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Studies on the structure of the carbohydrate moiety of rabbit γG-globulin—I degradation with glycosidases
lmmunochemistry. Pergamon Press 1969. Vol.6, pp. 739-749. Printed in Great Britain
STUDIES ON T H E STRUCTURE OF T H E CARBOHYDRATE MOIETY OF RABBIT y G - G L O B U L I N - I D E G R A D A T I O N W I T H GLYCOSIDASES* BURTON R. ANDERSEN Departments of Microbiology and Medicine, Northwestern University School of Medicine, Chicago, Ill. 60611, U.S.A.
(First received 13January 1969; in revisedform 17 February 1969) Abstract- Information about the structure of rabbit yG-globulin carbohydrate has been
obtained with the use of isolated glycosidases and concanavalin A. N-acetyl neuraminic acid and N-acetyl glucosamine were shown to be major terminal sugars. Only a small fraction of the galactose was in a terminal position. Mannose could not be detected in a terminal position and appeared to be internal to both N-acetyl glucosamine and galactose. The following anomeric linkages have been established: N-acetyl glucosamine and galactose have /3-linkages while mannose and fucose are c~-linked. Concanavalin A precipitation studies show that some of the non-terminal mannoses are linked to other sugars through carbon-2. INTRODUCTION I m p o r t a n t data about the c a r b o h y d r a t e structure o f rabbit 3/G-globulin have been p r o v i d e d by studies using the technique o f chemical d e g r a d a t i o n and analysis [1-4]. T h e type and a m o u n t o f the sugars [1-3] and some indication o f their location o n the 3/-globulin molecule[3,4] have been d e t e r m i n e d . T h e s e experiments, however, have not established the sugar sequence or the n a t u r e o f the glycosidic linkages. I have succeeded in isolating a n u m b e r o f specific glycosidases f r o m jack bean meal, snail hepatopancreas, and o t h e r sources. T h e action o f these enzymes on the c a r b o h y d r a t e moiety o f rabbit 3/G-globulin provides i n f o r m a t i o n about the sequence o f sugars and the anomeric configurations o f the glycosidic bonds. MATERIALS AND METHODS
Chemicals and substrates T h e following glycosides were obtained f r o m commercial sources: p-nitrophenyl-~-D-galactopyranoside,t p-nitrophenyl-/3-D-galactopyranoside,t p-nitrophenyl N-acetyl /3-D-galactosaminide (p-nitrophenyl 2-acetamido-2deoxy-/3-D-galactopyranoside),$ p-nitrophenyl ~-L-fucopyranoside,t p-nitrophenyl-/3-L-fucopyranoside,$ p-nitrophenyl-N-acetyl /3-D-glucosaminide (p*A preliminary report of some of this work was presented at a meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., April 1968. This study was supported in part by the Illinois Chapter of the Arthritis Foundation and U.S.P.H.S. General Research Support Grant 5-SO1-FR-05370-07. ?Aldrich Chemical Co., Milwaukee, Wisc. :~Pierce Chemical Co., Rockford, Ill. 739
740
B.R. ANDERSEN
nitrophenyl 2-acetamido-2-deoxy-/3-D-glucopyranoside),* p-nitrophenyl-a-Dmannopyranoside,* and methyl-a-D-mannopyranoside.t Melting points were determined in all cases, and they agreed with published values. Methyl-/3-Dmannopyranoside and a formamide extract of streptococcal cell walls (Z3) containing a-linked N-acetyl glucosamine [5] were generously supplied by Drs. Elwyn T. Reese and Jan Willers, respectively. The rabbit yG-globulin used in these experiments was obtained "from a commerical supplier.:~ Acrylamid~disk electrophoresis and immunoelectrophoresis were used to examine the preparation for contaminating proteins. Three proteins in addition to yG-globulin were found; one was albumin in a concentration of 1 per cent or less as measured by quantitative immunodiffusion studies. The other two proteins were not identified but were present in much lower concentrations than the albumin (probably < 0.1 per cent). Since the major contaminant, albumin, has a very low carbohydrate content (< 0.3 per cent), it is unlikely that measurable amounts of carbohydrate were contributed by these proteins. The carbohydrate content of the rabbit y-globulin used in this study is in general agreement with other reports [1,3]. The carbohydrate concentrations were the following: hexose 1.01 per cent, n-acetyl glucosamine 1.44 per cent, n-acetyl neuraminic acid 0.16 per cent, and fucose 0.10 per cent.
Enzyme assay Glycosidases, with the exception of neuramimdase, were assayed with p-nitrophenyl glycoside substrates. The reaction mixture consisted of the following: 0.1 ml of the enzyme test solution, 0.1 ml of the p-nitrophenyl glycoside at a concentration of 0.01 M, and 1.25 ml of the optimal buffer for the enzyme being tested. In most cases the buffer was 0.1 M citrate pH 4.5. After incubating for 1 hr at 37°C, 1.5 ml of 0.2 M Na2COn was added to the mixture. Glycosidase activity was indicated by the release of p-nitrophenol from the substrate and was measured by light absorption at 400 m#. The values of the change in optical density were used as the units of enzyme activity. A unit of neuraminidase activit$ was that amount which would liberate 1 gg of N-acetyl-neuraminic acid from a,-glycoprotein within 15 min at 37°C.
Source and preparation of enzymes A neuraminidase (Vibrio cholerae) preparation which was commercially obtained§ was tested and found to be free of other glycosidase activities./3Galactosidase (E. coli) which also was free of other glycosidases was generously provided by Dr. Martin Rachmeler. ot-Fucosidase was prepared from the hepatopancreas of the sea snail, Gastropoda Busycotypus. H The hepatopancreas was homogenized in a Waring blendor, and an aqueous suspension of this material was centrifuged to remove cell debris. The precipitate which formed at 50 per cent ammonium sulfate *Mann Research Laboratories, Inc., New York, N.Y. tPfanstiehl Laboratories, Inc., Waukegan, Ill. ~Pentex, Inc., Kankakee, Ill. §Hoechst Pharmaceuticals, Inc., Cincinnati, Ohio. ~Marine Biological Laboratory, Woods Hole, Mass.
The Structure of Rabbit y-Globulin Carbohydrate SO e4 (NH4)2SO 4
741
Precipitate
& CM Cellulose
•OI--OSM (luote Q- and [3- oaloctosidose ~-91ucosominidase trace Q-monnosidase
.2M (luate 0 - monnosidose [3 - glucosominidose trace ~-galactosidase
OEA(* Cellulose
DEAF- Sephadex
; "01M Cl- and [3" golactosidase - glucosominidose
•2 M (luote ~- glucosaminidose
~L
"5M P),ridine Acetate Eluate
DEAE - Sephodex
O - monnosidese
•01M Eluate -galactosidase
Fig. 1. The scheme for the isolation of glycosidases from jack bean meal. On some occasions one or more of the steps had to be repeated when the proper separation had not been achieved. See text for details. saturation was extensively dialysed. This preparation contained the following glycosidases: a-fucosidase 16/z/ml, fl-glucosaminidase > 30/.~/ml, 0t-galactosidase 1/z/ml and/3-galactosidase 6/x/ml. T h e r e was no measurable B-fucosidase activity. Attempts to completely separate the ct-fucosidase from the other glycosidases were unsuccessful. T h e scheme for enzyme isolation from jack bean meal* is shown in Fig. 1. Some of the steps are modifications of the procedure of Yu-Teh Li [6]. G r o u n d meal was first suspended overnight in distilled water and then centrifuged to remove insoluble material. T h e aqueous extract was b r o u g h t to 50 per cent a m m o n i u m sulfate saturation at 4°C, and the precipitate which f o r m e d was dissolved in and extensively dialysed against 0.01 M citrate pH 4.5 buffer. This preparation was a d d e d to carboxymethyl (CM) celluloset which had been equilibrated with the same buffer. Step-wise elutions with citrate buffers of constant pH (4-5) and increasing molarity provided two major fractions. T h e first fraction was eluted with 0.01-0.05 M citrate buffers. T h e CM-cellulose was then washed *Sigma Chemical Co., St. Louis, Mo. tCM-32 Whatman.
