Structure of the oligosaccharides of mouse immunoglobulin M secreted by the MOPC 104E plasmacytoma

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 201, No. 1, April 15, pp. 160-173, 1980

Structure

of the Oligosaccharides of Mouse lmmunoglobulin Secreted by the MOPC 104E Plasmacytomal RUTH BRENCKLE

Departments

of Internal

Medicine

AND

ROSALIND KORNFELD

and Biological Chemistry, Washington St. Louis, Missouri 63110

Received September

M

7, 1979; revised December

University

School of Medicine,

19, 1979

The structures of the oligosaccharides present in the mouse immunoglobulin M secreted by the plasma cell tumor MOPC 104E have been determined making use of glycopeptides derived from purified unlabeled immunoglobulin and from rH]mannose and [W]glucosaminelabeled immunoglobulin M. The glycopeptides were fractionated on columns of Bio-Gel P-6 and concanavalin A-Sepharose and high mannose type oligosaccharides were released from glycopeptide with clostridial endo-P-N-acetylglucosaminidase C,,. MOPC 104E immunoglobulin M was shown to contain four complex-type and one high mannose type oligosaccharide units per heavy chain. The glycopeptides with complex oligosaccharides were separated on concanavalin A-Sepharose into two classes with structures containing either two or three outer branches with the sequence -CNGNAol2 + 6Gal/31 + 4GlcNA@l + attached to the 2 position or to the 2 and 4 positions of mannose in a core with the structure Manal + G(Mana1 + 3)Manpl -f 4GlcNAcpl --f 4(Fucc~l + 6)GlcNAc + Asn. The high mannose oligosaccharides liberated by endo+N-acetylglucosaminidase C,, varied in size from Man,GlcNAc to Man,GlcNAc. A Man,GlcNAc with the structure ManLvl + G(Mancw1 + 3)Mancul -+ G(Mancu1 + 2Mancul --) S)Mar@l + 4GlcNAc was the predominant species. Some high mannose oligosaccharide was resistant to endo-P-N-acetylglucosaminidase C,, release and apparently has a “hybrid” or atypical structure.

Our current knowledge of the structure of immunoglobulins has been derived largely from the study of the homogeneous, monoclonal immunoglobulins secreted by plasma cell tumors arising spontaneously in man and chemically induced in mice. The structure of the oligosaccharides that are attached to the heavy chain of immunoglobulins has been determined in our laboratory for a number of human myeloma proteins of different types including IgG2 (l), IgM (2-4);

IgA (5, 6), and IgE (7, 8). In contrast the structures of the oligosaccharides on the heavy chains of mouse myeloma proteins have not yet been determined, although the mouse plasmacytomas have provided ideal systems for studying the synthesis, assembly, and secretion of immunoglobulins with radioactively labeled amino acids and sugars (9- 17). In particular, various mouse plasmacytomas including MOPC 104E which secretes an IgM, have been valuable in tracing the sequenceof events in glycosylation of immunoglobulins (10, 12, 14-18). Based on their studies with tunicamycin, which inhibited the glycosylation and subsequent secretion of MOPC 104E IgM, Hickman and Kornfeld (18) have postulated that blocking the glycosylation of immunoglobulins alters their physicochemical properties and inhibits their movement through the intracellular membraneous channels and this effect is most pronounced for more heavy glycosylated molecules. The carbohydrate composition of the MOPC 104E

’ This investigation was supported in part by Grants CA 08759, RR 00954, and IT 32 GM 07067 from the United States Public Health Service. * Abbreviations used: Ig, immunoglobulin; BSA, bovine serum albumin; solvent I, ethyl acetate/ pyridine/acetic acid/water (5151113); solvent II, 1-butanol/pyridine/water (41314); solvent III, pyridine/ ethyl acetate/water (113.611.15); glc, gas-liquid chromatography; SDS, sodium dodecyl sulfate; AC, acetyl; Con A, concanavalin A; VSV, vesicular stomatitis virus; Me, methyl; PBS, phosphate-buffered saline; HL, the subunit of IgM containing one heavy or H chain and one light or L chain. 0003-9861/80/050160-14$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved.

160

STRUCTURE

OF MOPC 104E IgM OLIGOSACCHARIDES

IgM suggests that it, like human IgM, contains multiple oligosaccharide chains, but the number and structure of these oligosaccharides have not been previously determined. In order to assesswhat features of the carbohydrate side chains are involved in allowing normal transport and secretion of MOPC 104E IgM it is necessary to know the structure of these oligosaccharides and therefore the present study was undertaken. In deducing the structure of the MOPC 104E IgM oligosaceharides, we have taken advantage of the opportunity provided by this system to radioactively label specific sugar residues in the oligosaccharides which facilitates the isolation and structural analysis of the glycopeptides. These studies, carried out in parallel with glycopeptides derived from purified unlabeled MOPC 104E IgM and from [3H]mannose- and [‘“Clglucosamine-labeled IgM, show that this mouse IgM contains four complex oligosaccharides and one high mannose type oligosaccharide per heavy chain. The complex oligosaccharides have structures of two types, those with two outer branches which are similar to the complex oligosaccharides of human immunoglobulins, and others with three outer branches which have not been previously reported in immunoglobulins. The high mannose oligosaccharides likewise have structures also seen in human IgM but in addition have some “hybrid” or atypical structures. EXPERIMENTAL

PROCEDURE

Materi&. D-[1-“C]glucosamine (45 mCi/mmol) was purchased from New England Nuclear Corporation and D-1Z”Hlmannose (2 Ciimmol) from Amersham. Medium 199 with Earle’s salts, penicillin, streptomycin, and fetal calf serum were purchased from Grand Island Biological Company. Ficoll, bovine serum albumin, S-glycolyl neuraminic acid, and ol-methylmannoside were from Sigma Chemical Company and Hypaque was from Winthrop Laboratories. Goat anti-mouse IgM and rabbit anti-goat-y-globulin were purchased from Gateway Immunosera Company, Cahokia, Illinois. The affinity adsorbent for MOPC 104E IgM consisting of the dextran from L. rneseatemides NRRL-B-1355 coupled to BSA-Sepharose ZB, prepared according to Hiramoto et al. (19), was a kind gift from Daniel Hansberg of the Microbiology Department of Washington University School of Medicine. Con A-Sepharose was from Pharmacia and Bio-Gel

