THE JOURNAL OF RIOLOGICAL CHEMISTRY Vol. 245, No. 7, Issue of April 10, pp. 1659-1669, 1970

printed in U.S.A.

Enzymes That Destroy Blood Group Specificity

I. PURIFICATION AND PROPERTIES OF Q-L-FUCOSIDASE FROM CLOSTRIDI UM PERFRINGENS*

(Received for publication, October 3, 1969)

DAVID AMINOFF AND KEN FURUKAWA'$

From the Departments of Internal Medicine (Simpson Memorial Institute) and Biological Chemistry,

the University of Michigan, Ann Arbor, Michigan 48104

SUMMARY

An a-L-fucosidase was purified approximately 600-fold from Clostridium perfringens; it is free of other glycosidases present in the crude extract. The fucosidase occurs in multiple “isozymic” forms with molecular weights greater than 200,000. It is an oc-(1 + 2)-specific L-fucosidase with action on oligosaccharides and glycoproteins, but with no action on simple methyl or nitrophenyl fucosides. Action on the H-specific hog submaxillary glycoprotein results in the loss of the serological specificity. The enzyme has a pH optimum of 6.0, with a Km of 0.175 mM and its activity is en- hanced by the presence of salts, the most effective being calcium chloride.

The literature is replete with reports of enzymes that degrade glycoproteins with blood group activity (l-3). Most of the potent sources of these enzymes are complex mixtures of gly- cosidases and proteinases, and in many cases are capable of destroying more than one blood group activity. Evidence has accumulated suggesting that the most active enzymes which destroy blood group activity are glycosidases. There is need, therefore, for pure glycosidases of known substrate specificity to confirm the role of the nonreducing terminal sugar in determin- ing blood group specificity (l-3), and secondly, as a corollary (3) to establish unequivocally the mechanism of enzymatic inactivation of the serological specificity. For instance, it has been shown that both N-acetylgalactosamine deacetylase and the oc-N-acetylgalactosaminidase of Clostridium tedium can destroy blood group A activity (4).

In most cases the purification of the enzymes was followed by the loss of serological activity on incubation with blood group active glycoproteins (3). A fair degree of enzymatic purification was obtained with this assay, but it has distinct limitations. These include (a) the serological technique, inhibition of hemag- glutination, is not suitable for quantitative enzymatic kinetic studies, (b) the loss of blood group activity can result potentially

* This work was supported by Grant AM 07305 from the National Institutes of Health and in part by a grant from the University of Michigan Cancer Research Institute.

$ Present address, Department of Legal Medicine, School of Medicine, Gunma University, Maebashi, Japan.

from a number of different enzymatic reactions thereby making interpretation of effectiveness of purification difficult in a mixture of such enzymes, and (c) the loss of blood group activity alone gives no clue as to the mechanism involved.

Synthetic phenolic glycosides have been widely employed by a number of investigators for the detection and isolation of glycosidases. Unfortunately, as will become apparent in this report, and as has been observed by others, the enzymes that degrade blood group substances usually have no action on these simple substrates (5).

The reason for the lack of extensive purification of enzymes that degrade blood group substances, therefore, is that of methodology, namely the paucity of available techniques for the determination of the free sugar specifically in the presence of other free sugars and of the same sugar glycosidically bound. With this limitation in mind, a method was recently developed for the determination of free fucose in the presence of the gly- cosidically bound sugar which was not significantly affected by the presence of other free sugars (6). This assay was used successfully for the screening of fucosidases in a number of biological extracts. The fucosidase from Clostridium perfringens was chosen for further purification because of its interesting specificity. A preliminary report of this investigation has already appeared (7).

EXPERIMENTAL PROCEDURE

Materials

Crystalline L-( -)-fucose (m.p. 139-141’), obtained from Pfanstiehl Laboratories Inc., Waukegan, Illinois, was chroma- tographically pure in three solvent systems and had a specific rotation of [cr]z3 -75.9’ f 0.2”.

Purified porcine submaxillary glycoproteins with A or H blood group specificities (8) were prepared by modifications of procedures described elsewhere (9). They had the following analytical values: A, 8.9% fucose and 20.1 y0 N-glycolyl neuramic acid; H, 9.4 and 20.8%, respectively. Purified porcine gastric mucin was prepared according to the procedure of Morgan and King (10). This is a mixture of glycoproteins obtained from a pool of stomachs from hogs of A and H phenotypes. Methyl-a- and @-L-fucofuranosides and methyl-or- and P-L-fucopyranosides were synthesized as described by Bhattacharyya and Aminoff

(f-3. We are most grateful to the following individuals for their

generous gifts of materials: 2’-fucosyl lactose, lactodifucotetraose,

1659

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1660 Enzymes That Destroy Blood Group Specificity. I Vol. 245, No. 7

lacto-N-fucopentaose I, lacto-N-fucopentaose II, lacto-N-difuco- hexaose I, lacto-N-difucohexaose II (crude) from Professor R. Kuhn (11) ; a-L-fucopyranosyl-(1 -+ 2)@-n-galactopyranosyl- (1 -+ 4)-N-acetyl-o-galactosaminitol (12) from Dr. E. H. Eylar; 3’-fucosyl-N-acetylglucosamine from Dr. Roger Jeanloz (13) ; 3’-fucosyl lactose and lacto-N-fucopentaose III from Dr. V. Ginsburg (14) ; p-nitrophenyl-a-, P-D-, and L-fucosides from Dr. James Conchie (15).

The following commercially available materials were used: reagents for polyacrylamide disc electrophoresis (Eastman) ; DEAE-cellulose (DE52) (Reeve Angel Company, New York, New York) ; Sephadex G-75, G-200 (Pharmacia) ; bacteriological growth media (Difco) ; Alumina Cy (Sigma) ; Celite (Johns- Manville) ; bentonite (Fisher) ; collodion bags for vacuum di- alysis (Schleicher and Schuell Company). It was necessary to wash the bags prior to use, first with 0.01 N NaOH, then with 0.01 N HCI, followed by distilled water and the phosphate buffer (0.01 M potassium phosphate buffer, pH 7.0, containing 0.025 M

KCI). Buffalo black, used for the staining of proteins in gel electrophoresis, was obtained from Allied Chemicals. The following synthetic nitrophenyl glycosides were obtained from Cycle Chemical Company; p-nitrophenyl-ar-n-glucoside, o-nitro- phenyl-@-n-glucoside, p-nitrophenyl-@-n-glucuronide, p-nitro- phenyl-ol-n- and fl-n-galactosides, p-nitrophenyl-P-n-xyloside, -a-n-mannoside, p-nitrophenyl-P-N-acetyl-n-glucosaminide, -& N-acetyl-n-galactosaminide, phenyl-cu-N-acetyl-n-glucosaminide. p-Nitrophenyl-cu-N-acetyl-n-galactosaminide was obtained from Dr. B. Weissmann (16).