742
B.R. ANDERSEN
with 0.1 M citrate and the wash solution discarded. The second fraction was eluted with 0.2 M citrate. The first CM-cellulose fraction (0.01-0.05 M eluate) which contained ~z- and /3-galactosidases,/3-glucosaminidase and a trace of o~-mannosidase was dialysed against 0.01 M phosphate pH 7.6 prior to adding diethyl amino ethyl (DEAE) cellulose* equilibrated with the same buffer. Two major fractions were obtained, the first contained those enzymes that did not bind to the DEAE-cellulose with the 0.01 M phosphate buffer, including a-galactosidase,/3-galactosidase, and a small amount of /3-glucosaminidase. After thoroughly washing the DEAEcellulose with the first buffer, the second fraction was eluted with 0"2 M phosphase pH 7.6. /3-Glucosaminidase was the major enzyme in this fraction, and when contaminating enzymes were present they could be removed by repeating the DEAE-cellulose step. The first fraction from the DEAE-cellulose was dialysed against a 0.01 M phosphate pH 7.0 buffer and mixed with DEAE-Sephadex A50 which had been equilibrated with the same buffer. Under these conditions/3-galactosidase failed to bind to the DEAE-Sephadex while the other enzymes were adsorbed. The second fraction from the CM-cellulose step which contained a-mannosidase,/3-glucosaminidase and some/3-galactosidase was dialysed against 0.01 M phosphate pH 7.0 and mixed with DEAE-Sephadex A50. Repeated washing with the same buffer removed all the contaminating/3-galactosidase, a-Mannosidase was eluted in pure form with a 0"5 M pyridine acetate buffer pH 6.0. Since p-nitrophenyl/3-mannopyranoside and p-nitrophenyl ~-glucosaminide were not available to check for/3-mannosidase and ~-glucosaminidase activities, there was some question whether the c~-mannosidase and /3-glucosaminidase enzymes had been completely isolated. The ~-niannosidase preparation was incubated with methyl o~-mannopyranoside and methyl /3-mannopyranoside. Mannose was cleaved only from the c~-anomer indicating that there was no contaminating/3-mannosidase in the preparation. The /3-glucosaminidase preparation was incubated with a formamide streptococcal cell wall extract (Z3) which contained terminal ~x-N-acetyl glucosamine[5]. No glucosamine was released after incubation for 2 weeks at 37°C indicating that the/3-glucosaminidase preparation was free of a-glucosaminidase activity.
Enzymatic treatment of rabbit y-globulin In experiments were neuraminidase was used it was always the first enzyme treatment because neuraminidase required a different buffer than the other enzymes and no other enzymes were needed to permit full activity of neuraminidase. The samples were incubated for 48 hr at 37°C in a buffer consisting of 0.05 M sodium acetate-acetic acid (pH 5-5) with 0.9 g NaCI and 0" 1 g CaC12 per liter. In most experiments 10/z of neuraminidase per mg of y-globulin was used. After neuraminidase treatment the y-globulin was dialysed against the buffer conditions required for the next enzyme. The buffer for all of the jack bean meal enzymes and the snail c~-fucosidase was 0" 1 M citrate pH 4.5. For the *DE-32 Whatman.
The Structure of Rabbit y-Globulin Carbohydrate
743
E. coli/3-galactosidase a 0.01 M tris (tris hydroxymethyl aminomethane) pH 7"8 buffer containing 0.01 M Mg C12, 0.01 M NaCI, and 0.01 M 2-mercaptoethanol was used. In all experiments where the samples were treated with more than one enzyme all of the enzymes except neuraminidase were used simultaneously in the reaction mixture. To prevent bacterial and fungal growth the samples were passed through a Millipore filter (0.45/z) and a drop of toluene was added to each. Incubation was at 37°C for a period of 2 weeks unless otherwise specified. In all experiments substrate and enzyme controls were simultaneously run to rule out the possibility of spontaneous release of sugars and autodigestion of enzymes.
Carbohydrate assays After glycosidase treatment the y-globulin was dialysed with many changes of distilled water to remove the free sugar. The dialysis baths were pooled, desalted with Amberlite MB-2, and brought to dryness. The y-blobulin was next dialysed against phosphate buffered (pH 7.0) saline. The free sugars from the dialysis bath were separated by descending paper chromatography with a pyridine, n-butanol, water (4 : 6 : 3) solvent system. A silver nitrate reducing sugar stain identified the sugars on pilot strips, while the remainder was eluted from the paper with water. The following methods were used for determinations of free and y-globulin bound sugars. After hydrolysis of the protein bound N-acetyl glucosamine with 4 N HC1 at 100°C for 6 hr, it was separated on Dowex 50 and measured by a modification of the Elson-Morgan procedure[7]. The thiobarbituric acid assay for neuraminic acid [8] was used after the bound neuraminic acid was released by acid hydrolysis (0.1 N H2SO4, 80°C, 1 hr). Free fucose was assayed by the DischeShettles procedure [9] while for protein bound fucose the Gyorky and Houck[10] modification was used. Free galactose and mannose separated by paper chromatography were measured by the reducing sugar method [11]. RESULTS Table 1 shows the results of various combinations of glycosidase treatment of rabbit yG-globulin. In all experiments involving two or more enzymes the enzymes were allowed to react simultaneously on the y-globulin with the exception of neuraminidase which was always used first. The range of values is given for those experiments which were done more than once. a-Fucosidase was excluded from the scheme because it could not be completely isolated from other glycosidases. In all experiments enzyme controls were run which invariably failed to show enzyme autodigestion or the release of sugars.