161

P-6 and P-4 (200-400 mesh) were from Bio-Rad Laboratories. The various oligosaccharide alcohols and methylated sugars used as standards for chromatography were those described earlier (20). Enzymes. Pronase and V. cholera neuraminidase were obtained from Calbiochem. Jack bean p-galac/3-N-acetylglucosaminidase, and ar-mantosidase, nosidase were prepared as described by Li and Li (21). Hen oviduct /3-mannosidase was purified by the method of Sukeno et a2. (22), and Clostridium perfrngens endo-pN-acetylglucosaminidase C,, was purified as described by Ito et al. (23). Diplococcal p-galactosidase, and endo-@N-acetylP-N-acetylglucosaminidase, glucosaminidase D were kindly supplied by Dr. Jacques Baenziger of Washington University School of Medicine. Bovine epididymal a-L-fucosidase was from Sigma. Chromatographic techniques. Descending paper chromatography was performed on Whatman No. 1 paper in the following solvent systems: solvent I, ethyl acetateipyridineiacetic acid/water (5/5/l/3); solvent II, 1-butanolipyridineiwater (41314);and solvent III, pyridineiethyl acetate/water (l/3.6/1.15 upper phase). Sugars were detected with the alkaline silver nitrate technique (24). Labeled compounds were localized either by radioscanning of the chromatogram with a TMC Vanguard Instrument Corporation Model 885 dual channel autoscanner, or by cutting l-cm strips, soaking them in 0.3 ml of water in scintillation vials, and counting the vials after addition of 3 ml of 3a70 scintillation counting fluid. Partially methylated [YH]mannose-labeled sugars were separated by thin layer chromatography and counted as described by Li et al. (20). N-Acetyl and N-glycolyl neuraminic acid were separated by thin layer chromatography on cellulose-coated plates (Avicel, 250~) prerun in 0.1 N HCI and dried before sample application and development in n-butanoll n-propanol/O. 1 N HCl (1/2/l) as described by Schauer (25). LabeIed compounds were located by scraping 0.5-c, segments of the cellulose from the plate into scintillation vials containing 0.4 ml water, adding 4 ml of 3a70, and counting in a scintillation counter. The standards were located by spraying the plate with the orcinol/FeCl, reagent as described by Schauer (25). Carbohydrate analysis. The monosaccharides present in unlabeled glycopeptides were quantitated as the trimethylsilyl derivatives of their methylglycosides by gas-liquid chromatography as previously described (7) and sialic acids were measured by the thiobarbituric acid method of Warren (26). Methods for structure analysis. The oligosaccharides and glycopeptides were methylated by the method of Hakomori (27) and the unlabeled permethylated products were subjected to acetolysis, reduction, and acetylation according to Stellner et al. (28) before glc-mass spectrometrie analysis as previously described (3). The labeled permethylated products were

162

BRENCKLEANDKORNFELD

hydrolyzed in 2 N H,SO, and analyzed by thin layer chromatography as reported by Li et al. (20). Labeled oligosaccharides were reduced with NaBH, and subjected to acetolysis by the methods previously described (3) or subjected to Smith periodate degradation as follows. Oxidation of the oligosaccharide in 50 ~1 of 0.05 M sodium acetate pH 4.6 eontaining0.08 M sodium metaperiodate for 24 h at 4°C was followed by addition of NaOH to bring the pH to 10 and reduction by the subsequent addition of 50 ~1 of sodium borohydride (26 mgiml). After 18 h at room temperature the reduction was terminated by addition of 10 ~1 of glacial acetic acid. Methanol containing 1% acetic acid was added and evaporated under vacuum five times to remove borate as methyl borate. The oxidized and reduced oligosaccharide was desalted by passage over a column of Amberlite MB-3 mixed bed resin and then subjected to mild acid hydrolysis in 0.1 N H,SO, at 80°C for 90 min. The hydrolysate was passed over a small column of Bio-Rad AG 3 x 4 (acetate form) to remove sulfate, and the eluate was evaporated to dryness and subjected to chromatography in solvent I. Glycosidase digestio7l.s. Glycopeptides and oligosaccharides were incubated with various glycosidases under the following conditions: V. cholera neuraminidase (10 units) in 30 ~1 of 0.05 M sodium acetate buffer/ 0.9% M NaCVO. 1% CaCl, pH 5.6 at 37°C for 24 h; jack bean @galactosidase and P-N-acetylglueosaminidase (0.05 unit) in 40 ~1 of 0.05 M sodium citrate buffer pH 4.6 for 24 to 48 h at 37°C; jack bean a-mannosidase (1 unit) in 25 /*I of 0.05 M sodium citrate buffer pH 4.6 for 18 h at 37°C; hen oviduct p-mannosidase (0.08 unit) in 50 ~1 of 0.05 M citrate-phosphate buffer pH 5.0 for 24 h at 37°C. Digestions with the glycosidases from Diplococcus pneum,onia were as described by Baenziger and Fiete (29) and with the endo+Nacetylglucosaminidase C,, and H as described by Li and Kornfeld (30). Bovine epididymal cY+fucosidase digestions were performed in 0.05 M sodium citrate buffer pH 4.6 for 60 h. MOPC lOhE plasmacytoma cells. The MOPC 104E plasmacytoma cells were a gift from Dr. R. G. Lynth and were maintained1 in the ascites of BALBic mice (18). Plasma cells were aspirated from ascites, diluted in ice-cold medium 199, separated from erythrocytes by a Ficoll-Hypaque gradient, and washed as previously described (31). Isolation of radioactively labeled MOPC 104E IgM. Washed MOPC 104E plasma cells (7 x 10’) were incubated in 21 ml of medium 199 containing 10% fetal calf serum, 100 units/ml of penicillin, 100 fig/ml of streptomycin, and 100 &i 2-rH]mannose in a 100mm Falcon plastic petri dish at 37°C in an atmosphere of 5% CO,-95% air. After 15 h the cells were removed by sedimentation and the medium placed in two l&ml heavy-walled glass centrifuge tubes previously soaked in a 0.5% solution of bovine serum albumin. The

secreted [3H]mannose-labeled IgM was immunoprecipitated by addition of 0.08 ml of goat anti-mouse IgM to each tube. After incubation for 3 h at 4”C, 1.75 ml of rabbit anti-goat y-globulin was added to each tube and the incubation was continued at 4°C overnight. The immunoprecipitates were collected by centrifugation, washed twice with 0.01 M Tris HCl0.15 M NaCl pH 7.4 (Tris-saline), and combined. [‘“CJGlucosamine-labeled IgM was isolated in the same manner from a smaller incubation mixture containing in 11 ml 1.7 x lo7 cells and 6 yCi [‘YJglucosamine of medium. Isolation of unlabeled MOPC 104E IgM. Ascites fluid was aspirated and the cells were removed by centrifugation. The MOPC 104E IgM which has antibody specificity for dextrans with oll,3-linked glucose residues was selectively adsorbed from the ascitic fluid by passage over an affinity column of L. nt?esenteroides dextran covalently attached to BSA-Sepharose as described by Hiramoto et al. (19). The column was washed with Tris-saline to remove unbound material and the IgM was elutetl with 0.1 M glycineHCl pH 2.3. From 18 ml of ascitic fluid 17.8 mg of IgM was obtained based on the optical density at 280 nm and the extinction coefficient of & = 16 used by Leon et al. (32). Polyacrylamide gel electrophoresis of the IgM in SDS under reducing conditions revealed, after staining with Coomassie blue, the presence of heavy and light chains and a faint contaminating band with mobility slightly faster than the heavy chain. The IgM was dialyzed against 0.1 M NH, HCOR, concentrated, and subjected to gel filtration on a column (1.5 x 56 cm) of Sephadex G-150 to remove this lower molecular weight contaminant. The fractions from the exclusion volume of the column were pooled and upon SDS-polyacrylamide gel electrophoresis in @mercaptoethanol gave only two bands that stained with Coomassie blue corresponding to the heavy and light chains. Gels stained for carbohydrate with periodic acid-Schiff’s reagent had a single band corresponding to the heavy chain of IgM. Pronase digestion of IgM. Glycopeptides were prepared from [SH]mannose-labeled, [‘4C]glucosaminelabeled, and unlabeled MOPC 104E IgM by ineubation of either the labeled immunoprecipitates or purified IgM with 10 pg Pronase/mg of protein to be digested in 0.05 M Tris-HClI0.005 M CaCl, pH 8 at 37°C under toluene. An equal amount of fresh Pronase was added at 12, 24, and 36 h and the reaction was stopped after 48 h by putting the reaction tubes in boiling water for 3 min. RESULTS