Blue dextran 2000, used as a marker in the gel chromatog- raphy, was obtained from Pharmacia. The following protein standards were used for the determination of the molecular weight of the fucosidase; fumarase (Dr. Vincent Massey), aldo- lase (E. F. Boehringer und Soehne), lactic acid dehydrogenase (Calbiochem), bovine serum albumin and cytochrome c (Mann). The n-fucose dehydrogenase, isolated from porcine liver, was obtained from Dr. Harry Schachter (17).

The buffers used in the determinations of the pH profiles were prepared as follows: (a) citrate phosphate (18), 0.4 M Na2HP0+ x ml, and 0.2 M citric acid (10-x) ml were mixed in varying pro- portions to give solutions of different pH; (b) acetate-Verona1 (19), 2 ml of a solution 0.143 M with respect to sodium acetate and to sodium diethylbarbiturate was mixed with 0.8 ml of 1.45 M lXaC1, and the pH was adjusted with 0.1 N HCI in a final vol- ume of 10 ml. All pH measurements were made at room tem- perature.

Methods

The following methods were employed: total fucose with a IO-min heating period (20); free fucose (6) ; total sialic acid by modification of the Svennerholm procedure (21) with I-butanol instead of isoamyl alcohol for the development of color (22) ; free sialic acid by the thiobarbituric acid procedure (23) ; protein by absorbance at 280 nm (24) or by the microbiuret procedure

(25). Electrophoresis in polyacrylamide gel slabs was performed by

a modification of the procedure of Davis (26) with the use of a 10.5% standard gel in borate buffer, pH 9. The electrophoretic separations were carried out at 4-6” with 50 ma per slab (27).

Paper partition chromatography was carried out in the follow- ing solvent systems: A, 1-butanol-ethanol-water (4 : 1: 1) ; B, l-butanol-pyridine-water (6:4:3) ; and C, Solvent B run on

dried borate (0.025 M, pH 9.0)-impregnated paper (28). Alka- line silver nitrate was used for the detection of reducing sugars, and Ehrlich’s reagent for hexosamines and N-acetylhexosamines (29, 30).

Enzyme Assay

Unless otherwise indicated, incubation mixtures contained the following components: ammonium sulfate, 130 pmoles; CaC12, 5 pmoles (at pH S.O), and 0.5 pmoles of glycosidically bound fucose in a total volume of 0.5 ml. The substrate em- ployed for the routine tests was a purified glycoprotein isolated from hog submaxillary glands with blood group H-specificity (8). Incubations were conducted at 37” for 15 min, and the reactions were stopped by heating the mixtures for 1 min at 100”. A 400~~1 aliquot was then quantitatively transferred to Conway units for the determination of fucose released (6).

When the substrate contained both fucose and sialic acid and it was desired to assay for both the fucosidase and sialidase ac- tivities, the same incubation mixture was used with a final vol- ume of 550 ~1. Of this, 400 ~1 were used for the determination of released fucose (6) and 100 ~1 for released sialic acid (23). Control incubation mixtures contained all the components with the deletion of either substrate or enzyme.

A unit of fucosidase (or sialidase) activity was defined as the amount of enzyme that released 1 pmole of fucose (or sialic acid) per hour from the hog submaxillary glycoprotein with H-speci- ficity. Specific activity was expressed in terms of micromoles of sugar released per hour per mg of protein.

Purijkation of Fucosidase

Growth of Cells-C. perfringens, Type 33-48, obtained from Mr. George S. Fearnehough (Department of Microbiology, University of Michigan), was maintained and grown in cooked meat medium (Difco).

For the preparation of enzyme the organism was grown in a Todd-Hewitt broth medium of the following composition (grams per liter): Todd-Hewitt broth powder (Difco), 35; K~HPOI. 3Hz0, 2.36; NaCI, 2.5; glucose, 1.5; cysteine HCl.lHzO, 0.05. The glucose and cysteine HCl were each autoclaved separately from the broth and both added to the medium just before the inoculation. A 0.1% inoculum from the meat broth stock cul- ture was used and growth maintained at 37” for 72 hours.

The cultures were grown in 2-liter flasks containing 1.5 liters of medium. At the end of 72 hours, the cultures were chilled to 4” and the cells were removed by centrifugation in a refriger- ated Sorvall or Sharples centrifuge. All subsequent steps in the purification were carried out in the cold room at 4”.

Ammonium Sulfate Fractionation-The culture supernatant solution (94.2 liters) was adjusted to 80% saturation (516 g of solid ammonium sulfate per liter). After 16 hours at 4”, the precipitate was collected by centrifugation in a Sharples ultra- centrifuge at 17,000 rpm. The pellet was dissolved in 5,500 ml of phosphate buffer. The resulting solution could be lyophilized or stored frozen for at least 2 years without loss of activity. To effectively separate the fucosidase from the sialidase, it was neces- sary to refractionate with solid ammonium sulfate collecting the precipitates between narrow concentration differences of am- monium sulfate. The best fraction was obtained between 52.3% saturation (307 g per liter) and 54.5% (322.4 g per liter) with a fucosidase to sialidase ratio of 15 (Table I). Only this fraction was utilized for further purification of the fucosidase, despite

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Issue of April 10, 1970 D. Aminoj’ and K. Furukawa 1661

the low yield, since we have found it extremely difficult to sep- arate the fucosidase from the sialidase in subsequent, steps.