N-Acetyl neuraminic acid Neuraminidase removed a major portion of the N-acetyl neuraminic acid from rabbit y-globulin. Table 2 shows the amounts removed with varying concentrations of enzyme. Although 68 per cent was the greatest amount of neuraminic acid lost, more enzyme and longer incubation would probably have increased the loss. Since the neuraminidase preparation was free of other glyco-
B. R. ANDERSEN
744
Table 1 Per cent of sugar removed from rabbit yG-globulin N-Acetyl neuraminic acid (%)
Enzyme treatment* NA GL GA MA NA,GL NA,GA NA,MA GL,GA GL,MA GA,MA NA,GL,GA NA,GL,MA NA,GA,MA GL,GA,MA NA,GL,GA,MA
N-Acetyl glucosamine (%)
Galactose (%)
Mannose (%)
0 13-25 0 0 25 0 0 15-23 16 0 30 15 0 17 35-45
0 0 1-8 0 0 1 0 10-15 0 8 10-15 0 16 10-15 20-30
0 0 0 0 0 0 0 0 0 1 0 6 1 3 10-20
55-65 0 0 0 55-65 55-65 55-65 0 0 0 55-65 55-65 55-65 0 55-65
*NA = neuraminidase; GL =/3-D-glucosaminidase; GA =/3-D-galactosidase; MA = a-D-mannosidase. Table 2. Effect of neuraminidase (V. cholera) on whole rabbit yG-globulin Neuraminidase Units*/mg RGG
Hours of incubation at 37°C
Per cent reduction in neuraminic acid 0
0
24
0"1
24
3%
1
24
29%
10 20
24 48
58% 68%
*Units=/zg. of N-acetylneuraminic acid liberated in 15 min at 37° from arglycoprotein. sidases a n d no o t h e r sugars were released w h e n n e u r a m i n i d a s e alone was used, it can be c o n c l u d e d that N-acetyl n e u r a m i n i c acid is a terminal sugar. It m a y be the t e r m i n u s o f a single straight chain or o f a side o r b r a n c h e d chain.
N-A cethylD-glucosamine I n the e x p e r i m e n t shown in Fig. 2 the time r e q u i r e d for the/3-glucosaminidase reaction to r e a c h c o m p l e t i o n was a b o u t 7 days. I n this e x p e r i m e n t 3"2/z o f /~-glucosaminidase p e r m g o f y-globulin was used. T h e e n z y m e activity did not decrease significantly d u r i n g this 28 day e x p e r i m e n t a n d t h e r e f o r e could not be responsible for the cessation o f N-acetyl glucosamine cleavage. Because the o t h e r
The Strui:ture of Rabbit y-Globulin Carbohydrate Q el 0
745
30
4D
n*
/
Q) im
E 0 el
0
f.) :3 m
20
f.9 D
0
,<
I0
/
0 ,'-"-
0
0
k,~,l hr
$
I
I
I
l
2
7
14
28
Time (Deysl
Fig. 2. Rate of release of N-acetyl glucosamine from rabbit yG-globulin by //-glucosaminidase at 37°C. glycosidases probably have equally slow reaction rates and because terminal groups must be removed first before they can exert their effect, I choose to use a reaction time of 2 weeks for most of the experiments described below. Since fl-glucosaminidase removes N-acetyl glucosamine without the benefit of other glycosidases a portion of this sugar must be in a non-reducing terminal position. When 1.5-2.5 ~ fl-glucosaminidase per mg of y-globulin was used 13-25 per cent of the N-acetyl glucosamine was cleaved (Table 1). The variability in sugar loss was not correlated with differences in enzyme activity but was probably due at least in part to variability of the hexosamine assay. Some increase in N-acetyl glucosamine cleavage was associated with the combined use of fl-glucosaminidase, neuraminidase and fl-galactosidase (Table 1). This effect may be due to either steric interference by N-acetyl neuraminic acid and galactose or to the terminal linkage of these sugars to some N-acetyl glucosamines. The absence of a-glucosaminidase activity in the fl-glucosaminidase preparation supports the conclusion that the N-acetyl glucosamine of rabbit y-globulin is fl-linked. D-Galactose Only a small amount (1-8 per cent) of galactose was removed from yglobulin when fl-galactosidase from jack bean meal was used alone (Table 1). The amount of enzyme for this experiment was 1.0 t~/mg y-globulin. When/~galactosidase was paired with the other three enzymes only/3-glucosaminidase was associated with an increase in fl-galactosidase activity resulting in the release of 10-15 per cent of the y-globulin galactose. This suggests that some of the
746
B.R. ANDERSEN
galactose is penultimate to N-acetyl glucosamine. In e x p e r i m e n t s using m o r e c o m p l e x combinations o f enzymes there was a tendency for increased galactose loss to be associated with loss o f N-acetyl glucosamine. O n e exception to this was the greater release o f galactose with the combined use o f neuraminidase, /3-galactosidase, and ~-mannosidase. Although the ~-galactosidase f r o m jack bean meal was never completely isolated, experiments using mixtures o f ~- and /3-galactosidases showed no greater cleavage o f galactose than those which used/~-galactosidase alone. An isolated ~-galactosidase which was p r e p a r e d f r o m a culture of Aspergillus niger failed to release galactose f r o m rabbit y-globulin. Galactose, therefore, is /31inked at least in part with no evidence thus far o f any a-linkages. /3-Galactosidase f r o m E. coli used either alone or following neuraminidase and /3-glucosaminidase t r e a t m e n t was unable to r e m o v e any galactose f r o m rabbit y-globulin. T h e e n z y m e activity of/3-galactosidase (E. coli) for this experim e n t was over 10 times the level o f activity used for most o f the jack bean/3galactosidase studies. H u g h e s and Jeanloz[12] r e p o r t e d a similar e x p e r i e n c e where/3-galactosidase (E. coli) had no effect o n a serum glycoprotein (orosomucoid) while/3-galactosidase f r o m a n o t h e r source was effective.
D-Mannose T h e r e was no release o f mannose when ~-mannosidase (1-1 k~/mg y-globulin) was used alone (Table 1). Removing some o f the N-acetyl glucosamine with /3-glucosaminidase had no effect on o~-mannosidase activity, c~-Mannosidase in Table 3. Effect of crude c~-fucosidase preparation (snail hepatopancreas) on a glycopeptide preparation from rabbit yG-globulin Incubation conditions Time (days)
Temperature (°C)
14 56 116
37 37 45
Per cent reduction in fucose (%) 2
8 15
combination with/3-galactosidase released a small a m o u n t o f mannose (1 per cent). In o t h e r e n z y m e combinations only small amounts o f mannose (1-6 per cent) were r e m o v e d by 0~-mannosidase with the exception o f the e x p e r i m e n t which used all f o u r o f the enzymes with a resulting release o f 10-20 per cent o f the mannose. T h e s e data suggest that mannose was m o r e inaccessible or internally located than the o t h e r sugars in this series. T h e release o f mannose with an a - m a n n o s i d a s e free o f detectable 13-mannosidase d e m o n s t r a t e d that mannose was an ~-anomer.