Fractionation

of the IgM Glycopeptides

The Pronase digests of either labeled or unlabeled MOPC 104E IgM were subjected to gel filtration on columns of Bio-Gel P-6 as

STRUCTURE

400

h

18

i 2oom IO

20

30

40

163

OF MOPC 104E IgM OLIGOSACCHARlDES

50

60

70

FRACTION FIG. 1. Gel filtration of [“Hlmannose-labeled IgM glycopeptides. The samples were filtered on a column (1.5 x 96 cm) of Bio-Gel P-6 (200-400 mesh) in 0.1 1~ NH,HCO,, 2-ml fractions were collected, and 25 ~1 of each fraction was counted. (A) The Pronase digest of 1”HJmannose-labeled IgM; fractions were pooled as sho\vn. (B) Pool 2 fractions, after lyophilization and treatment with endo-P-N-acetylglucosaminidase C,,; fractions constituting 2A and 2B were pooled as shown. V,, and V,,,,, are the elution positions on the Bio-Gel P-6 column of bovine serum albumin and galactose, respectively, in a separate experiment.

when the smallest glycopeptides, in peaks 3 and 4 from the first Bio-Gel P-6 column (Fig. lA), were treated with endo-&N-acetylglucosaminidase CII, all the radioactivity in each was recovered in released oligosaccharides after filtration on Bio-Gel P-6 (data not shown). The glycopeptides in peak 1 were further fractionated by affinity chromatography on a column of Con A-Sepharose. This lectin binds glycopeptides with high mannose oligosaccharides with highest affinity and those with complex oligosaccharides containing two mannose residues unsubstituted at C3, 4, and 6 with intermediate affinity, but does not bind glycopeptides containing fewer than two such interacting mannose residues in their complex oligosaccharides (34). As shown in Fig. 2,73% of the radioactivity in peak 1 was not bound by the Con A-Sepharose, 27% was eluted with 10 mM a-methyl mannoside, and none was eluted with 100 mM haptene sugar, indicating the presence of glycopeptides having complex oligosaccharides with

IOOO-

’

lLlmM

WOmM

1

I

m MeMan

BOO -

shown in Fig. 1A for the [“Hlmannoselabeled IgM digest. The largest glycopeptides, in peak 1, were shown to contain only complex oligosaccharide chains as described below. The smaller glycopeptides in peak 2 were shown to be of two types; some containing complex oligosaccharides and others containing high mannose oligosaccharides. The peak 2 glycopeptides were subjected to treatment with C. perfringens endo-P-N-acetylglucosaminidase Ci,, which cleaves the di-N-acetylchitobiose unit of glycopeptides having high mannose oligosaccharides with at least Man1 --$ 3Manl 4 6(Man1 + 3)Manl+ 4GlcNAcl-+ 4GlcNAc + Asn in their structure (33). When the cleavage products were separated by gel filtration on Bio-Gel P-6, as shown in Fig. lB, 24% of the radioactivity eluted in the position of released oligosaccharide chains (peak 2B) and 76% of the radioactivity (peak 2A) remained unchanged in size, eluting in the same region as before. In contrast,

E 600 v 400 200 -

5

IO

IS

20

25

FRACTION

FIG. 2. Affinity chromatography of [“Hlmannoselabeled glycopeptide fraction 1 on Con A-Sepharose. Fraction 1 from Bio-Gel P-6 was lyophilized and a portion was chromatographed on a column of Con ASepharose (0.8 x 7 cm) equilibrated in Dulbecco’s phosphate-buffered saline pH 7.4 containing 1 mM CaCl, and 1 mM Mg Cl, (PBS-Ca” Mg2+). The sample was percolated into the gel bed and flow was stopped for 10 min. Then elution continued with 8 ml PBS-Ca’+Mg”, 8 ml PBS-Ca”Mg’+ containing 10 mM a-methyl mannoside, and finally 8 ml PBSCa’+iMg*+ containing 100 mM a-methyl mannoside. One-milliliter fractions were collected and 50 ~1 of each was counted. Fractions 2-5 were pooled as 1A and fractions 11-14 were pooled as 1B.

164

BRENCKLEANDKORNFELD TABLE I SUGAR

COMPOSITION

OF

MOPC 104E IgM

AND ITS GLYCOPEPTIDES

Total nmol” (residue/H chain)* Sugar Fucose Mannose Galactose N-AcetylgIucosamine N-Glycolylneuraminic acid”

IN

1

2

2A

2B

316 (6.3) 939 (18.8) 524 (10.5)

223 (4.4) 653 (13) 565 (11.3)

0 285 (5.7) 25.6 (0.5)

0 80.5 (1.6) 22 (0.4)

0 200 (4.0) 7 (0)

885 (17.7)

937 (18.7)

128.5 (2.6)

81 (1.6)

54 (1.1)

311 (6.2)

322 (6.4)

0

0

0

u Values are averages of two to four separate determinations. b Calculated from 4.744 mg IgM and assuming a molecular weight of 95,000 per HL which equals 50 nmol HL in Pronase digest. c Measured by the thiobarbituric acid color test; other sugars by glc.

three or more outer chains (lA), lesser amounts with two outer chains (lB), and none having high mannose oligosaccharides. Although the same pattern of fractionation was observed for the glycopeptides derived from unlabeled MOPC 104E IgM, there were some differences. No very small glycopeptides like peaks 3 and 4 (Fig. 1) were detected and no complex glycopeptides appeared in peak 2 as shown by the absence of fucose and sialic acid (Table I). Consequently a greater proportion of the peak 2 material was susceptible to cleavage by endo-fi-N-acetylglucosaminidase C,, and recovered as free oligosaccharide in 2B. The occurrence of [3H]mannose-labeled complex glycopeptides small enough to fractionate in peak 2 could be due to some of their outer chain sialic acid and galactose residues being cleaved by glycosidases released from damaged cells during the incubation in culture medium. Sugar Composition of MOPC lO4E IgM and its Glycopeptides