Xephadex G-76 Treatment-The fraction, precipitated between 52.3 and 54.5% ammonium sulfate saturation, was dissolved in 200 ml of the phosphate buffer, and 196 ml were applied to a Sephadex G-75 (particle size 40 to 120 ~.r) column (8.4 X 75 cm) previously equilibrated with phosphate buffer. The enzymes were then eluted with the same buffer at a flow rate of 10 ml per hour, and 20-ml fractions were collected for protein and enzyme

assays. Both fucosidase and sialidase activities appeared in the first of the three major peaks of material absorbing at, 280 nm (Fig. 1). All tubes containing fucosidase activity were pooled (650 ml) for subsequent, fractionation.

Alumina Cy-Celite Chromatography-Alumina Cy and Celite were washed with phosphate buffer, mixed in the ratio of 1:4 (v/v), and packed in a column, 4 x 30 cm. Part, of the Sephadex G-75 eluate (250 ml) was applied to the column and washed with phosphate buffer (950 ml) until the absorbance of the effluent

70-

60 -

1000 2000 3000 4c Effluent volume (ml)

ZO

5.0

at, 280 nm was less than 0.025. Stepwise elution with ammonium sulfate was then achieved with the use of ammonium sulfate at,

TABLE I

Refractionation with ammonium sulfate I

L ‘00

5.0 E

8 1.0 N Fraction

?3

3.0 E? 6

e :: Original

20 2 1 2

1.0 3 4 5 6 7

FIG. 1. Elution pattern of 52.3 to 54.5yo ammonium sulfate re- fractionated material from Sephadex G-75. Protein concentra- tion (o- - -0) was determined by absorbance at 280 nm, fucosi- dase activity (O--O) by the Conway unit assay, and sialidase activity (A---A) by the thiobarbitnric acid assay.

8 9

10 11

_-

1.2

I .o

0.2

wH4)2SOa saturation

%

O-80.0

O-35.0 35.0-39.0 39.0-42.2 42.2-45.0 45.0-48.6 48.6-50.0 50.0-52.3 52.3-54.5 54.5-56.6 56.6-60.1 60.1-80.0

- I

--

-

Fucosidase

Total units

182,984 38,600

306 600

0 8,400

10,206 27,620 26,520

8,118 0 0

-

-(

-(

-(

‘0

Specific activity

0.68 1.3 3.02 3.6 0.04 0.5 0.09 0.2

1.23 0.2 2.86 0.4 4.09 2.0 4.31 15.1 1.06 3.2

latio of fucosidase to sialidase

10000 Effluent volume (ml)

FIG. 2. Alumina C-&elite chromatography of Sephadex G-75 eluate. Protein concentration (o-- - l ), fucosidase (O-O), and sialidase (A--A) activities were determined as in Fig. 1. The arrows indicate points at which the ammonium sulfate con- centration (To, w/v) was changed. Areas I to XIX designate tubes pooled for further investigation.

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1662 Enzymes That Destroy Blood Group XpeciJicity. I Vol. 245, No. 7

TABLE II

Summary of Alumina Cy-Celite chromatography fractions (I) (2) (3) (4) (6) (4) (6) VII VII VIII VIII

:NH&SO< concen- tration

% (4~) ml

Before 250 0 864 0.75 955 1.00 338 1.00 686 1.00 492 1.00 338 1.00 510 1.25 1120 1.50 750 1.75 410 1.75 1000 2.00 2800 2.50 1600 3.00 305 3.00 665 3.00 2050 4.00 195 4.00 750 4.00 1620

Fucosidase

Eluate Total xotein

Total units

Sephadex I

II III IV V

VI VII

VIII IX X

XI XII

XIII XIV

xv XVI

XVII XVIII

XIX

w 1075

61 162

24 69 25 10 15 22 7 4

20 28

5275 26 57 54

268 324 446

1330 1052 390 123 460 140

%

100

0.5 1.1 1.0 5.1 6.1 8.5

25.2 20.0

7.4 2.3 8.7 2.7

9 53

2 7

40 0.8 66 1.3

144 2.7 14 0.3 60 1.1 16 0.3

Total 518 5010

14.2

12: 18.8 10.4 15.0 15.3

0.7 2.5

ip3iC ctivity

4.91 0.42 0.3 2.3 3.9

13.2 1-4.6 38.6 $7.7 55.8 30.8 23.0 5.0

4.5 1.3

7.0 8.6

TABLE III

Purification of wfucosidase - -

Fraction l&al units

-

_-

-

Specific activity

Purifi- cation factor

-

c Re-

:ovely

182,984 0.68 1 100 1.3

26,520 4.31 6 15 15.1 13,715 4.91 7 8 14.2 3,460 88.6 130 2 1,287.5 3,170 176 702 2 cc 1,335 418 615 0.’ m

-

1. (NHd)zSOb (O-80yo saturation).

2. (NHdG304 (52.3- 54.5% saturation).

3. Sephadex G-75. . 4. Alumina Cr eluate 5. Bentonite 6. Vacuum dialysis.. 4

FIG. 3. Purity at various stages in the purification of ol-L-fu- cosidase as monitored by polyacrylamide gel electrophoresis of 0.6 unit of enzyme at pH 9.0 and stained with Buffalo black. 1, 0 to 80% ammonium sulfate precipitate; 2, 52.3 to 54.5% ammonium sulfate refractionated material; S, Sephadex G-75 eluate; 4, alu- mina Cr eluate Fractions VII and VIII; and 6, material after bentonite treatment and concentration by vacuum dialysis.

FIG. 4. As for Fig. 3, but histochemically stained for glycosi- dases with hog H-specific submaxillary glycoprotein as substrate.

progressively increasing concentrations up to 4% dissolved in phosphate buffer and at a flow rate of 75 ml per hour. Fractions of 30 ml each were collected for protein and enzyme assays. The stepwise elution pattern obtained from Cy is shown in Fig. 2. The major fucosidase activity was eluted at 1 to 1.25% ammo- nium sulfate (w/v). This treatment removed the last traces of sialidase activity (Table II).

Negative Absorption with Betionit+Bentonite (31) was washed with the phosphate buffer until the supernatant was completely clear after centrifugation. Preliminary batchwise absorption

experiments with bentonite indicated that the protein con- taminant is preferentially absorbed; in the presence of excess bentonite, fucosidase will also be absorbed. It is therefore necessary to carry out an initial “titration” to determine the optimum amount of bentonite necessary to use for each batch of CT eluate. With CT eluate VII (Table II), 5 ml of packed

bentonite were required per 100 ml of eluate. The CT eluate was added to the packed bentonite, well mixed for 10 min at 4”, and centrifuged at 9000 rpm for 20 min in a refrigerated Sorvall centrifuge.