L-Fucose In preliminary studies it was noted that the fucosidase (snail) p r e p a r a t i o n was very limited in its ability to r e m o v e fucose f r o m whole rabbit y-globulin
The Structure of Rabbit y-Globulin Carbohydrate
747
even when other glycosidases had removed substantial amounts of the carbohydrate. In order to minimize steric interference from the protein portion of the y-globulin molecule a pepsin-pronase digestion as described by Clamp and Putnam[13] was used to remove most of the protein. In spite of pretreatment with pepsin and pronase and the simultaneous cleavage of a portion of the galactose, mannose, N-acetyl neuraminic acid, and N-acetyl glucosamine only 15 per cent of the fucose was removed after incubating for 116 days at 45°C (Table 3). To be certain that the fucose released was due to the specific enzymatic action of a-fucosidase a number of controls were run. There was no evidence of spontaneous release of fucose under the same temperature and buffering conditions as used in the enzyme reaction. The a-fucosidase showed no evidence of containing /3-fucosidase activity. P-Nitrophenyl fl-fucopyranoside was not measurably effected by incubation with the enzyme for 2 weeks at 37°C. These observations indicate that fucose is a-linked. Concanavalin A The method of Agrawal and Goldstein [14] was used to prepare concanavalin A from jack bean meal. The isolated concanavalin A was mixed with native rabbit yG-globulin in a phosphate buffer (pH 7-0) saline solution. The solution was incubated at 37°C for 1 hr and then overnight at 4°C. A heavy precipitate developed. In order to be certain that the precipitate consisted of yG-globulin rather than the trace contaminants described earlier, the precipitate was washed 3 times and dissolved with 0.1M methyl a-D-mannopyranoside. Immunoelectrophoretic studies of the redissolved precipitate were performed using 0.1 M methyl a-D-mannopyranoside in the agar to prevent reprecipitation of concanavalin A. Antisera against rabbit yG-globulin and whole rabbit serum both reacted with the redissolved precipitate and demonstrated that concanavalin A was reacting primarily with yG-globulin. The studies of So and Goldstein [15] have shown that the specificity of concanavalin A includes the hydroxyl groups of carbons-3,4 and 6 of glucosamine and mannose. Since a-linkage is another requirement for reaction with concanavalin A the N-acetyl glucosamine of y-globulin is not likely to be involved. This was further supported by an experiment in which 25 per cent of the N-acetyl glucosamine was enzymatically removed without any loss in the amount of precipitate which formed with concanavalin A. Mannose is an a-anomer but since it is not a terminal sugar it must be linked through its carbon-2 position in order to keep the hydroxyl groups on carbons 3, 4 and 6 free to react with concanavalin A.
DISCUSSION The first phase of this experiment was the detection and isolation of glycosidases which would specifically cleave single non-reducing terminal sugars from rabbit yG-globulin. To accomplish this I used synthetic p-nitrophenyl glycoside substrates which corresponded to the types of sugars known to be present in y-globulin. Since the enzymes were isolated using these synthetic substrates as indicators of enzyme activity, the final enzyme preparations would undoubtedly have specificity requirements which would reflect the nature of those substrates. They would react only with a specific glycoside in a particular anomeric position.
748
B. R. ANDERSEN
Since the aglycon group of the synthetic substrate is non-carbohydrate in nature the isolated enzyme would probably have a binding site whose specificity depended only on the terminal sugar. The effect of the isolated enzymes on rabbit y-globulin supports these conclusions since in no instance was a sugar released by an enzyme of different specificity or was there any evidence of the release of oligosaccharides. These observations also exclude the possibility that epimerization or transglycosidation was occurring. The heterogeneity of antibody carbohydrate was a major factor in limiting the interpretation of my data. Press [2] was the first to demonstrate the presence of carbohydrate heterogeneity. Smyth and Utsumi[4] have shown that one of the two carbohydrate units (C1) present on the H polypeptide chain and described by Utsumi and Karush [3] was present on only 35 per cent of the molecules. The heterogeneity could be due solely to the variable presence of this one carbohydrate moiety. However, it could also include variability of the structure of the carbohydrate units. It is because of this heterogeneity that glycosidase degradation of rabbit yG-globulin can only provide the average results in the total population of molecules. The anomeric configurations of the glycosidic bonds in the antibody carbohydrate have been established by this study. Fucose and mannose are o-linked, and galactose and N-acetyl glucosamine are/3-linked. Although these structures can only be stated with certainty for that portion of the sugars actually cleaved by the specific enzyme it is unlikely that the anomeric linkages for a given sugar are mixed. Knowledge of the types and numbers of terminal sugars is an important step in evaluating the degree of branching, heterogeneity, and the sequence of sugars. Since significant amounts of N-acetyl neuraminic acid and N-acetyl glucosamine were removed from y-globulin by their specific glycosidases without the benefit of other glycosidases they must be major terminal groups. The small amount of galactose cleaved by y-galactosidase might either be due to carbohydrate heterogeneity in which only some antibody molecules have terminal galactose or to inefficiency of the/3-galactosidase. I favor the former explanation because the /3-galactosidase was able to cleave galactose very readily in the presence of other enzymes. Since no trace of mannose was cleaved until other sugars were removed it is unlikely that there is any terminal mannose. Fucose may be a terminal sugar although no data are available from this study to verify the point. Other laboratories[16] have shown that fucose is usually a terminal sugar in other serum glycoproteins but as yet there is no direct evidence about its position in antibody carbohydrate. Although it was not possible in this study to establish complete sugar sequence because of carbohydrate heterogeneity some generalizations can be made about the relationship of certain sugars to other sugars. Galactose cleavage was generally improved by the loss of N-acetyl glucosamine suggesting that some of' the galactose is penultimate to N-acetyl glucosamine. Mannose is probably more internally located than galactose since only the combination of neuraminidase, /3-galactosidase/3-glucosaminidase, and ct-mannosidase will permit the removal of significant amounts of mannose.
The Structure of Rabbit y-Globulin Carbohydrate
749
T h e ability of non-terminal mannose to react with concanavalin A suggests that some of the mannoses are linked t h r o u g h their carbon-2 position since carbons 3, 4 and 6 are required for precipitation with concanavalin A. T h e studies of Nolan and Smith [1] have shown that N-acetyl ~'lucosamine is involved in the carbohydrate-protein linkage. Since this is probably an amide linkage/3-glucosaminidase would be unable to remove those sugars. This probably accounts in part for the incomplete removal of N-acetyl glucosamine by /3-glucosaminidase. Smyth and Utsumi [4] have also shown that N-acetyl galactosamine is responsible for the carbohydrate protein linkage of one of the carbohydrate moieties. F u r t h e r progress in determining carbohydrate structure might be achieved by isolating the two carbohydrate moieties described by Utsumi ,and Karush [3]. Using these preparations one could eliminate at least some of the heterogeneity. In addition the enzymes used in sequence rather than in combination might further clarify the relationships between sugars. Finally, an effective fucosidase must be obtained to establish the position of fucose in the carbohydrate structure. Acknowledgements-The author would like to thank Miss Deborah Day, Miss Sharon
Lazzaro, and Miss Eileen Lasko for their excellent technical assistance, and Dr. Peter Z. Allen, Dr. Georg F. Springer and Dr. Wilton E. Vannier for many helpful discussions.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
REFERENCES Nolan C. and Smith E.,J. biol. Chem. 237,446 (1962). Press E., Symp. Molec. & Cell. Basis of Antibody Formn, p. 93, Prague (1965). Utsumi S. and Karush F., Biochemistry 4, 1766 (1965). Smyth D. and Utsurni S., Nature, Lond. 216, 332 (1967). Willers J. and Alderkamp G.,J. gen. Microbiol. 49, 41 (1967). Li Yu-Teh,J. biol. Chem. 242, 5474 (1967). Boas N.,J. biol. Chem. 204, 553 (1953). Aminoff D., B iochemJ. 81,384 (1961 ). Dische Z. and Shettles L.,J. biol. Chem. 175,595 (1948). Gyorky G. and HouckJ., Can.J. Biochem. 43, 1807 (1965). ParkJ. and Johnson M.,J. biol. Chem. 181,149 (1949). Hughes R. and Jeanloz R.,Biochemistry 3, 1535 (1964). ClampJ. and Putnam F.,J. biol. Chem. 239, 3233 (1964). Agrawal B. and Goldstein I., Biochem.J. 96, 23C (1965). So L. and Goldstein I.,J. biol. Chem. 243, 2003 (1968). Spiro R., New Engl.J. Med. 269, 566 (1963).