Table I shows the sugar composition of the purified unlabeled MOPC 104E IgM, isolated from the ascitic fluid of tumorbearing mice, expressed as the total nanomoles present in the amount of IgM subjected to Pronase digestion and also expressed as the number of residues of each sugar per heavy chain. Also shown is the

sugar composition and residues per H chain of glycopeptides 1,2,2A and oligosaccharide 2B isolated as described above. Glycopeptide fraction 1 contains, in addition to mannose and N-acetylglucosamine, all the fucose and N-glycolyl neuraminic acid and most of the galactose recovered in peaks 1 and 2 on Bio-Gel P-6 gel filtration, indicating that it contains complex-type oligosaccharide chains. Glycopeptide fraction 2 contains primarily mannose and N-acetylglucosamine and a small amount of galactose, consistent with its containing high mannose type oligosaccharides. The amount of each sugar recovered in glycopeptide fractions 1 plus 2 is about equivalent to the amount present in the starting IgM except for some apparent loss of fucose and gain in N-acetylglueosamine. These differences are probably related to inaccuracies in the sugar analysis on intact IgM. Under the methanolysis conditions used for the glc analysis there is some variability in the completeness of cleavage of the N-acetylglucosamine + asparagine linkage, especially with intact proteins, which probably caused an underestimate of the IgM N-acetylglucosamine. Conversely, the fucose measurement on the IgM is probably an overestimate due to the presence of an interfering peak on the glc which obscured one of the pair of fucose peaks, requiring that the calculation be based solely on the area of the major fucose species.

STRUCTURE TABLE

OF MOPC 104E IgM OLIGOSACCHARIDES

II

SUGARCOMPOSITIONAND METHYLATION ANALYSIS OFUNLABELEDGLYCOPEPTIDES 1A AND 1B FROMCON A-SEPHAROSE 1A

1B

ResiSugar Fucose 2,3,4-Tri-Me Mannose 3,4,6-Tri-Me 3,6-Di-Me 2,4-Di-Me Galactose 2,3,4,6-Tetra-Me 2,3,4-Tri-Me N-Acetylglucosamine 3,6-Di-Me 3-Mono-Me

nmol

dues”

141

1 1 3 1 1 1 3 1 2 5 4 1

389

414

Resinmoles 63.6 209

153

dues” 1 1 3 2 0 1 2 1 1 4 3 1

165

mannose oligosaccharide containing the other N-acetylglucosamine residue. The composition of 2B is compatible with its containing about 0.6 mol/H chain of an oligosaccharide of 6-7 mannose residueslmol and the composition of 2A is consistent with its containing about 0.4 moUH chain of a hybrid oligosaccharide with 4 mannose, 2 outer chain N-acetylglucosamine and 1 outer galactose residue and 2 core N-acetylglucosamine residues. Structural Studies of the Complex Oligosaccharide Chains of Glycopeptides IA and 1B

Table II shows the sugar composition and methylation analysis of unlabeled glyco597 258 peptides 1A and 1B obtained from glycopeptide fraction 1 by affinity chromatography on Con A-Sepharose. In each case the mannose content was set equal to three n Calculated by setting mannose equal to 3 residues. residues. 1A contained three galactose and five N-acetylglucosamine residues, indicaSince the complex oligosaccharides from tive of three outer chains whereas 1B con[3H]mannose-labeled fraction 1 each con- tained only two galactose and four Ntain three mannose residues (see below), acetylglucosamine residues, indicative of and the unlabeled complex glycopeptide two outer chains. Methylation of each fraction 1 contains 13 mannose residues/H glycopeptide, followed by g&mass specchain, it seems reasonable to conclude trometric analysis of the constituent meththat MOPC 104E IgM contains 4 complex ylated sugar species, showed that 1A conoligosaccharides. Similarly the sugar tained one mannose residue substituted at composition of glycopeptide fraction 2 is positions 3 and 6, another substituted at most compatible with a single high mannose positions 2 and 4, and a third substituted oligosaccharide containing two N-acetylat position 2 only. In contrast, 1B contained glucosamine residues in the core, variable one mannose residue substituted at positions amounts of mannose averaging about 6 3 and 6 and the two others substituted residues, and some molecules which in at position 2 only, consistent with its afaddition contain outer chain N-acetylglucofinity for Con A-Sepharose. Both glycosamine and galactose similar to the hybridpeptides contained a single terminal fucose type oligosaccharides found by Kobata and residue, and a single terminal galactose co-workers in ovalbumin (35). In fact, when residue. Glycopeptide 1A contained two glycopeptide 2 was treated with endo+galactose residues substituted at position N-acetylglucosaminidase CI1 and passed 6 whereas 1B had one such galactose residue. over Bio-Gel P-6 the fraction recovered in Limitations of material dictated that esthe original elution position of peak 2 (2A) sentially all of 1A and 1B be used for methcontained essentially all of the “extra” ylation and therefore none was used for sialic N-acetylglucosamine and galactose. All the acid determination but studies described fractions beyond that point, including the below for labeled 1A and 1B indicate that oligosaccharide region, were pooled for 2B, N-glycolyl neuraminic acid must be attached thus including the peptide stub with 1 N- to these substituted galactose residues. acetylglucosamine residue of the cleaved Both 1A and 1B also contain a single residue chitobiose unit, as well as the released high of N-acetylglucosamine substituted at posi-

166

BRENCKLEANDKORNFELD

tions 4 and 6. Experiments carried out on labeled 1A and lB, as described below, indicate that this is the N-acetylglucosamine linked to asparagine and bearing a fucose at C6 and the other N-acetylglucosamine residue of the chitobiose core at C4. All the remaining N-acetylglucosamine residues are substituted at C4 in both 1A and 1B. Since glycopeptide fraction 1 which gave rise to 1A and 1B contained 4 complex oligosaccharide chains/H chain, and the ratio of 1A to 1B is 1.8611.0, this would correspond to MOPC 104E IgM having 2.6 oligosaccharides of type 1A and 1.4 oligosaccharides of type 1B per heavy chain. There is precedence for the occurence at a single glycosylation site of complex oligosaccharide chains containing both two and three outer chains (e.g., VSV Gprotein) and this would explain the noninteger distribution of 1A and 1B observed in this case. The 2.6 mol of 1A should contain 5.2 mol of sialic acid and the 1.4 mol of 1B should contain 1.4 mol of sialic acid to account for the galactose residues substituted at C6 in each and the sum, 6.6 mol, is in good agreement with the 6.4 residues of Nglycolylneuraminic acid per H chain found in glycopeptide fraction 1. Additional structural studies were carried out on the [3H]mannosemlabeledglycopeptides 1A and 1B. Since rH]mannose is converted to [3H]fucose via GDP-mannose it is expected that fucose residues will also be labeled. Both acid hydrolysis (2 N HCl, 3 h, 1OO’C)and a-fucosidase digestion of the glucopeptides released approximately 25% of the 3H label in a sugar identified as fucose by paper chromatography in solvent III. The other 75% of the 3H in the acid hydrolysate was identified as mannose. Figure 3 shows the results obtained when glycopeptide fraction 1 and glycopeptides 1A and 1B were methylated, hydrolyzed, and the methylated sugars separated by thin layer chromatography. As shown in Fig. 3A, glycopeptide fraction 1 gave rise to 3,6- and 2,4-dimethyl mannose and 3,4,6trimethyl mannose as expected. A fourth radioactive peak (d) migrating in the same position as standard 2,3,4,6-tetramethylmannose was observed and is presumed to be 2,3,4-trimethyl fucose since it was

400 I

I

‘A D

b

I\

200 -.

