Structure of the High Mannose Oligosaccharides of a Human IgM Myeloma Protein I. THE MAJOR OLIGOSACCHARIDES OF THE TWO HIGH MANNOSE GLYCOPEPTIDES*

the oligosaccharides

The structures of the predominant high mannose oligosaccharides present in a human IgM myeloma protein (Patient Wa) have been determined. The IgM glycopeptides, produced by pronase digestion, were fractionated on DEAE-cellulose and CM-cellulose which resolved two high mannose-type glycopeptides. Amino acid analysis shows that glycopeptide I contains Asn, Pro, Ala, Thr, and His and glycopeptide II contains Asn, Val, and Ser, which are the same amino acids found in the sequences around Asn 402 and Asn 563 respectively, to which high mannose oligosaccharides are attached in IgM (Patient Ou) (Putnam, F. W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (1973) Science 182, 287-290).
The high mannose glycopeptides in IgM (Wa) exhibit heterogeneity in the oligosaccharide portion. Structural analysis of the major oligosaccharides indicates that the simplest structure is: Mancvl-6Manal -+ 6ManjIl + 4GlcNAc/Il+ 4GlcNAc t al,3 t a193 1 Man Man Asn The larger oligosaccharides present have additional mannose residues linked (Y 1 + 2 to terminal mannose residues in the above structure.
Glycopeptide I contains primarily Mans and Mane species, while glycopeptide II contains Mane and Mans species. The two Mans oligosaccharides have different branching patterns.
Human IgM immunoglobulins contain both complex and high mannose-type oligosaccharides linked to asparagine residues on the heavy chain of the protein. Amino acid sequence and carbohydrate analysis performed on the human IgM (Ou) by Putnam and co-workers, has shown that this myeloma protein contains two high mannose oligosaccharides, one attached to Asn 402, and the other to Asn 563 (1, 2). Structural studies carried out by Hickman et al. (3) on a high mannose glycopeptide derived from the Asn 563 region of a different human IgM myeloma protein indicated that the oligosaccharide moiety was heterogeneous in respect to the number of mannose residues per chain. The methods used in that study provided partial characterization of the oligosaccharide structure of the glycopeptide, but the mixture of high mannose chains was not separated. Subsequently the enzyme * This investigation was supported in part by Grants CA 08759 and RR 00954 from the United States Public Health Service. The material in this paper constitutes a portion of a thesis to be submitted (by A. C.) in partial fulfillment of the requirements for the degree of Doctor of Philosophy. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. endo+N-acetylglucosaminidase was isolated from several sources and shown to have the very useful property of cleaving the N,N'-diacetylchitobiose linkage in the core of most high mannose glycopeptides, thereby releasing the oligosaccharide chains and leaving the linkage iV-acetylglucosamine residue attached to asparagine in the peptide (4). Thus, it is now possible to release such high mannose oligosaccharide chains, to separate them according to size, and, by employing a variety of techniques including acetolysis, methylation and periodate oxidation, to determine their complete structure. Using this approach, we have now determined the structures of the high mannose oligosaccharides that occur on two glycopeptides isolated from the human IgM myeloma protein (Wa) and shown that both glycopeptides display heterogeneity in their oligosaccharide moieties. To the remaining sample, 100 al of NaBH, (50 mg/ml in 0.05 N NaOH) was added. The mixtures were kept at 30°C for 6 h, then 25 or 75 d of glacial acetic acid were added to destroy excess borohydride.
The samples were evaporated with 1% acetic acid in methanol five times on a rotary evaporator (Buchler Instruments, New York) in order to remove methylborates.
The samples were then combined, and the reduced oligosaccharides were separated from buffer salts and digested peptide material on a column of Amberlite MB3 (10 x 0.9 cm). The oligosaccharides were eluted with water. After recovery of the released oligosaccharides, peptide-containing material was eluted from the column with 0.2 M ammonium bicarbonate. Hydrolysis of the reduced oligosaccharides followed by borate electrophoresis showed that all of the label was incorporated into glucosaminitol.
Acetolysis-Fifty nanomoles of oligosaccharide was subjected to acetolysis as described by Tai et al. (12), except that the incubation period was shortened to 12 h at 35°C and, at the end of the incubation period, the sample was extracted directly into 0.35 M sodium bicarbonate.
Periodate Oxidation-Periodate oxidation of the glycopeptides was performed as described by Baenziger et al. (8).