STUDIES ON T H E STRUCTURE OF T H E CARBOHYDRATE MOIETY OF RABBIT y G - G L O B U L I N - I D E G R A D A T I O N W I T H GLYCOSIDASES* BURTON R. ANDERSEN Departments of Microbiology and Medicine, Northwestern University School of Medicine, Chicago, Ill. 60611, U.S.A.
(First received 13January 1969; in revisedform 17 February 1969) Abstract- Information about the structure of rabbit yG-globulin carbohydrate has been
obtained with the use of isolated glycosidases and concanavalin A. N-acetyl neuraminic acid and N-acetyl glucosamine were shown to be major terminal sugars. Only a small fraction of the galactose was in a terminal position. Mannose could not be detected in a terminal position and appeared to be internal to both N-acetyl glucosamine and galactose. The following anomeric linkages have been established: N-acetyl glucosamine and galactose have /3-linkages while mannose and fucose are c~-linked. Concanavalin A precipitation studies show that some of the non-terminal mannoses are linked to other sugars through carbon-2. INTRODUCTION I m p o r t a n t data about the c a r b o h y d r a t e structure o f rabbit 3/G-globulin have been p r o v i d e d by studies using the technique o f chemical d e g r a d a t i o n and analysis [1-4]. T h e type and a m o u n t o f the sugars [1-3] and some indication o f their location o n the 3/-globulin molecule[3,4] have been d e t e r m i n e d . T h e s e experiments, however, have not established the sugar sequence or the n a t u r e o f the glycosidic linkages. I have succeeded in isolating a n u m b e r o f specific glycosidases f r o m jack bean meal, snail hepatopancreas, and o t h e r sources. T h e action o f these enzymes on the c a r b o h y d r a t e moiety o f rabbit 3/G-globulin provides i n f o r m a t i o n about the sequence o f sugars and the anomeric configurations o f the glycosidic bonds. MATERIALS AND METHODS
Chemicals and substrates T h e following glycosides were obtained f r o m commercial sources: p-nitrophenyl-~-D-galactopyranoside,t p-nitrophenyl-/3-D-galactopyranoside,t p-nitrophenyl N-acetyl /3-D-galactosaminide (p-nitrophenyl 2-acetamido-2deoxy-/3-D-galactopyranoside),$ p-nitrophenyl ~-L-fucopyranoside,t p-nitrophenyl-/3-L-fucopyranoside,$ p-nitrophenyl-N-acetyl /3-D-glucosaminide (p*A preliminary report of some of this work was presented at a meeting of the Federation of American Societies for Experimental Biology, Atlantic City, N.J., April 1968. This study was supported in part by the Illinois Chapter of the Arthritis Foundation and U.S.P.H.S. General Research Support Grant 5-SO1-FR-05370-07. ?Aldrich Chemical Co., Milwaukee, Wisc. :~Pierce Chemical Co., Rockford, Ill. 739
740
B.R. ANDERSEN
nitrophenyl 2-acetamido-2-deoxy-/3-D-glucopyranoside),* p-nitrophenyl-a-Dmannopyranoside,* and methyl-a-D-mannopyranoside.t Melting points were determined in all cases, and they agreed with published values. Methyl-/3-Dmannopyranoside and a formamide extract of streptococcal cell walls (Z3) containing a-linked N-acetyl glucosamine [5] were generously supplied by Drs. Elwyn T. Reese and Jan Willers, respectively. The rabbit yG-globulin used in these experiments was obtained "from a commerical supplier.:~ Acrylamid~disk electrophoresis and immunoelectrophoresis were used to examine the preparation for contaminating proteins. Three proteins in addition to yG-globulin were found; one was albumin in a concentration of 1 per cent or less as measured by quantitative immunodiffusion studies. The other two proteins were not identified but were present in much lower concentrations than the albumin (probably < 0.1 per cent). Since the major contaminant, albumin, has a very low carbohydrate content (< 0.3 per cent), it is unlikely that measurable amounts of carbohydrate were contributed by these proteins. The carbohydrate content of the rabbit y-globulin used in this study is in general agreement with other reports [1,3]. The carbohydrate concentrations were the following: hexose 1.01 per cent, n-acetyl glucosamine 1.44 per cent, n-acetyl neuraminic acid 0.16 per cent, and fucose 0.10 per cent.
Enzyme assay Glycosidases, with the exception of neuramimdase, were assayed with p-nitrophenyl glycoside substrates. The reaction mixture consisted of the following: 0.1 ml of the enzyme test solution, 0.1 ml of the p-nitrophenyl glycoside at a concentration of 0.01 M, and 1.25 ml of the optimal buffer for the enzyme being tested. In most cases the buffer was 0.1 M citrate pH 4.5. After incubating for 1 hr at 37°C, 1.5 ml of 0.2 M Na2COn was added to the mixture. Glycosidase activity was indicated by the release of p-nitrophenol from the substrate and was measured by light absorption at 400 m#. The values of the change in optical density were used as the units of enzyme activity. A unit of neuraminidase activit$ was that amount which would liberate 1 gg of N-acetyl-neuraminic acid from a,-glycoprotein within 15 min at 37°C.
Source and preparation of enzymes A neuraminidase (Vibrio cholerae) preparation which was commercially obtained§ was tested and found to be free of other glycosidase activities./3Galactosidase (E. coli) which also was free of other glycosidases was generously provided by Dr. Martin Rachmeler. ot-Fucosidase was prepared from the hepatopancreas of the sea snail, Gastropoda Busycotypus. H The hepatopancreas was homogenized in a Waring blendor, and an aqueous suspension of this material was centrifuged to remove cell debris. The precipitate which formed at 50 per cent ammonium sulfate *Mann Research Laboratories, Inc., New York, N.Y. tPfanstiehl Laboratories, Inc., Waukegan, Ill. ~Pentex, Inc., Kankakee, Ill. §Hoechst Pharmaceuticals, Inc., Cincinnati, Ohio. ~Marine Biological Laboratory, Woods Hole, Mass.
The Structure of Rabbit y-Globulin Carbohydrate SO e4 (NH4)2SO 4
741
Precipitate
& CM Cellulose
•OI--OSM (luote Q- and [3- oaloctosidose ~-91ucosominidase trace Q-monnosidase
.2M (luate 0 - monnosidose [3 - glucosominidose trace ~-galactosidase
OEA(* Cellulose
DEAF- Sephadex
; "01M Cl- and [3" golactosidase - glucosominidose
•2 M (luote ~- glucosaminidose
~L
"5M P),ridine Acetate Eluate
DEAE - Sephodex
O - monnosidese
•01M Eluate -galactosidase
Fig. 1. The scheme for the isolation of glycosidases from jack bean meal. On some occasions one or more of the steps had to be repeated when the proper separation had not been achieved. See text for details. saturation was extensively dialysed. This preparation contained the following glycosidases: a-fucosidase 16/z/ml, fl-glucosaminidase > 30/.~/ml, 0t-galactosidase 1/z/ml and/3-galactosidase 6/x/ml. T h e r e was no measurable B-fucosidase activity. Attempts to completely separate the ct-fucosidase from the other glycosidases were unsuccessful. T h e scheme for enzyme isolation from jack bean meal* is shown in Fig. 1. Some of the steps are modifications of the procedure of Yu-Teh Li [6]. G r o u n d meal was first suspended overnight in distilled water and then centrifuged to remove insoluble material. T h e aqueous extract was b r o u g h t to 50 per cent a m m o n i u m sulfate saturation at 4°C, and the precipitate which f o r m e d was dissolved in and extensively dialysed against 0.01 M citrate pH 4.5 buffer. This preparation was a d d e d to carboxymethyl (CM) celluloset which had been equilibrated with the same buffer. Step-wise elutions with citrate buffers of constant pH (4-5) and increasing molarity provided two major fractions. T h e first fraction was eluted with 0.01-0.05 M citrate buffers. T h e CM-cellulose was then washed *Sigma Chemical Co., St. Louis, Mo. tCM-32 Whatman.
