (16) have given rise to several different mutantswith defects in glycosylation. Four of the glycosylation-de-fective Thy-l- lymphoma mutants belong to different com-

plementation groups (13); the mannose-6-P receptor mutantB4-2-1 has been shown in this paper to define a new CHOcomplementation group Lecl5; and three of the four com-

plementing low-density lipoprotein receptor CHO mutantsprobably represent additional glycosylation-defective groups

since they are affected in genes other than the low-densitylipoprotein receptor structural gene (15) and, in addition,they exhibit distinctive LecR phenotypes (D. Kingsley andM. Krieger, personal communication). Clearly mutants incarbohydrate biosynthesis are prevalent among survivors ofselections against membrane molecules. For those in searchof new glycosylation mutations as well as for those whowould prefer to eliminate such mutants, it is important torapidly identify this class of mutation.The simplest method for identifying a likely glycosylation

mutant is by the LecR P-test (Table 1). Most carbohydratealterations lead to changes in the surface binding of at leasttwo different plant lectins which may be detected by theLecR P-test or by a lectin-binding assay. However, theresults must be interpreted with caution. First, as observedfor the Lecl5 phenotype (Table 1), its glycosylation defectdoes not lead to dramatic changes in resistance to the panelof five lectins usually tested. Second, the Lec3 phenotype,which is characterized by fivefold resistance to WGA (26),does not correlate with decreased WGA binding at the cellsurface (32). In addition, depending on the temperature ofassay, altered lectin binding might reflect an internalizationdefect (20). Therefore, although the most common propertyof glycosylation mutants is a dramatic change in LecR andLecB properties (28, 38), more subtle changes may also becaused by different glycosylation defects.The LecR (and LecB) phenotype is particularly useful for

tentative assignment of a new isolate to a mutant group (26).However, final assignment must await complementationanalysis. First, the LecR phenotype is dependent on the totalarray of structures available at the cell surface and this willvary with different cell lines and, therefore, with the mutantsderived from them. For example, the Thy-lE lymphomamutants are CON A-resistant whereas the Lecl5 CHOmutants are not (Table 1), and yet both appear to have arisenfrom mutations affecting the same glycosylation enzyme (3,33). In addition, determination of the LecR phenotype willdepend on the source of lectins and serum used in the P-test(26). Finally, mutants which appear quite different by P-testmay belong to the same complementation group (e.g., Lecland LeclA; Lecl3 and Lecl3A; 26). Hopefully mutantswhich appear identical by P-test but belong to differentcomplementation groups will also be found in the future.As outlined in Results, the previously published proper-

ties and the LecR phenotypes (Table 1) of the independentisolates are consistent, for the most part, with their comple-mentation group assignments (Tables 2 to 5). Two excep-tions were noted, however. The abrin-resistant CHO-Klcells originally predicted on the basis of phenotypic proper-ties (18) to be like clone 13 (complementation group 8) are

actually members of complementation group 1 (Table 3). Apossible explanation of these results is that the originalisolates were contaminated with Lecl mutants which ulti-mately became the predominant cells in the population. This

TABLE 5. Complementation analyses with independent mutants687, 699, and Cl1

No. of colonies in WGACell lines (jig/ml) Comple-crossed mentation

0 3

687bx Lecl >800 >800x Lec8 >800 0 +

699bx Lecl >800 >800x Lec8 >800 0 +

C1x Lecl 779 696x Lec2 782 781x Lec3 627 1 +x Lec8 579 0 +

a Cell lines 687, 699, and Cl were fused with Gat-LecR lines fromcomplementation groups 1, 2, 3, and 8. Hybrids were selected from 105 cellsplated in deficient medium containing 0, 1, 2, or 3 ,ug of WGA per ml. After 8days, plates were stained and colonies were counted.

b The fusion frequency was very high in these crosses. The number ofrevertants in the parental populations was 0l-5, and the frequency ofspontaneous hybfids was approximately 20 per 10' cells plated.