Vacuum DialysisThe resulting supernatant was concentrated by vacuum dialysis in collodion bags against several changes of phosphate buffer in an ice bath at the rate of 25 ml in 8 hours. The enzyme is extremely labile at this stage of the purification

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CRUDE CLOSTRfDlLJM EXTRACT 1 PURIFIED FUCOSIDASE H

-H-l--- -+H+ 8 - -tt ;;_I - * *-- -2E +++-- -

IO -H+-- -ii+ +-- - +c

12 ‘..:,.;;.,;:;y1’ + - - - - ++-- -T +-- - .‘,:~.!,.,..‘p:.,’ + - - - - f-- - -

14 - -

16 - I

24 - I 26 .,..., :.,,, ..,,: - - - - - . . . . ;:., . . . --- - - ;,:.-;,i ,:t ‘y>; +-- - - + _ _ _ _ 26 -

30 ’ - -

32 ‘.,;y;?;$;:)L:: 1 1 1 : 1 -

34 -

FIG. 5. Identification of nature of glycosidase bands obtained on acrylamide gel electrophoresis. Fuc, L-fucose; Gal, o-galac- tose; NGN, N-glycolyl neuraminic acid; GalNAc, N-acetyl-n- galactosamine.

and invariably there is some loss of activity (Table III). Other methods of concentrating the enzymes were attempted but these are not as reproducible nor do they give as good recovery of the enzymes.

Purity of Fucosidase

Gel Ebctrophoresis-The course of fractionation at the various stages of purification (Table III) was followed by electrophoresis on polyacrylamide gels (26, 27) and stained for protein (Fig. 3).

The glycosidases in the various fractions were shown histo- chemically on the acrylamide gels by a modification of the for- mazan staining technique for reducing sugars (32), devised specifically to meet the requirements of a substrate with a very large molecular weight (33). The substrate was incorporated in the acrylamide prior to gelation. By use of this modified technique with the H-specific hog submaxillary glycoprotein as substrate, it was possible to show a number of formazan-staining bands in both the crude and the highly purified enzyme fractions (Figs. 4 and 5).

Since only fucose could be detected in the reaction products of the incubation (see below), the presence of several bands would imply that we have multiple isozymic forms of fucosidase. The following experiment was carried out in order to establish this. The crude extract and pure enzymes were run in parallel. One section of each was stained for glycosidases while the remainder of the slab was sliced horizontally into l-mm sections. Each I-mm section was then incubated separately with more substrate overnight, and the reaction products tested chromatographically for the sugars released (28-30) and serologically for the loss of H activity.

Of the glycosidases detected in the crude extract, galactosidase, sialidase, and fucosidase, only fucosidase appeared in the purified preparation. The principal fucosidase activity occurred in Slices 7 to 9, (Fig. 5), with a simultaneous maximal H-destroying

GalNAc

3

0 Gal

00000000 (I) (2) (3) (4) (5) (6) (7) (8)

FIG. 6. Diagrammatic presentation of paper chromatographic results of fucosidase action on H-specific hog submaxillary glyco- protein. Crude enzyme only (2)) pure enzyme only (S), substrate only (4), substrate plus crude enzyme (5), substrate plus pure en- zyme (6), and substrate plus pure enzyme plus fucose (7). (1) and (8) are standards. Whatman No. 1 paper treated with 0.025 M sodium tetraborate was used. The solvent system was l- butanol-pyridine-water (6:4:3) run for 22 hours by descending procedure. The paper was stained with alkaline silver nitrate reagents. GaZNAc, N-acetyl-n-galactosamine, Fuc, L-fucose; Gal, n-galactose, NAN, N-acetyl neuraminic acid; NGN, N- glycolyl neuraminic acid.

activity. Minor fucosidase and H-destroying activity occurred in Slices 11 and 12 and 19 and 20 with trace activities in 92, d7 and 88.

Action on Phenyl- and o- and p-Nitrophenyl GlycosidesThe following phenyl, o-nitrophenyl or p-nitrophenyl glycosides were tested: (Y- and fl-n-glucosides, /3-n-glucuronide, o(- and P-n-galac- tosides, P-n-xyloside, a-n-mannoside, Q- and O-n-N-acetylgalac- tosaminides, and o(- and P-n-N-acetylglucosaminides. The reaction mixture consisted of 0.6 unit of the fucosidase from the crude or pure enzyme, 5 pmoles of CaC12, 130 pmoles of ammo- nium sulfate, and 0.5 pmole of the substrate in a total volume of 0.5 ml. These were incubated for 15 min and 16 hours at 37”, and heated in a boiling water bath for 1 min to stop the reaction.

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1664 Enzymes That Destroy Blood Group SpeciJcity. I Vol. 245, No. 7

i

r: 06 :'

p

05 :

0.1

60 70 SO 90 100 110 120 130 Effluent volume (ml)

FIG. 7. Sephadex G-200 gel filtration of fucosidase and some standard proteins. Fucosidase (O--O) was assayed by Conway unit method. Blue dextran (a- - -0) was estimated by extinc- tion at G25 nm. Fumarase (CI- - -0) was determined by change of the absorbance at 300 nm. Lactic acid dehydrogenase (a- - -A) was assayed according to procedure of Neilands (46).

For the determination of hydrolysis of the p- and o-nitrophenyl glycosides, 0.5 ml of 0.97 N NazC03 was added to the incubations, mixed well, and spun to remove the precipitated salt. The extent of hydrolysis was determined photometrically, with p- nitrophenol as the standard. With the phenyl glycosides, 0.5 ml of 1 N Folin reagent was added to the incubation mixture and spun down. To 0.3 ml of the supernatant was added 1.7 ml of 0.4 M Na&O, and the resulting blue color after 30 min at room temperature was compared at 650 nm with phenol as the standard.