742
B.R. ANDERSEN
with 0.1 M citrate and the wash solution discarded. The second fraction was eluted with 0.2 M citrate. The first CM-cellulose fraction (0.01-0.05 M eluate) which contained ~z- and /3-galactosidases,/3-glucosaminidase and a trace of o~-mannosidase was dialysed against 0.01 M phosphate pH 7.6 prior to adding diethyl amino ethyl (DEAE) cellulose* equilibrated with the same buffer. Two major fractions were obtained, the first contained those enzymes that did not bind to the DEAE-cellulose with the 0.01 M phosphate buffer, including a-galactosidase,/3-galactosidase, and a small amount of /3-glucosaminidase. After thoroughly washing the DEAEcellulose with the first buffer, the second fraction was eluted with 0"2 M phosphase pH 7.6. /3-Glucosaminidase was the major enzyme in this fraction, and when contaminating enzymes were present they could be removed by repeating the DEAE-cellulose step. The first fraction from the DEAE-cellulose was dialysed against a 0.01 M phosphate pH 7.0 buffer and mixed with DEAE-Sephadex A50 which had been equilibrated with the same buffer. Under these conditions/3-galactosidase failed to bind to the DEAE-Sephadex while the other enzymes were adsorbed. The second fraction from the CM-cellulose step which contained a-mannosidase,/3-glucosaminidase and some/3-galactosidase was dialysed against 0.01 M phosphate pH 7.0 and mixed with DEAE-Sephadex A50. Repeated washing with the same buffer removed all the contaminating/3-galactosidase, a-Mannosidase was eluted in pure form with a 0"5 M pyridine acetate buffer pH 6.0. Since p-nitrophenyl/3-mannopyranoside and p-nitrophenyl ~-glucosaminide were not available to check for/3-mannosidase and ~-glucosaminidase activities, there was some question whether the c~-mannosidase and /3-glucosaminidase enzymes had been completely isolated. The ~-niannosidase preparation was incubated with methyl o~-mannopyranoside and methyl /3-mannopyranoside. Mannose was cleaved only from the c~-anomer indicating that there was no contaminating/3-mannosidase in the preparation. The /3-glucosaminidase preparation was incubated with a formamide streptococcal cell wall extract (Z3) which contained terminal ~x-N-acetyl glucosamine[5]. No glucosamine was released after incubation for 2 weeks at 37°C indicating that the/3-glucosaminidase preparation was free of a-glucosaminidase activity.
Enzymatic treatment of rabbit y-globulin In experiments were neuraminidase was used it was always the first enzyme treatment because neuraminidase required a different buffer than the other enzymes and no other enzymes were needed to permit full activity of neuraminidase. The samples were incubated for 48 hr at 37°C in a buffer consisting of 0.05 M sodium acetate-acetic acid (pH 5-5) with 0.9 g NaCI and 0" 1 g CaC12 per liter. In most experiments 10/z of neuraminidase per mg of y-globulin was used. After neuraminidase treatment the y-globulin was dialysed against the buffer conditions required for the next enzyme. The buffer for all of the jack bean meal enzymes and the snail c~-fucosidase was 0" 1 M citrate pH 4.5. For the *DE-32 Whatman.
The Structure of Rabbit y-Globulin Carbohydrate
743
E. coli/3-galactosidase a 0.01 M tris (tris hydroxymethyl aminomethane) pH 7"8 buffer containing 0.01 M Mg C12, 0.01 M NaCI, and 0.01 M 2-mercaptoethanol was used. In all experiments where the samples were treated with more than one enzyme all of the enzymes except neuraminidase were used simultaneously in the reaction mixture. To prevent bacterial and fungal growth the samples were passed through a Millipore filter (0.45/z) and a drop of toluene was added to each. Incubation was at 37°C for a period of 2 weeks unless otherwise specified. In all experiments substrate and enzyme controls were simultaneously run to rule out the possibility of spontaneous release of sugars and autodigestion of enzymes.
Carbohydrate assays After glycosidase treatment the y-globulin was dialysed with many changes of distilled water to remove the free sugar. The dialysis baths were pooled, desalted with Amberlite MB-2, and brought to dryness. The y-blobulin was next dialysed against phosphate buffered (pH 7.0) saline. The free sugars from the dialysis bath were separated by descending paper chromatography with a pyridine, n-butanol, water (4 : 6 : 3) solvent system. A silver nitrate reducing sugar stain identified the sugars on pilot strips, while the remainder was eluted from the paper with water. The following methods were used for determinations of free and y-globulin bound sugars. After hydrolysis of the protein bound N-acetyl glucosamine with 4 N HC1 at 100°C for 6 hr, it was separated on Dowex 50 and measured by a modification of the Elson-Morgan procedure[7]. The thiobarbituric acid assay for neuraminic acid [8] was used after the bound neuraminic acid was released by acid hydrolysis (0.1 N H2SO4, 80°C, 1 hr). Free fucose was assayed by the DischeShettles procedure [9] while for protein bound fucose the Gyorky and Houck[10] modification was used. Free galactose and mannose separated by paper chromatography were measured by the reducing sugar method [11]. RESULTS Table 1 shows the results of various combinations of glycosidase treatment of rabbit yG-globulin. In all experiments involving two or more enzymes the enzymes were allowed to react simultaneously on the y-globulin with the exception of neuraminidase which was always used first. The range of values is given for those experiments which were done more than once. a-Fucosidase was excluded from the scheme because it could not be completely isolated from other glycosidases. In all experiments enzyme controls were run which invariably failed to show enzyme autodigestion or the release of sugars.
N-Acetyl neuraminic acid Neuraminidase removed a major portion of the N-acetyl neuraminic acid from rabbit y-globulin. Table 2 shows the amounts removed with varying concentrations of enzyme. Although 68 per cent was the greatest amount of neuraminic acid lost, more enzyme and longer incubation would probably have increased the loss. Since the neuraminidase preparation was free of other glyco-
B. R. ANDERSEN
744
Table 1 Per cent of sugar removed from rabbit yG-globulin N-Acetyl neuraminic acid (%)
Enzyme treatment* NA GL GA MA NA,GL NA,GA NA,MA GL,GA GL,MA GA,MA NA,GL,GA NA,GL,MA NA,GA,MA GL,GA,MA NA,GL,GA,MA
N-Acetyl glucosamine (%)
Galactose (%)
Mannose (%)
0 13-25 0 0 25 0 0 15-23 16 0 30 15 0 17 35-45
0 0 1-8 0 0 1 0 10-15 0 8 10-15 0 16 10-15 20-30
0 0 0 0 0 0 0 0 0 1 0 6 1 3 10-20
55-65 0 0 0 55-65 55-65 55-65 0 0 0 55-65 55-65 55-65 0 55-65
*NA = neuraminidase; GL =/3-D-glucosaminidase; GA =/3-D-galactosidase; MA = a-D-mannosidase. Table 2. Effect of neuraminidase (V. cholera) on whole rabbit yG-globulin Neuraminidase Units*/mg RGG
Hours of incubation at 37°C
Per cent reduction in neuraminic acid 0
0
24
0"1
24
3%
1
24
29%
10 20
24 48
58% 68%
*Units=/zg. of N-acetylneuraminic acid liberated in 15 min at 37° from arglycoprotein. sidases a n d no o t h e r sugars were released w h e n n e u r a m i n i d a s e alone was used, it can be c o n c l u d e d that N-acetyl n e u r a m i n i c acid is a terminal sugar. It m a y be the t e r m i n u s o f a single straight chain or o f a side o r b r a n c h e d chain.