would be consistent with the known likelihood of isolatingmixed colonies in lectin selections and with the fact thatLecl and Lec8 mutants are present in similar numbers inCHO populations (25). The other exception was Cl whichexhibits a unique LecR phenotype strongly indicative of anew glycosylation mutation. Coinplementation analysis re-vealed, however, that Cl fails to complement mutants fromboth complementations groups 1 and 2 and, thus, it is at leasta double mutant. Therefore, the novel LecR phenotype of Clcells is the result of a combination of glycosylation defectsand probably not of interest with regard to the identificationof a new gene affecting glycosylation.The examples discussed above show that much time and

effort will be saved in characterizing a mutant if it is firstsubjected to complementation analysis. Other Chinese ham-ster mutants reported in the literature to be affected inglycosylation reactions include the WgaR mutant 1021 (2)which has recently been shown to belong to complementa-tion group 2 (7); the Con AR CHO mutants described byWright and colleagues (40, 41) (which appear to be identicalto the mutants isolated by R. M. Baker [5] and previouslyassigned [31] to complementation group 5); the conditional-lethal mutant of DON hamster cells characterized by Tennerand Scheffler (35, 36) (which is complex but might possess aprimary defect in glycosylation affecting a new locus); andseveral tunicamycin-resistant CHO lines, two of which ap-pear to be affected in glycosylation-related genes (6, 17, 34).One mutant is thought to have an N-acetylglucosaminyl-transferase enzyme resistant to inhibition by tunicamycin(17), whereas another appears to overproduce the enzymeand might have undergone amplification at this locus (6).Neither of these mutants might be expected to exhibit analtered LecR phenotype, and they are probably not repre-sented among the CHO glycosylation mutants we havedefined. However, since the effects of tunicamycin in vivoare not well characterized, it is possible that selections fortunicamycin resistance would give rise to other types ofglycosylation mutants. Finally, there are two LecR pheno-types of CHO cells which do not result from changes inglycosylation enzymes. Ray and Wu have described a RicR

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928 STANLEY

CHO mutant which is defective in the internalization of RIC(20) and Ono et al. (19) have a RicR CHO line which exhibitsan altered 60S ribosomal subunit.To summarize, membrane mutants with primary defects in

glycosylation may be rapidly identified by the LecR P-testand subsequent complementation analysis. Unfortunately,the complementation groups identified for CHO mutantsmay not be useful in determining the novelty of glycosyla-tion mutations in other cell types since cross-species hybridsare notoriously unstable and rapidly lose chromosomesduring culture (8). However, the 11 recessive complementa-tion groups of LecR CHO cells are extremely useful foranalyzing new CHO membrane mutants. Interestingly, themajority of the independent isolates investigated in thisstudy fell into complementation group 1. Hirschberg et al.(11) also obtained a Lecl mutant from a screen for cells withreduced incorporation of [3H]-fucose and two other groupshave described CHO mutants with typical Lecl phenotypes(19, 20). This is one of the most likely glycosylation mutantsto be selected from a CHO population because of its highdegree of resistance to many different lectins (26) andbecause of the comparatively high mutation rate of this locus(approximately 10-6 mutations/cell per generation; 30). Toavoid the repeated isolation of this mutant type, it is possibleto pregrow parental cells in 5 p.g of CON A per ml, to includeCON A in the selection medium, or to add CON A oncesmall colonies have formed on selection plates to inhibittheir further expansion (25, 27). Each of these approachesselects against Lecl cells and for all LecR phenotypes whichare not hypersensitive to the toxicity of CON A. Suchstrategies have been used to specifically isolate many of theless common glycosylation mutants from CHO populations(26).

ACKNOWLEDGMENTSI am indebted to Barbara Dunn for excellent technical assistance

and to all those who contributed cell lines (see Materials and Meth-ods). Thanks are also extended to Carlos Hirschberg for suggestionson the original manuscript.

This work was supported by Public Health Service grant RO130645 from the National Cancer Institute. P.S. is the recipient of afaculty award from the American Cancer Society. Partial supportfrom Core Cancer Grant 3PO CA13330 is also acknowledged.

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