5

IO

4

15

cm

FIG 3. Thin layer chromatography of partially methylated sugars from rH]mannose-labeled glycopeptides. (A) Fraction 1; (B) glycopeptide IA; (C) glycopeptide IB; and (D) core oligosaccharide (MansGlcNAc) derived from fraction 1 by endo-/SN-acetylglucosaminidase D after removal of outer chain sugars with glycosidases. a, b, c, and e correspond to 3,6-di-, 2,4,-di-, 3,4,6,-k-, and 2,3,4,&tetramethyl mannose, respectively, and d to 2,3,4-trimethyl fucose. The arrows indicate the position of the following standards: (1) 2,3-di-Me-Man; (2) 2,4-di-Me-Man; (3) 3,4,6-tri-MeMan; (4) 2,3,4-tri-Me-Man; (5) 2,3,6-tri-Me-Man; and (6) 2,3,4,6-tetra-Me-Man.

not present when glycopeptide fraction 1 was subjected to mild acid treatment and gel filtration on Sephadex G-25 to remove released sialic acid and fucose before methylation. Figure 3B shows that glycopeptide 1A gave rise to equal amounts of 3,6-di-, 2,4,-di-, and 3,4,6-trimethyl mannose and 2,3,4-trimethyl fucose, and panel C shows that glycopeptide 1B gave rise to 2,4,-di-, and 3,4,6trimethyl mannose and 2,3,4trimethyl fucose in a ratio of approximately 1:2:1. To further characterize the core region an aliquot of [3H]mannose-labeled glycopeptide fraction 1 was desialyzed with mild acid treatment and then digested with a mixture of @-galactosidase, /3-N-acetylglucosaminidase, and endo-P-N-acetylglucosaminidase D from Diplococcus Pneumonia. The latter enzyme will cleave the chitobiose

STRUCTURE

OF MOPC 104E IgM OLIGOSACCHARIDES

167

unit of complex type oligosaccharides with inidase D digestion. Since the peptide the structure Mancul + 3(Mancul * 6)- band also contained some [“Hlmannose it Man01 + 4GlcNAcPl + 4GlcNAc + Asn must have been contaminated with some inafter the outer chains are removed (23,36). completely cleaved core glycopeptides. The digestion products were separated by These results, combined with the fact that unlabeled 1A and 1B each contain a single paper chromatography and a radioactive oligosaccharide with a mobility faster N-acetylglucosamine residue substituted at than (Man),GlcitolNAc and slower than C4 and CC;(Table II) and the fact that cyMan,GlcitolNAc was isolated. As shown in fucosidase can release all the fucose from Fig. 3D, methylation of this oligosaccharide these glycopeptides, indicate that the core gave rise to 2,4-dimethyl mannose and glycopeptide in both 1A and 1B has the 2,3,4,6-tetramethyl mannose in a ratio of structure ManLvl + 3(Mancul -r 6)Manpl + approximately 1:2, indicating that the 4GlcNAc/jl + 4(Fucal + 6)GlcNAc ---;r mannose residues substituted at positions peptide. 2 and 4 and at position 2 in glycopeptide The sequence of the outer chain sugar fraction 1 had been converted to terminal, residues was established by glycosidase unsubstituted residues by removal of the digestions of either [“Hlmannose-labeled glycopeptide fraction 1 or [‘4C]glucosamineouter chains. When the core (Man),GlcNAc was treated with a-mannosidase and the labeled glycopeptides 1A and 1B isolated on digest was subjected to paper chromatog- Con A-Sepharose as described for ]?H]raphy, 70% of the radioactivity had the mannose-labeled material. The latter glycomobility of free mannose and 30% migrated peptides should have 14Cin both the sialic in the same position as Man,GlcNAc. In acid and N-acetylglucosamine residues. contrast, digestion with both (Y- and p- Treatment of 14C-labeled 1A and 1B with mannosidase converted almost all the radio- V. cholera neuraminidase followed by activity in Man,GlcNAc to free mannose. filtration of each digest on Bio-Gel P-6 Thus the core region of the complex oligosac- revealed that 22 and 20% of their 14C, charides must have the structure Mancul -+ respectively, had been released into a peak 3(Mancul ---, 6)Manpl -+ 4GlcNAcPl + of lower molecular weight material eluting 4GlcNAc. like sialic acid. The 14Csugar was identified In order to establish the location of the as N-glycolyl neuraminic acid by thin layer terminal fucose residue, [“Hlmannosechromatography. Subsequent incubation of labeled glycopeptides 1A and 1B were each residual glycopeptide with /3-N-acetyldigested with neuraminidase, P-galacto- glucosaminidase followed by Bio-Gel P-6 sidase, @-N-acetylglucosaminidase, and filtration revealed that no small molecular diplococcal endo-/3-N-acetylglucosaminidase weight radioactivity was released and the D. Upon paper chromatography in solvent residual glycopeptides were recovered and I 64 and 76%, respectively, of the radio- finally treated with a mixture of P-galactoactivity in the digestion mixtures of 1A sidase and /3-N-acetylglucosaminidase. and 1B migrated in a peak with the same Upon Bio-Gel P-6 gel filtration both digests mobility as Man,GlcNAc and separated now contained a peak of low molecular from a broad slower-moving band of radio- weight 14Cmaterial eluting in the region of activity. Subsequent acid hydrolysis of the N-acetylglucosamine and the residual 14C tetrasaccharide peaks and separation of the glycopeptide had shifted to a later elution constituent sugars by paper chromatography volume. The 14C sugar was identified as in solvent III revealed that 94% of the N-acetylglucosamine by paper chromatogradioactivity was contained in [3H]mannose raphy in solvent III. This series of experiand only 6% in [3H]fucose. In contrast, ments indicated that the oligosaccharides 77% of the radioactivity in the slower moving of 1A and 1B contain the sequenceNGNA + broad band was [3H]fucose suggesting that P P this band contained Fuc + GlcNAe -+ Gal + GlcNAc + and no chains terminate peptide moieties as the other reaction in N-acetylglucosamine. Similarly, [3H]product of the endo-P-N-acetylglucosammannose-labeled glycopeptide fraction 1