Only a trace of activity was detected with p-nitrophenyl-/?-N- acetyl-o-glucosaminide after 16 hours of incubation. This was very small compared to the strong reactivity shown by the crude enzyme preparation after 15 min of incubation.

Sialidase-To determine the presence of sialidase, purified hog H-specific submaxillary glycoprotein (9.4% fucose and 20.8% N-glycolyl neuraminic acid) was used as the substrate. Incuba- tions were set up in the usual manner and the fucose and sialic acid released were determined chemically (6, 23). No sialic acid was detected even after 16 hours of incubation when over 95% of the total fucose was released. The absence of sialidase was further confirmed by the chromatographic data as described under “Characterization of Reaction Product” (Fig. 6).

Protease Activity-To test for protease activity, crude porcine submaxillary glycoprotein still contaminated with proteins was considered the most suitable typical substrate. The crude glycoprotein, 1 mg, was incubated with 0.05 mg of trypsin, Pronase, or 0.5 unit of fucosidase from the crude or pure prep- aration (Steps 1 and 6, Table III) in acetate-Verona1 buffer, pH 6.0, in a total volume of 0.5 ml. After 8 hours at 37”, 1 ml of the ninhydrin reagent? was added and the tubes heated for 12 min in a boiling water bath, cooled in ice water, and after 5 min

1 The ninhydrin reagent consists of 25 parts of 0.04% ninhydrin in 0.5 M citrate buffer. DH 5.5. mixed with 12 parts of analvtical grade glycerol. The method described is a private communica- tion from Dr. H. Tager, Department of Biological Chemist,ry, University of Michigan, and represents a modification of the pro- cedure described by Lee and Takahashi (34).

4.0 45 50 5.5 6.0 65 70 7.5 80

PH

FIG. 8. Effect of pH on enzyme activity. O--O, acetate- Veronal; O----O, acetate-Verona1 plus GaGlz (.Ol M); X--X, citrate-phosphate.

of aeration the colors obtained were read at 570 am with glycine as standard. Under these conditions of test, trypsin released ninhydrin-reacting material equivalent to 2.3 pmole of glycine, Pronase, 2.9 wmoles; crude fucosidase, 0.6 pmole and the pure fucosidase, 0 pmole.

Action on Serological Activity of Blood Group Substances-As has been observed by Stack and Morgan (35), crude Clostridium welchii culture filtrates can inactivate A, B, and O(H) blood group specificities. We have confirmed this and, moreover, have shown that our crude C. perfringens filtrates also contain Lea-destroying activity (36). It was of interest, therefore, to compare the effect of the purified fucosidase on the various sero- logical activities. These results will be reported in greater detail elsewhere,2 but suffice it to state at this stage that only the H- destroying activity could be detected in the purified preparation.

Characterization of Reaction Product

Hog submaxillary glycoprotein of H-specificity contains N- acetyl-2-deoxy-Z-amino-n-galactose, n-galactose, N-glycolyl neu- raminic acid, and n-fucose (37-40). Of these components, only n-fucose would yield acetaldehyde on oxidation with periodate. This product was detected and determined in the Conway unit assay (6). Identification of fucose, as the sugar released by the enzyme, was confirmed by paper partition chromatography of the incubation mixture after the removal of salts by precipitation with 4 volumes of ethanol.

Solvent system C gave the most effective separation of all the carbohydrate components anticipated (Fig. 6). The crude en- zyme preparation released N-glycolyl neuraminic acid and fucose, while the purified enzyme released only one reducing spot that migrated like fucose. Admixture of the incubation products of

2 K. Furukawa and D. Aminoff, to be published.

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Issue of April 10, 1970 D. Amino$ and K. Furukawa 1665

the purified enzyme with substrate and known n-fucose resulted in no increase in the number of spots (Fig. 6).

Further characterization of the reaction product as n-fucose was made with the DPN+-dependent L-fucose dehydrogenase from porcine liver (17). The amount of n-fucose released on incubation of H-active hog submaxillary glycoprotein with the fucosidase at 15 min and 5 hours was determined by the Conway unit method (6) and by the DPN+-dependent n-fucose dehy- drogenase (41). In both cases the dehydrogenase assay ac- counted for 80 to 85% of the n-fucose as determined by the peri- odate method. In view of the established specificity of the dehydrogenase (17), one may conclude that the enzyme from C. perfringens is an n-fucosidase.

Determination of Molecular Weight

The behavior of the cu-fucosidase on gel filtration through Sephadex G-200 was undertaken to determine whether the elec- trophoretically demonstrable isozymic pattern is reflected in a like number of enzymatically active molecular subunits, and also to determine the order of magnitude of molecular weight of the enzyme (42, 43). Sephadex G-200 (particle size 40 to 120 p) was allowed to swell in 0.01 M potassium phosphate buffer, pH 7.0, containing 0.025 M KCl. The deaerated suspension was packed under gravity in a column, 2.4 x 50 cm. The concen- trated T-VIII, bentonite-treated fucosidase preparation, con- taining 40 units of the enzyme, was mixed with a number of proteins of known molecular weight and applied to the gel as a solution in 2 ml of the same buffer. The elution rate was 14 ml per hour and 2.3-ml fractions of eluate were collected in a fraction collector at 4”. Blue dextran 2000 (1 mg) was used to indicate the void volume, and the following proteins were used as standards: fumarase, 1.2 mg (molecular weight 185,000 to 225,000) ; aldolase, 1 mg (molecular weight 140,000 to 150,000) ; lactic acid dehydrogenase, 0.0815 mg (rabbit muscle, molecular weight 130,000 to 140,000) ; bovine serum albumin, 4 mg (molec- ular weight 65,000 to 70,000) ; cytochrome c (horse heart, molec- ular weight 12,400) (43). The position of the elution peaks of the fucosidase, blue dextran and standard proteins was deter- mined either by direct absorption at 280 nm (bovine serum al- bumin), 412 nm (cytochrome c), and 625 nm (blue dextran), or by the appropriate enzyme assay for fumarase (44), aldolase (45), and lactic acid dehydrogensse (46).

Fig. 7 shows the relevant results obtained with the blue dex- tran, fumarase, lactic acid dehydrogenase, and the fucosidase. The broadness of the peak obtained with the fucosidase as well as the fact that it eluted faster than any of the protein standards precludes an accurate assessment of its molecular weight beyond the statement that it appears to be greater than that of fumarase, 185,000 to 225,000 (43).