N-A cethylD-glucosamine I n the e x p e r i m e n t shown in Fig. 2 the time r e q u i r e d for the/3-glucosaminidase reaction to r e a c h c o m p l e t i o n was a b o u t 7 days. I n this e x p e r i m e n t 3"2/z o f /~-glucosaminidase p e r m g o f y-globulin was used. T h e e n z y m e activity did not decrease significantly d u r i n g this 28 day e x p e r i m e n t a n d t h e r e f o r e could not be responsible for the cessation o f N-acetyl glucosamine cleavage. Because the o t h e r
The Strui:ture of Rabbit y-Globulin Carbohydrate Q el 0
745
30
4D
n*
/
Q) im
E 0 el
0
f.) :3 m
20
f.9 D
0
,<
I0
/
0 ,'-"-
0
0
k,~,l hr
$
I
I
I
l
2
7
14
28
Time (Deysl
Fig. 2. Rate of release of N-acetyl glucosamine from rabbit yG-globulin by //-glucosaminidase at 37°C. glycosidases probably have equally slow reaction rates and because terminal groups must be removed first before they can exert their effect, I choose to use a reaction time of 2 weeks for most of the experiments described below. Since fl-glucosaminidase removes N-acetyl glucosamine without the benefit of other glycosidases a portion of this sugar must be in a non-reducing terminal position. When 1.5-2.5 ~ fl-glucosaminidase per mg of y-globulin was used 13-25 per cent of the N-acetyl glucosamine was cleaved (Table 1). The variability in sugar loss was not correlated with differences in enzyme activity but was probably due at least in part to variability of the hexosamine assay. Some increase in N-acetyl glucosamine cleavage was associated with the combined use of fl-glucosaminidase, neuraminidase and fl-galactosidase (Table 1). This effect may be due to either steric interference by N-acetyl neuraminic acid and galactose or to the terminal linkage of these sugars to some N-acetyl glucosamines. The absence of a-glucosaminidase activity in the fl-glucosaminidase preparation supports the conclusion that the N-acetyl glucosamine of rabbit y-globulin is fl-linked. D-Galactose Only a small amount (1-8 per cent) of galactose was removed from yglobulin when fl-galactosidase from jack bean meal was used alone (Table 1). The amount of enzyme for this experiment was 1.0 t~/mg y-globulin. When/~galactosidase was paired with the other three enzymes only/3-glucosaminidase was associated with an increase in fl-galactosidase activity resulting in the release of 10-15 per cent of the y-globulin galactose. This suggests that some of the
746
B.R. ANDERSEN
galactose is penultimate to N-acetyl glucosamine. In e x p e r i m e n t s using m o r e c o m p l e x combinations o f enzymes there was a tendency for increased galactose loss to be associated with loss o f N-acetyl glucosamine. O n e exception to this was the greater release o f galactose with the combined use o f neuraminidase, /3-galactosidase, and ~-mannosidase. Although the ~-galactosidase f r o m jack bean meal was never completely isolated, experiments using mixtures o f ~- and /3-galactosidases showed no greater cleavage o f galactose than those which used/~-galactosidase alone. An isolated ~-galactosidase which was p r e p a r e d f r o m a culture of Aspergillus niger failed to release galactose f r o m rabbit y-globulin. Galactose, therefore, is /31inked at least in part with no evidence thus far o f any a-linkages. /3-Galactosidase f r o m E. coli used either alone or following neuraminidase and /3-glucosaminidase t r e a t m e n t was unable to r e m o v e any galactose f r o m rabbit y-globulin. T h e e n z y m e activity of/3-galactosidase (E. coli) for this experim e n t was over 10 times the level o f activity used for most o f the jack bean/3galactosidase studies. H u g h e s and Jeanloz[12] r e p o r t e d a similar e x p e r i e n c e where/3-galactosidase (E. coli) had no effect o n a serum glycoprotein (orosomucoid) while/3-galactosidase f r o m a n o t h e r source was effective.
D-Mannose T h e r e was no release o f mannose when ~-mannosidase (1-1 k~/mg y-globulin) was used alone (Table 1). Removing some o f the N-acetyl glucosamine with /3-glucosaminidase had no effect on o~-mannosidase activity, c~-Mannosidase in Table 3. Effect of crude c~-fucosidase preparation (snail hepatopancreas) on a glycopeptide preparation from rabbit yG-globulin Incubation conditions Time (days)
Temperature (°C)
14 56 116
37 37 45
Per cent reduction in fucose (%) 2
8 15
combination with/3-galactosidase released a small a m o u n t o f mannose (1 per cent). In o t h e r e n z y m e combinations only small amounts o f mannose (1-6 per cent) were r e m o v e d by 0~-mannosidase with the exception o f the e x p e r i m e n t which used all f o u r o f the enzymes with a resulting release o f 10-20 per cent o f the mannose. T h e s e data suggest that mannose was m o r e inaccessible or internally located than the o t h e r sugars in this series. T h e release o f mannose with an a - m a n n o s i d a s e free o f detectable 13-mannosidase d e m o n s t r a t e d that mannose was an ~-anomer.