168

BRENCKLE NGNA

a2,6

AND KORNFELD NGNA

I

I

02.6

Gal

Gal

Gal

NGNA

cr2,6 I Gal

Gal

El,4 i Gl CNAC

51,4 I Gl CNAC

I

I

61.4

81,4

GlcNAc

GlcNAc I !31,4

81.4 al,6 Fuc---+GlcNAc

Fuc~~cNAc 1 An

I 4 ASn 1E

1A

FIG. 4. The structures

proposed for glycopeptides

was incubated with various combinations of glycosidases and the products separated by paper chromatography in solvent I. Treatment with a-mannosidase alone or cr-mannosidase plus P-N-acetylglucosaminidase released no free mannoseindicating the absence of either terminal mannose residues or GlcNAc + Man sequences. Treatment with a-mannosidase, P-Nacetylglucosaminidase, and p-galactosidase to degrade those chains terminated in galactose gave rise to only 8.6% of the 3H as free mannose (0.34 residue). Finally, digestion with all three glycosidases plus V. cholera neuraminidase released 58% of the 3H as free mannose (2.3 residues) indicating cleavage of all the a-linked mannoses. The residual glycopeptide region contained 42% of the 3H or 1.7 residues which can be assigned to the core /3-mannose and [3H]fucose attached to the N-acetylglucosamine residues in the core. On the basis of these studies the structures shown in Fig. 4 are proposed for glycopeptides IA and 1B. Structural Studies of the High Mannose Oligosaccharide Fraction 2B

All the structural work was performed on the pH]mannose-labeled oligosaccharide

1A and 1B.

2B released from glycopeptide fraction 2 with endo-P-N-acetylglucosaminidase C,,. When 2B was reduced with NaBH, and subjected to paper chromatography in solvent I, it separated into four different oligosaccharide fractions corresponding in size to (Man),-,GlcitolNAc as shown in Fig. 5A. The material in the paper was eluted as indicated and to confirm the size assignments an aliquot of each fraction was subjected to paper chromatography in solvent II with standard oilgosaccharides of various sizes as shown in Fig. 5B. The most abundant was oligosaccharide 2B III which contained 66% of the [3H]mannose and migrated as a symmetrical peak with the mobility of Man,GlcitolNAc. Oligosaccharide 2B IV (22%) migrated as a broad but symmetrical peak identified as Man,GlcitolNAc. Oligosaccharide 2B II (8%) was mainly Man,GlcitolNAc but contained some Man,GlcitolNAc and oligosaccharide 2B I (4%) migrated as a symmetrical peak like Man,GlcitolNAc. When each oligosaccharide was treated with a-mannosidase and subjected to paper chromatography for 16 h in solvent I, each was completely degraded to a mixture of 1 part Man,GlcitolNAc to 7.5, 5.5, 5.1, and 4.8 parts of free mannose for oligosaccharides 28 I, 2B II, 2B III, and 2B IV, respectively. Further

STRUCTURE

169

OF MOPC 104E IgM OLIGOSACCHARIDES

FIG. 5. Descending paper chromatography of the pH]mannose-labeled oligosaccharide alcohols in 2B. (A) The material in 2B from B&Gel P-6 (Fig. 1B) was reduced with NaBH,, desalted, and subjected to paper chromatography in solvent I for 4% days. The radioscan tracing of the chromatogram is shown and the segments indicated were eluted as fractions I, II, III, and IV. (B) A portion of each fraction (I-IV) was subjected to paper chromatography in solvent II for 3 days and l-cm segments of each paper strip were counted. The arrows indicate the migration position of the following standards: (1) Man,GlcitolNAc; (2) Man,GlcitolNAc; and (3) Man,GlcitolNAc.

analyses were performed only on the more abundant oligosaccharides 2B III and 2B IV. When 2B III and 2B IV were methylated, hydrolyzed, and the methylated mannose species separated by thin layer chromatography the results shown in Table III were obtained. 2B III and 2B IV were also subjected to acetolysis, which preferentially cleaves the 1 -+ 6 linkages between mannose residues giving rise to fragments of different sizes determined by the branching pattern of the oligosaccharide. Since some under- and over-degradation occurs during the acetolysis reaction the results can only be evaluated qualitatively. When the fragments from acetolysis were separated by paper chromatography the results shown in Fig. 6 were obtained. Figure 6A shows that three major fragments with mobilities of

tetra-, di-, and monosaccharides were obtained from oligosaccharide 2B III and a structure indicating the cleavage sites which could give rise to these fragments is also shown. The methylation results for oligosaccharide 2B III are in agreement with this branching pattern. In contrast, oligosaccharide 2B IV gave rise to four major cleavage fragments corresponding in size to tetra-, tri-, di-, and monosaccharides as shown in Fig. 6B. A mixture of two Man,GlcitolNAc structures, as shown, is required to account for this pattern of cleavage as well as for the various methylated mannose species (Table III) obtained in noninteger amounts. The complete structures proposed for oligosaccharides 2B III and 2B IV are shown in Fig. 7. The branching structure proposed for oligosaccharide 2B III, which is the only Man,GlcitolNAc structure susceptible to endo/3-N-acetylglucosaminidase C,, cleavage that could give rise to the acetolysis and methylation patterns obtained, was further corroborated by Smith periodate degradation. This procedure should oxidize and remove all mannose residues with vicinal hydroxyl groups, i.e., all but the branching mannose residues in the proposed 2B III al 6 P structure, giving rise to a Man &Man -+ xylosaminitol. When 5700 cpm of oligosaccharide 2B III, which amounts to 950 cpmlmannose residue, was subjected to Smith degradation as described under TABLE

III

METHYLATION OF~LIGOSACCHARIDES 2B III AND2B Iv Methylated mannose species 2,3,4,6-Tetra-Me 3,4,6-Tri-Me 2,4,6-T&Me 2,4-Di-Me

2B III cpm (residues)” 371 (3) 125 (1)

20 (0) 252 (2)

2B IV cpm (residues) 431 59 48 256

(2.7) (0.4) (0.3) (1.6)

a The counts per minute representing a single mannose residue were determined by dividing the total cpm recovered in all methylated mannose species in each case by the number of mannose residues per molecule of oligosaccharide, that is, by 6 for 2B III and by 5 for 2B IV.