Properties of cr-Fucosidase

Stability of Enzyme-The fucosidase is stable to freezing at all stages of purification, but is inactivated on repeated freezing and thawing. Lyophilization results in inactivation of the purified preparations. The only procedure to give satisfactory concen- tration of the enzyme without appreciable loss of activity is vacuum dialysis against phosphate buffer, as discussed above. Simple dialysis against distilled water results in a rapid loss of activity. Dialysis against buffers at various pH values results in preciprtation of the enzyme at pH values below 5.5, with con- siderable inactivation.

Y I 2 3 4

Bound fucose (M ~10~) in H submaxillary glycoproteln [sl

FIG. 9. Effect of substrate (H-specific hog submaxillary glyco- protein) concentration on reaction rate. O---U, substrate only, A; O--O, substrate plus enzyme, B; O--O, corrected values for B - A, C; A--A, plot of corrected [S]/V against [S].

Kinetic Studies

Effect of pH-The following incubation mixtures were set up in order to determine the pH profile of the enzyme: 100 ~1 of the enzyme, 1 unit; 100 ~1 of the buffer of the appropriate pH, 100 ~1 of the hog H submaxillary glycoprotein substrate con- taining 0.45 pmole of bound fucose in a final volume of 0.5 ml. The amount of fucose released was determined after 15 min at 37” and the results expressed in terms of units of activity as compared to that determined by the standard conditions (1 unit) in ammonium sulfate (0.26 M) and CaClz (0.01 M) previously adjusted to pH 6.0.

The results are shown in Fig. 8. Maximal activity is obtained at pH 5.8 in sodium acetate-Verona1 buffer and 6.3 in the citrate- phosphate buffer. Enhancement of activity and a slight in- crease in pH optimum to 6.0 occurs with acetate-Verona& in the presence of CaC&, 0.01 M. Under these conditions, the activity is the same as with CaClz and ammonium sulfate pre- viously adjusted to pH 6.0 (final concentration 0.01 and 0.26 M,

respectively) as used in the routine assay. Effect of Substrate Concentration-Incubating various concen-

trations of the same substrate for 15 min with the optimal con- centrations of CaClz and ammonium sulfate, alone and with the enzyme, resulted in Curves A and B, Fig. 9. The substrate gives a persistent and constant “blank” which is directly pro- portional to the amount of substrate present. Correction for this interference results in Curve C (Fig. 9) which gives an indi- cation of substrate inhibition at high concentrations. When plotted according to the method of Lineweaver and Burk (47) as modified by Hanes (48), the K,,, obtained is 0.175 mM. How- ever, this is only a tentative and an apparent K, value on ac- count of the high substrate blank and the fact that the substrate is a high molecular weight polymer with multiple substrate sites.

E$ect of Enzyme Concentration and Period of Incubation-The rate of hydrolysis of the H-specific submaxillary glycoprotein was proportional to the amount of enzyme (Fig. 10) and period of incubation (Fig. ll), in the range normally employed.

Inhibitors and CofactorsThe effect of a number of metals and miscellaneous compounds was tested at 10 mM concentration, unless otherwise stated, for their ability to enhance or inhibit

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Enzymes That Destroy Blood Group Specificity. I Vol. 245, No. 7

V I I 8 I 1 I 0 2 4 6 6 IO

Protein o&l)

FIG. 10. Effect of enzyme concentration on reaction rate. In- cubation mixtures were made up in the assay procedure described in the text and containing CaClz and ammonium sulfate at pH 6.0 and 0.5 pmole of bound fucose. The fucose released was deter- mined in Conway units after 15 min of incubation.

Minutes Incubation

FIG. 11. Effect of time on the amount of fucose released. In- cubation mixtures were made up in the assay procedure described in the text and containing CaClz and ammonium sulfate at pH 6.0, 0.5 rmole of bound fucose, and 5.25 pg of enzyme.

the release of fucose from the H-specific hog submaxillary glyco- protein. The incubations were carried out in Veronal-acetate buffer, pH 6.0, and the amount of fucose released in 15 min in the buffer alone was taken as 100% for purposes of comparison. The compounds under investigation had no effect on the assay for free fucose, unless otherwise indicated.

Sodium, magnesium, and calcium chlorides, as well as am- monium sulfate, enhance the activity of the enzyme. The rela- tionship of enzymatic activity to the concentration of the salt was investigated in greater detail in the case of NaCl, CaC12, and ammonium sulfate, as additives to 0.009 M acetate-Verona1 buffer, pH 6.0. The results are expressed in terms of a per- centage of the activity shown in the presence of buffer alone. The enhancing effect of CaClz is illustrated in Fig. 12. The ammonium sulfate stimulation reaches a plateau of 0.1 M, with little further stimulation. Sodium chloride enhances and, in higher concentrations, inhibits the fucosidase activity.

The following compounds, at a final concentration of 0.01 M,

t I I /

0.2 0.4 0.6 0.6 I .o 1.2 1.4 16 ti F~nol concentration of salt CM)

1 !2

FIG. 12. Effect of different ions on rate of reaction, expressed as a percentage of activity in 0.009 M sodium acetate-Verona1 buffer, pH 6.0, without any other additives. O--O, CaCL; e-0, ammonium sulfate; O-O, NaCl. All salt solutions wereprevi- ously adjusted to pH 6.

FIG. 13. Extent of fucose released from the hog A- and H-spe- cific submaxillary glycoproteins. The experimental details are given in the text.

I I

I 2 3 4 HOUrS

FIG. 14. Effect of various additives to the amount of fucose re- leased from hog A- and H-specific submaxillary glycoproteins. The experimental details are given in the text.

have no significant effect on the fucosidase activity: Na2SOd, Na2HP04, KCl, CoC12, spermine hydrochloride (0.003%), sper- midine phosphate (0.003 %), merthiolate (0. 1 %, w/v), toluene (lo%, v/v), and chloroform (10% v/v).