L-Fucose In preliminary studies it was noted that the fucosidase (snail) p r e p a r a t i o n was very limited in its ability to r e m o v e fucose f r o m whole rabbit y-globulin
The Structure of Rabbit y-Globulin Carbohydrate
747
even when other glycosidases had removed substantial amounts of the carbohydrate. In order to minimize steric interference from the protein portion of the y-globulin molecule a pepsin-pronase digestion as described by Clamp and Putnam[13] was used to remove most of the protein. In spite of pretreatment with pepsin and pronase and the simultaneous cleavage of a portion of the galactose, mannose, N-acetyl neuraminic acid, and N-acetyl glucosamine only 15 per cent of the fucose was removed after incubating for 116 days at 45°C (Table 3). To be certain that the fucose released was due to the specific enzymatic action of a-fucosidase a number of controls were run. There was no evidence of spontaneous release of fucose under the same temperature and buffering conditions as used in the enzyme reaction. The a-fucosidase showed no evidence of containing /3-fucosidase activity. P-Nitrophenyl fl-fucopyranoside was not measurably effected by incubation with the enzyme for 2 weeks at 37°C. These observations indicate that fucose is a-linked. Concanavalin A The method of Agrawal and Goldstein [14] was used to prepare concanavalin A from jack bean meal. The isolated concanavalin A was mixed with native rabbit yG-globulin in a phosphate buffer (pH 7-0) saline solution. The solution was incubated at 37°C for 1 hr and then overnight at 4°C. A heavy precipitate developed. In order to be certain that the precipitate consisted of yG-globulin rather than the trace contaminants described earlier, the precipitate was washed 3 times and dissolved with 0.1M methyl a-D-mannopyranoside. Immunoelectrophoretic studies of the redissolved precipitate were performed using 0.1 M methyl a-D-mannopyranoside in the agar to prevent reprecipitation of concanavalin A. Antisera against rabbit yG-globulin and whole rabbit serum both reacted with the redissolved precipitate and demonstrated that concanavalin A was reacting primarily with yG-globulin. The studies of So and Goldstein [15] have shown that the specificity of concanavalin A includes the hydroxyl groups of carbons-3,4 and 6 of glucosamine and mannose. Since a-linkage is another requirement for reaction with concanavalin A the N-acetyl glucosamine of y-globulin is not likely to be involved. This was further supported by an experiment in which 25 per cent of the N-acetyl glucosamine was enzymatically removed without any loss in the amount of precipitate which formed with concanavalin A. Mannose is an a-anomer but since it is not a terminal sugar it must be linked through its carbon-2 position in order to keep the hydroxyl groups on carbons 3, 4 and 6 free to react with concanavalin A.
DISCUSSION The first phase of this experiment was the detection and isolation of glycosidases which would specifically cleave single non-reducing terminal sugars from rabbit yG-globulin. To accomplish this I used synthetic p-nitrophenyl glycoside substrates which corresponded to the types of sugars known to be present in y-globulin. Since the enzymes were isolated using these synthetic substrates as indicators of enzyme activity, the final enzyme preparations would undoubtedly have specificity requirements which would reflect the nature of those substrates. They would react only with a specific glycoside in a particular anomeric position.
748
B. R. ANDERSEN
Since the aglycon group of the synthetic substrate is non-carbohydrate in nature the isolated enzyme would probably have a binding site whose specificity depended only on the terminal sugar. The effect of the isolated enzymes on rabbit y-globulin supports these conclusions since in no instance was a sugar released by an enzyme of different specificity or was there any evidence of the release of oligosaccharides. These observations also exclude the possibility that epimerization or transglycosidation was occurring. The heterogeneity of antibody carbohydrate was a major factor in limiting the interpretation of my data. Press [2] was the first to demonstrate the presence of carbohydrate heterogeneity. Smyth and Utsumi[4] have shown that one of the two carbohydrate units (C1) present on the H polypeptide chain and described by Utsumi and Karush [3] was present on only 35 per cent of the molecules. The heterogeneity could be due solely to the variable presence of this one carbohydrate moiety. However, it could also include variability of the structure of the carbohydrate units. It is because of this heterogeneity that glycosidase degradation of rabbit yG-globulin can only provide the average results in the total population of molecules. The anomeric configurations of the glycosidic bonds in the antibody carbohydrate have been established by this study. Fucose and mannose are o-linked, and galactose and N-acetyl glucosamine are/3-linked. Although these structures can only be stated with certainty for that portion of the sugars actually cleaved by the specific enzyme it is unlikely that the anomeric linkages for a given sugar are mixed. Knowledge of the types and numbers of terminal sugars is an important step in evaluating the degree of branching, heterogeneity, and the sequence of sugars. Since significant amounts of N-acetyl neuraminic acid and N-acetyl glucosamine were removed from y-globulin by their specific glycosidases without the benefit of other glycosidases they must be major terminal groups. The small amount of galactose cleaved by y-galactosidase might either be due to carbohydrate heterogeneity in which only some antibody molecules have terminal galactose or to inefficiency of the/3-galactosidase. I favor the former explanation because the /3-galactosidase was able to cleave galactose very readily in the presence of other enzymes. Since no trace of mannose was cleaved until other sugars were removed it is unlikely that there is any terminal mannose. Fucose may be a terminal sugar although no data are available from this study to verify the point. Other laboratories[16] have shown that fucose is usually a terminal sugar in other serum glycoproteins but as yet there is no direct evidence about its position in antibody carbohydrate. Although it was not possible in this study to establish complete sugar sequence because of carbohydrate heterogeneity some generalizations can be made about the relationship of certain sugars to other sugars. Galactose cleavage was generally improved by the loss of N-acetyl glucosamine suggesting that some of' the galactose is penultimate to N-acetyl glucosamine. Mannose is probably more internally located than galactose since only the combination of neuraminidase, /3-galactosidase/3-glucosaminidase, and ct-mannosidase will permit the removal of significant amounts of mannose.
The Structure of Rabbit y-Globulin Carbohydrate
749
T h e ability of non-terminal mannose to react with concanavalin A suggests that some of the mannoses are linked t h r o u g h their carbon-2 position since carbons 3, 4 and 6 are required for precipitation with concanavalin A. T h e studies of Nolan and Smith [1] have shown that N-acetyl ~'lucosamine is involved in the carbohydrate-protein linkage. Since this is probably an amide linkage/3-glucosaminidase would be unable to remove those sugars. This probably accounts in part for the incomplete removal of N-acetyl glucosamine by /3-glucosaminidase. Smyth and Utsumi [4] have also shown that N-acetyl galactosamine is responsible for the carbohydrate protein linkage of one of the carbohydrate moieties. F u r t h e r progress in determining carbohydrate structure might be achieved by isolating the two carbohydrate moieties described by Utsumi ,and Karush [3]. Using these preparations one could eliminate at least some of the heterogeneity. In addition the enzymes used in sequence rather than in combination might further clarify the relationships between sugars. Finally, an effective fucosidase must be obtained to establish the position of fucose in the carbohydrate structure. Acknowledgements-The author would like to thank Miss Deborah Day, Miss Sharon
Lazzaro, and Miss Eileen Lasko for their excellent technical assistance, and Dr. Peter Z. Allen, Dr. Georg F. Springer and Dr. Wilton E. Vannier for many helpful discussions.
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
REFERENCES Nolan C. and Smith E.,J. biol. Chem. 237,446 (1962). Press E., Symp. Molec. & Cell. Basis of Antibody Formn, p. 93, Prague (1965). Utsumi S. and Karush F., Biochemistry 4, 1766 (1965). Smyth D. and Utsurni S., Nature, Lond. 216, 332 (1967). Willers J. and Alderkamp G.,J. gen. Microbiol. 49, 41 (1967). Li Yu-Teh,J. biol. Chem. 242, 5474 (1967). Boas N.,J. biol. Chem. 204, 553 (1953). Aminoff D., B iochemJ. 81,384 (1961 ). Dische Z. and Shettles L.,J. biol. Chem. 175,595 (1948). Gyorky G. and HouckJ., Can.J. Biochem. 43, 1807 (1965). ParkJ. and Johnson M.,J. biol. Chem. 181,149 (1949). Hughes R. and Jeanloz R.,Biochemistry 3, 1535 (1964). ClampJ. and Putnam F.,J. biol. Chem. 239, 3233 (1964). Agrawal B. and Goldstein I., Biochem.J. 96, 23C (1965). So L. and Goldstein I.,J. biol. Chem. 243, 2003 (1968). Spiro R., New Engl.J. Med. 269, 566 (1963).