170

BRENCKLEANDKORNFELD

Experimental Procedures and the product was isolated by paper chromatography in solvent I, a single radioactive oligosaccharide with the mobility of a trisaccharide was observed. The trisaccharide eluted from the paper chromatogram contained 920 cpm or approximately 50% of the theoretical yield expected for an oligosaccharide containing two mannose residues; a reasonable recovery in view of the losses expected in the several steps, especially in elution of the chromatogram. Methylation of the trisaccharide gave rise to 2,3,4,6tetra- and 2,3,4-trimethyl mannose, indi10 20 30 40 cating that the branch point mannose cm residues were linked al + 6. The high FIG. 6. Descending paper chromatography of the mannose oligosaccharides released from acetolysates of rH]mannose-labeled oligosaccharides. (A) 2B III and (B) 2B IV. The chromatograms glycopeptides 3 and 4 by endo-p-N-acetylglucosaminidase C,, were also reduced and were developed in solvent I for 25 h and l-cm segsized by paper chromatography and shown ments of each paper strip were counted. The locato consist of 1 part Man,GlcitolNAc and tions are shown for the following standards (1) 2 parts Man,GlcitolNAc in both cases al- Man,GlcitolNAc; (2) Man,GlcitolNAc; and (3) mannose. though no further structural studies were performed. on paper chromatography as expected for Man,GlcitolNAc (Fig. 8B) and constituted The Oligosaccharides in Glycopeptide 39% of the 3H 2A subjected to enzyme Fraction 2A digestion. Another 13% of the radioactivity, As indicated earlier [3H]mannose-labeled in fractions 29-32 on Bio-Gel P-4, probably glycopeptide fraction 2A contained a signifi- corresponds to [3H]fucose attached to the cant amount of smaller complex glycopep- GlcNAc peptide from which the oligosactides. They were shown to be present, charide had been cleaved. The residual and effectively removed, by treating the large glycopeptide 2A” contained 48% of the [3H]mannose-labeled 2A with a mixture radioactivity and, being freed of contamof diplococcal /3-galactosidase, P-N-acetyl- inating complex of oligosaccharide chains, glucosaminidase, and endo-p-N-acetylshould be equivalent to the glycopeptide glucosaminidase D followed by gel filtration 2A obtained from unlabeled MOPC 104E on a column of Bio-Gel P-4 as shown in Fig. IgM. The sugar composition of 2A (Table I) 8A. The oligosaccharide fraction, pooled as and the resistance of 2A and 2A” to endoindicated and reduced with NaBH,, migrated P-N-acetylglucosaminidases C,,, D, and H Mall

Mall

I

I

Man

Man /

\

?lan

Man

/

I

I la193

t

t

Man

I

Man

Man al.6

\/

al,3 \

Man I al,6

Man 61.4

61,4

I GlcitolNAc

GlcitolNAc

GlcitolNAc

a(0.7) Oligosaccharide

al,2 I

al.6

al,3

Man

28

FIG. 7. The structures

III

Oligosaccharide

proposed for oligosaccharides

+ 28

b(0.3) IV

2B III and 2B IV.

STRUCTURE

OF MOPC 104E IgM OLIGOSACCHARIDES

FRACTION /

5

?

lo

4

3

15

20

25

30

35

cm

FIG. 8. Removal of the complex oligosaccharides from [“Hlmannose-labeled glgcopeptide 2A. (A) Gel filtration of 2A after digestion with diplococcal P-galactosidase, B-N-acetylglucosaminidase, and endo-P-Nacetylglucosaminidase D on a column (1.5 x 98 cm) of Bio-Gel P-4 (200-400 mesh) eluted with 0.1 M NH,HCO,,. Two-milliliter fractions were collected and 0.1 ml of each was counted. V,, and V,,, are the elution positions of bovine serum albumin and galactose. The fractions containing the released oligosaccharide were pooled as shown. (B) Paper chromatography of an aliquot of the pooled oligosaccharide fraction from the Bio-Gel P-4 column after reduction with NaBH,. The chromatogram was developed in solvent I for 3 days and l-cm segments of the paper were counted. The arrows indicate the positions of the following standards: (I) Man,GlcitolNAc; (2) Man,GlcitolNAc; (3) Man,GlcitolNAc; and (4) Man,GlcitolNAc.

suggest that it contains a high mannose oligosaccharide with an atypical hybrid structure. DISCUSSION

The carbohydrate composition of MOPC 104E IgM myeloma protein reported here differs somewhat from that reported by previous workers. For example, Miller (37) found 2.1 fucose, 13.7 mannose, 8.5 galactose, 13.9 N-acetylglucosamine, and 2.8 sialic acid residues per HL subunit whereas Melchers (38) found 4 fucose, 17 mannose, 5galactose, 13 N-acetylglucosamine, N-glycolyl neuraminic acid + (not quantitated) and in addition 1 N-acetylgalactosamine and 1.5 glucose residues. Some of these variations may reflect the differences in methods used for carbohydrate analysis and in the values taken for the molecular weight of the IgM

171

and for the molar extinction of the IgM at 280 nm. As indicated in Table I the present study is most consistent with the presence of 5 oligosaccharide chains on the MOPC 104E IgM heavy chain, 4 of the complex type and 1 of the high mannose type. Very recently Kehry et al. (39) have reported the amino acid sequence of the MOPC 104E p-chain and proposed that it contains 6 oligosaccharide chains, 5 attached to Asn residues in the constant region and 1 attached to Asn 57 in the variable region. They further suggest that 3 are high mannose oligosaccharides and 3 are complex. Since the carbohydrate composition of these glycopeptides has not yet been reported it is not possible to account for the discrepancies between our findings and theirs. The structure of the complex oligosaccharides with two outer chains in MOPC 104E IgM (1B) is like that previously found in human IgG, IgE, IgA, and IgM (6) except that the sialic acid is N-glycolyl neuraminic acid in the mouse IgM rather than N-acetyl neuraminic acid. However, the structure of the complex oligosaccharides with three outer chains (1A) has not previously been found to occur in immunoglobulins although essentially the same structure containing sialic acid linked (r2,3 to the three terminal galactose residues occurs on the G protein of vesicular stomatitis virus (VSV) grown in BHK,, cells (40). Complex oligosaccharides of somewhat different structure, but containing three outer chains, have also been reported in fetuin (29) cul acid glyeoprotein (41) and calf thymocyte plasma membranes (42). In both VSV and human (~1 acid glycoprotein (41) the same glycosylation site m individual protein molecules may carry a complex oligosaccharide with either two or three outer chains, which also seems to be the case for the MOPC 104E heavy chain. The structure of the high mannose oligosaccharide 2B III is identical to that of the Man, oligosaccharide attached to Asn 563 in the p-chain of the human IgM (Wa) (3) and to the Man6 oligosaccharide in ovalbumin (43) and Chinese hamster ovary cell membranes (44). Mans oligosaccharides with the structure of oligosaccharide 2B IVa also occur commonly in other glycoproteins and this structure has

172

BRENCKLE

AND KORNFELD

been shown to be a key intermediate in the biosynthetic “processing” of high mannose oligosaccharides by a specific a1,2-mannosidase (45). Thus 2B IVa could have arisen from 2B III by this mechanism before secretion of the MOPC 104E IgM. In contrast, the structure of oligosaccharide 2B IVb is not typical and it may have arisen from 2B III after secretion of the IgM due to the action of an a-mannosidase released into the incubation medium by damaged cells. In this connection it is also noteworthy that the [3H]mannose-labeled high mannose oligosaccharides from fractions ZB, 3, and 4 have a combined average of 5.75 mannose residues per chain, whereas the composition of unlabeled fraction 2B gives an average of 7.4 mannose residues per chain, further suggesting some degree of extracellular degradation of the [3H]mannose-labeled high mannose chains of IgM occurred in the culture medium. ACKNOWLEDGMENT We are very grateful to Dr. Scot Hickman for his kind assistance in teaching us techniques for the culture of MOPC 104E cells and isolation of IgM.