Spermine hydrochloride and spermidine phosphate were tested because of the alleged (49) stimulating effect of these compounds on glycosidases. Toluene, chloroform, and merthiolate, the common bacteriostatic agents used for prolonged incubations, were tested in order to determine their effect on the enzyme.

p-Chloromercuribenzoate, iodoacetamide, and EDTA inhibit

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Issue of April 10, 1970 D. Aminof and K. Furukawa 1667

the fucosidase to a limited extent (50 to 70%) when tested at a not be determined, however, since oxidation of these compounds final concentration of 0.01 M in 0.014 M acetate-Verona1 buffer. with periodate under the conditions of assay for free fucose re- There is marked inhibition by salts of heavy metals (0.01 M): sults in the release of acetaldehyde. Since the fucosidase has Fe++, Fe+++, Hg++, Zn++, Cu++. The results with cysteine no action on the simple methyl and nitrophenyl fucosides (see HCl and oxidized glutathione are not too dependable in view of “Substrate Specificity”), it was of interest to determine whether their significant interference in the Conway unit assay for free these would inhibit the fucosidase. In this experiment, the fucose, but they also appear to markedly inhibit the enzyme. H-specific hog submaxillary glycoprotein (at 1 mM of bound

It has been established that lactones inhibit the corresponding fucose) was incubated with the enzyme in the presence of CaClz

glycosidase (50). The effect of D- and n-fuconolactones could and ammonium sulfate at pH 6.0, in the standard test condition

TABLE IV

Substrate specificity of rr-L-jucosidase

Substrate structure

Porcine submaxillary glycoprotein (H) ........ Human ovarian cyst glycoprotein (Lea). ...... Methyl-ol-n-fucopyranoside. ................... Methyl-P-n-fucopyranoside. ................... Methyl-cu-n-fucofuranoside. .................... Methyl-@-L-fucofuranoside ..................... p-Nitrophenyl-a-n-fucopyranoside. ............ p-Nitrophenyl-a-n-fucopyranoside ............. p-Nitrophenyl-P-n-fucopyranoside ............. p-Nitrophenyl-P-n-fucopyranoside ............. or-n-Fucopyranosyl-p-azophenyl-0-BSAa ........

3’-Fucosyl-N-acetylglucosamine ................

2’-Fucosyllactose .............................

3’-Fucosyllactose .............................

. . . .

. . . .

. . . .

. . . .

Trisaccharide from porcine submaxillary glycoprotein

Lactodifucotetraose. . . . . . . . . . . . . . . . . . . . .

Lacto-N-fucopentaose I. . . . . . . . . . . . . . . . . . . . . . . . . .

.

.

. ’

. (

.

, .

Gal 153 GlcNAc 1 8, 3 Gal l-%4 Glc

T 2

CYFUC

Lacto-N-fucopentaose II. . . . . . . . . . . . . . . . . . Gal 1 --% 3 GlcNAc 1 -@+ 3 Gal 1 --% 4 Glc

T 4

orFuc

Lacto-N-fucopentaose III.. . . . . . . . . . . . . . . . . . Gal 1 -k 4 GlcNAc 1 L 3 Gal 1 -% 4 Glc

T 3

orFuc

Lacto-A’-difucohexaose I. . . . . . . . . . . . . . . . .

La&o-N-difucohexaose II. . . . . . . . . . . . . . . . . . . . . . . . ,

Gal 1 --% 3 GlcNAc l-% 3 Gal 1 L 4 Glc

T 2 T 4

aFuc aFuc

Gal 1 8, 3 GlcNAc 1 -% 3 Gal 1 -% 4 Glc

T 4 T 3

olFuc olFuc

--

Fuc 1 P, 3 GlcNAc

Fuc 1 * 2 Gal 1 -% 4 Glc

Gal 1 8, 4 Glc

T 3

cuFuc

Fuc 1 -% 2 Gal l-&4 Gal.OLNAc

Fuc 1 -% 2 Gal 18,4 Glc

T 3

olFuc

-

- Fucose released in

15 min 5 hrs

% 35

0 2 0 1 0 0 0 0 1 0

0

21

2

36

5

12

2

2

2

90 6 0 0 0 0 0 0

0 4

2

79

10

92

39

80

4

9

7

14

0 The abbreviations used are: BSA, bovine serum albumin; Fuc, n-fucose; GlcNAc, N-acetyl-n-glucosamine; Gal, n-galactose; Glc, n-glucose; Gal’OLNAc, N-acetyl-n-galactosaminitol.

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with or without the addition of the appropriate fucoside (1 mM) under test. No significant inhibition was observed. The re- sults would also suggest that there has been no transglycosylation involved with the simple glycosides acting as acceptors.

Substrate Specificity

The following experiment was done in order to determine the extent of release of fucose from hog H-submaxillary glycoprotein. The incubation mixtures in a total volume of 0.5 ml contained 1 unit of fucosidase, 5 pmoles of CaC&, 130 pmoles of ammonium sulfate, and 1 mg of H-active glycoprotein containing 0.63 pmoles of bound fucose. The incubation mixtures were held at 37” and at predetermined time intervals the tubes were re- moved, boiled for 1 min, and 400-J aliyuots removed for the determination of fucose released (6). There is a very rapid release of fucose, as shown in Fig. 13, and the reaction is essen- tially complete in 1 hour. The experiment was repeated with hog A-active submaxillary glycoprotein, 1 mg containing 0.48 pmole of bound fucose. A similar rapid release of fucose occurs within 1 hour and again the reaction is essentially complete. However, in this case only 20% of the total fucose is released.

In order to determine the reason for this incomplete release of fucose, incubations were put up as indicated above and kept at 37” for 2 hours. The course of the fucose released over the next 2 hours was followed after incubation with the following addi- tives: (a) more substrate, 0.48 pmole of A-glycoprotein, (b) more of the pure enzyme, 1 unit, (c) addition of the crude enzyme from C. perfringens, 0.75 unit, and (d) no additives. From the results obtained, shown diagrammatically in Fig. 14, we can infer that the pure enzyme has not been inactivated during the 2 hours of incubation, but rather that it is fully active and capable of re- leasing all the fucose from the fresh substrates added. Addition of more fresh pure fucosidase releases no further amount of fucose, confirming that the reaction was essentially complete at the end of the 2 hours and implying that all the bound fucose that is susceptible to release with this fucosidase has indeed been released. The fact that more fucose is released on incuba- tion with the crude enzyme, from both the A-specific, and to a lesser extent, H-specific glycoproteins, suggests that these glyco- proteins either contain fucose bound in yet another form (to account for the observed increment of fucose released with the crude C. perfringens), or, as is more likely in the case of the A- specific glycoprotein, there is steric hindrance attributable to the close proximity of the terminal N-acetyl-n-galactosamine, the A-determinant, attached cr(1 + 3) to the same galactose residue to which the fucose is attached cr(1 -+ 2), or both. The observed 20% fucose released would represent incomplete oligosaccharide chains within the glycoprotein macromolecule. Such chains have been detected chemically in the hog submaxillary A-glyco- protein (40).

It would appear from these kinetic data that the fucosidase is highly specific. The nature of that specificity was determined by its action on a number of simple methyl and phenyl fucosides and on milk oligosaccharides of known structure. Table IV summarizes the results of incubation for 15 min and 5 hours to reflect relative rate and extent of susceptibility to hydrolysis. The fucosidase has no action on the simple fucosides, a- or /?-, D-,

or I~-, methyl- or p-nitrophenyl, pyranoside, or furanoside. Of the milk oligosaccharides tested, the most readily suscepti-

ble appear to be 2’-fucosyl lactose and lacto-N-fucopentaose I. In both cases the L-fucose is bound to galactose, in an a-(1 + 2) linkage (11). In both cases, however, the rate of release of

fucose from these oligosaccharides is slower than with the large molecular weight glycoprotein substrate. The trisaccharide ob- tained by Katzman and Eylar (12) from porcine submaxillary glycoprotein on alkaline borohydride hydrolysis behaves in exactly the same manner as the original I-I-specific glycoprotein.

The enzyme has a very limited action on 3’-fucosyl lactose. The results obtained with the lactodifucotetraose are misleading. Lactodifucotetraose contains 2 fucose residues per molecule, only 1 of which, the (~(1 -+ 2) residue, is susceptible to hydrolysis. Hence, the 39% of the total fucose released after 5 hours would represent 78% of the cr(l + 2).linked fucosyl residues. This is of the same order of magnitude obtained with the other a(1 --t 2) fucosyl oligosaccharides. The lack of activity on ol(l 4 4)- bound fucose is evident in fucopentaose II, and reflected in the fact that it has very little action on the Lea-active glycoprotein from human ovarian cyst fluid. The activity with lacto-N- difucohexaose I is inexplicable at present. It should be of the same order of magnitude as the lactodifucotetraose.

DISCUSSION

A number of fucosidases of mammalian origin have been de- scribed in the literature (51, 52) and in limpet (53), abalone livers (54, 55), Helix pomatia (56), Rhodopseudomonas palustris (57), and, phage-induced, in Klebsiella aerogenes (58). In a few cases there has been a limited amount of purification beyond the initial identification (51, 54, 55). In most studies, the substrate used was a simple nitrophenyl fucoside for ease of following the purification of the enzymes. In only a few cases, however, was the substrate specificity of the enzyme determined. Where tested, these fucosidases had no action on the blood group active glycoproteins (5).

Where enzymes capable of destroying the H- or Lea-blood group specificity of glycoproteins had been described (3, 5), it was presumed that the loss of serological activity was attributa- ble to the loss of fucose. But since the release of more than one sugar was detectable, the complete correlation of st#ructure and serological specificity was not possible. Again, where tested, these enzymes had no action on simple glycosides, and thus their purification was greatly handicapped for the lack of a suitable rapid chemical assay (3).

In order to overcome these limitations, it was necessary to devise a specific assay to determine the free fucose in the presence of the glycosidically bound sugar (6). The purification and isolation of the specific ~y(l + 2)+fucosidase was achieved by the use of a substrate in which all the fucose is bound in that form only, namely the hog H submaxillary glycoprotein. An- other valuable innovation introduced in these investigations (33) is the development of a comprehensive procedure for the histo- chemical detection of glycosidases adaptable to large molecular weight substrates. This is a necessary tool in the preliminary survey for detection of potential glycosidases acting on the same substrate, to follow the purification of the glycosidase under investigation, ultimately to establish its purity, and, finally, to show the presence of multiple forms of the enzyme as evidenced in this particular a-L-fucosidase.

This clear cut demonstration of multiple isozymic forms in a glycosidase with well defined substrate position specificity, ~(1 + 2)+fucosidase, is very intriguing. The isozymes may quite possibly represent end-products of partial degradation of the fucosidase by amino- or carboxypeptidases present in the crude bacterial culture filtrates.

It is possible that the same type of isozymic pattern will be

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Issue of April 10, 1970 D. Aminoff and K. Furukawa

detected in other glycosidases after extensive purification, as has already been suspected in the sialidases (22, 59). Apart from its intrinsic interest, it does present technical difficulties in iso- lating a given type of glycosidase free of all other straggling iso- zymes of other glycosidases, and providing us the assurance that all the isozymes of the given glycosidase under investigation are indeed harvested in the final preparation.

Turning now from the enzymes to the substrates upon which they act, there are indications that perhaps not all the fucosyl residues in the complex glycoproteins studied are a!-(1 --+ 2). This is strongly suggested by the nature of the kinetic studies on the fucose released from the various submaxillary glycopro- teins when treated with the crude and purified enzymes. The data, however, could also be explained, to a certain extent, by steric hindrance caused by sugars adjacent to the potentially susceptible fucose. Investigations are under way to resolve these problems and their significance will be discussed elsewhere (30).

The isolation of pure glycosidases provides us with valuable tools for structural studies of complex heteropolysaccharides and conjugated glycoproteins and glycolipids. Since many of these compounds have interesting and important biological properties, either in solution or at cellular membrane surfaces, these can now be studied at the molecular level correlating biological activity with structure.

Acknowledgments-We are deeply indebted to the able tech- nical assistance of the Misses Cheryl Lethemon, Marianne P. Morrow, and Leslie Wang.

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David Aminoff and Ken FurukawaPERFRINGENS

-l-FUCOSIDASE FROM CLOSTRIDIUMαPROPERTIES OF Enzymes That Destroy Blood Group Specificity: I. PURIFICATION AND

1970, 245:1659-1669.J. Biol. Chem. 

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