REFERENCES 1. KORNFELD, R., KELLER, J., BAENZIGER, J., AND KORNFELD, S. (19’71) J. Biol. Chem. 246, 3259-3268. 2. HICKMAN, S., KORNFELD, R., OSTERLAND, C. K., AND KORNFELD, S. (1972) J. Biol. Chem. 247, 2156-2163. 3. CHAPMAN, A., AND KORNFELD, R. (1979) J. Biol. Chem. 254, 816-823. 4. CHAPMAN, A., AND KORNFELD, R. (1979) J. Biol. Chenz. 254,824-828. 5. BAENZIGER, J., AND KORNFELD, S. (1974) J. Biol. Chem. 249, 7260-7269. 6. BAENZIGER, J., AND KORNFELD, S. (19’74) J. Biol. Chem. 249, 7270-7281. 7. BAENZIGER, J., KORNFELD, S., AND KOCHWA, S. (1974) J. Biol. Chem. 249, 1889-1896. 8. BAENZIGER, J., KORNFELD, S., AND KOCHWA, S. (1974) J. Biol. Chem. 249, 1897-1903. 9. POTTER, M. (1972) Physiol. Rev. 52, 631-719. 10. BEVAN, M. J., PARKHOUSE, R. M. E., WILLIAMSON, A. R., AND ASKONAS, B. A. (1972) Progr. Biophys. Mol. Biol. 25, 131-162. 11. BERGMAN, L. W., AND KUEHL, W. M. (1978) Biochemistry 17, 5174-5180.

12. WAECHTER, C. J., AND LENNARZ, W. J. (1976) Annu. Rev. Biochem. 45, 95-112. 13. BAUMAL, R., POTTER, M., AND SCHARFF, M. D. (1971) J. Exp. Med. 134, 1316-1334. 14. BERGMAN, L. W., AND KUEHL, W. M. (1979) J. Biol. C&m. 254, 5690-5694. 15. TABAS, I., SCHLESINGER, S., AND KORNFELD, S. (1978) J. Biol. Chem. 253, 716-722. 16. MELCHERS, F. (1971) Biochemistry 10, 653659. 17. PARKHOUSE, R. M. E., AND MELCHERS, F. (1971) Biochem. J. 125, 235-240. 18. HICKMAN, S., AND KORNFELD, S. (1978) J. Zmmunol. 121, 990-996. 19. HIRAMOTO, R., GHANTA, V. K., MCGHEE, J. R., SCHROHENLOHER, R., AND HAMLIN, N. M. (1972) ~rn?~~~ochern~st~ 9, 1251-1253. 20. LI, E., TABAS, I., AND KORNFELD, S. (1978) J. Biol. Chem. 253, 7762-7770. 21. LI, Y. T., AND LI, S. C. (1972) fin Methods in Enzymology (Ginsburg, V., ed.), Vol. 28, pp. 702-713, Academic Press, New York/ London. 22. SUKENO, T., TARENTINO, A. L., PLUMMER, T. H., AND MALEY, F. (1972) Biochemistry 11, 1493-1501. 23. ITO, S., MURAMATSU, T, AND KOBATA, A. (1975) Arch. Biochem.. Biophys. 171, 78-86. 24. TREVELYAN, W. E., PROCTER, D. P., AND HARRISON, J. S. (1950) Nature (London) 166, 444-445. 25. SCHAUER, R. (1978) in Methods in Enzymology (Ginsburg, V., ed.), Vol. 50, pp. 64-89, Academic Press, New York/London. 26. WARREN, L. (1959) J. Biol. Chem. 234, 1971-1975. 27. HAKOMORI, S. (1964) J. Biochem. (Tokyo) 55, 205-208. 28. STELLNER, K., SAITO, H., AND HAKOMORI, S. (1973) Arch. Biochem. Biophys. 155, 464472. 29. BAENZIGER, J. U., AND FIETE, D. (1979) J. Biol. C&m. 254, 789-795. 30. Lr, E., AND KORNFELD, S. (1979) J. Biol. Chem. 254, 2754-2758. 31. HICKMAN, S., KULCZYCKI, A., JR., LYNCH, R. G., AND KORNFELD, S. (1977) J. Biol. Chem. 252, 4402-4408. 32. LEON, M. A., YOUNG, N. M., AND MCINTIRE, K. R. (1970) Biochemistry 9, 1023-1030. 33. TAI, T., YAMASHITA, K., AND KOBATA, A. (1977) BiocLm. Biophys. Res. Commun. 78, 434-441. 34. KRUSIUS, T., FINNE, J., AND RAUVALA, H. (1976) FEBS Lett. 71, 117-120. 35. YAMASHITA, K., TACHIBANA, Y., AND KOBATA, A. (1978) J. Biol. Chem. 253, 38623869.

STRUCTURE

OF MOPC 104E IgM OLIGOSACCHARIDES

36. ITO, S., MURAMATSU, T., AND KOBATA, A. (1975) Biochem. Biophys. Res. Commun. 63, 938-944. 37. MILLER, F. (1971) J. Immunol. 107, 11611167.

38. MELCHERS, F. (1972) Biochemistry

11, 2204-

2208.

39. KEHRY, M., SIBLEY, C., FUHRMAN, J., SCHILLING, J., AND HOOD, L. E. (1979) Proc. Nat. Acad. Sci. USA 76, 2932-2936.

40. READING, C. L., PENHOET, E. E., BALLOU, C. E. (1978) J. Biol. Chem. 5600-5612.

41. FOURNET, B., MONTREUIL, J., STRECKER, G., DORLAND,L., HAVERKAMP,J., VLIEGENTHART, J. F. G., BINETTE, J. P., AND SCHMID, K. 17, 5206-5214. (1978) Biochemistry 42. KORNFELD, R. (1978) Biochemistw 17, 14151423. 43. TAI, T., YAMASHITA, K., OGATA-ARAKAWA, M., KOIDE, N., MURAMATSU, T., IWASHITA, S., INOUE, Y., AND KOBATA, A. (1975) b. Biol.

44. LI,

AND 253,

173

Chem.

250, 8569-8575.

E., AND KORNFELD, S. (1979) J. Biol.

Chem.

254, 1600- 1605.

45. TABAS, I., AND KORNFELD, S. (1979) J. Biol. Chem.

254, 11655-11663.