Smith Periodate
Degradation--Samples were oxidized as described by Spiro (13). Hydrolysis of the samples was carried out in 0.1 N HzS04 at 80°C for 80 min. Hydrolysates were desalted over columns (0.5 x 1.5 cm) of Dowex AG 3-X4 (100 to 200 mesh, acetate form).
Methylation Analysis-The oligosaccharides and glycopeptides were methylated by the method of Hakomori (14). The permethylated products were subjected to acetolysis, reduction, and acetylation as described by Stellner et al. (15). The alditol acetates of the partially methylated sugars were separated and analyzed on a Finnegan gas chromatograph-mass spectrometer (model 3200 or 3300). Separation was achieved on columns of either 1% OV-17 on Gas-chrom Q or 3% ECNSS-M on Gas-chrom Q (Applied Science). ECNSS-M was operated at 17O'C for 20 min and then programmed at 4"C/min to 240°C to separate 2,3,4-and 2,4,6-trimethylmannose.
OV-17 runs were programmed at 4'C/min from 140 to 240°C. Mass spectra were recorded with a separator temperature of 24O"C, ionization potential of 70 eV, ionization current of 60 PA, and ion source temperature of 27O'C. Injection temperature was 260°C. Identification of sugar derivatives was achieved by comparison of retention times and mass spectra with those known for standard compounds (16)(17)(18). Molar ratios of sugar derivatives from methylation of intact glycopeptides, released oligosaccharide alcohols, and periodate fragments were determined by measuring the area under the peaks on the flame detector scan obtained by gas chromatography using an F&M 402 gas chromatograph with either a 1% OV-17 column or 3% ECNSS-M column at 140 + B"C/min (use of the ECNSS-M column with this program provided separation of the 2,4-dimethyl-mannose peak from a non-sugar contaminant which led to anomalous ratios in the OV-17 runs).
When reduced oligosaccharides were methylated (Tables IV and  V), the two derivatives of 4-monosubstituted N-acetylglucosaminitol were observed.
Materials--NaBaH, was purchased from New England Nuclear.
Other chemicals were reagent grade and were purchased from commercial sources. Mannoses.
["Hlglucosaminitol standards were purified from Chinese hamster ovary cell glycopeptides by E. Li, who determined their composition and structure.'

Fractionation
of the pronase-digested IgM glycopeptides on DEAE-and CM-cellulose columns resulted in six major glycopeptide fractions (Fig. 1). Fractions A, B, C, and D consist of complex type glycopeptides containing mannose, N-acetylglucosamine, galactose, fucose, and (except for Fraction A) sialic acid. The structural analysis of these glycopeptides will be the subject of a future report.
Fractions I and II are high mannose-type glycopeptides, as shown by their monosaccharide and amino acid compositions (Table I). In order to determine the sequence of sugars and the linkages involved in these two glycopeptides, they were subjected to methylation analysis, exoglycosidase digestion, and periodate oxidation. The methylation results shown in Table II indicate that both glycopeptides contain 3 terminal mannose residues, and that none of the N-acetylglucosamine is terminal. Both glycopeptides contain fractional residues of mannose substituted at position 2, indicating heterogeneity of the oligosaccharides.
Based on the total number of mannose residues and the pattern of methylated mannose derivatives, 2 disubstituted mannose residues (branch points) are expected for each glycopeptide.
The value of 2.5 residues of 2,4-dimethylmannose in glycopeptide I is most likely due to a non-sugar contaminant that co-chromatographs with 2,4dimethylmannose.
To confirm this explanation, the glycopeptides were subjected to periodate oxidation, which destroys sugars containing vicinal hydroxyls. The disubstituted mannoses and the 2 N-acetylglucosamine residues should be the only sugars resistant to oxidation. Results in Table III show that in each case 2 residues of mannose and both residues of N-acetylglucosamine survived periodate oxidation.

Exoglycosidase Digestion of Intact
Glycopeptides-a-Mannosidase removed all but 1 of the mannose residues present in each glycopeptide (Table III). The remaining mannose residue was partially removed with ,f3-mannosidase. Treatment of the a-mannosidase-digested core with both /3mannosidase and /3-N-acetylglucosaminidase, but not with p-N-acetylglucosaminidase alone, resulted in release of N-acetylglucosamine and mannose. These results, in conjunction with the methylation results, indicate that the structure of the product of a-mannosidase digestion in each glycopeptide is: The Mans [3H]GlcitolNAc standard was derived from a glycopeptide with the structure shown in the discussion as the common oligosaccharide structure (20).   ' The proportions were determined by setting 3,6-dimethyl-N-acetylglucosamine = 2.0 residues. Separation of Oligosaccharides Released from Glycopeptides I and II---Results obtained by methylation of the intact glycopeptides suggested that heterogeneity existed in the oligosaccharide portion of the glycopeptides. Therefore, glycopeptides I and II were digested with endo-P-N-acetylglncosaminidase Cri, which cleaves the di-N-acetylchitobiose core in most high mannose glycopeptides. The released oligosaccha-rides were reduced with NaB3H4 and fractionated on a Bio-Gel P-4 column, As shown in fig. 2, both glycopeptide I and glycopeptide II gave rise to two radioactive oligosaccharide fractions. Each of the four oligosaccharide fractions was then subjected to descending paper chromatography in Solvent I for 4% days to further resolve the component oligosaccharide chains. In Fig. 3, the radioactivity profiles of the chromatograms reveal that the major oligosaccharide components originating from glycopeptide II co-migrated with MansGlcitolNAc (IIA), and MancGlcitolNAc (IIB). The oiigosaccharide chains in Fraction IB separated into two components, which co-migrated with MansGlcitolNAc (IB-2) and MansGlcitolNAc (IB-1). Fraction IA was resolved into four components which co-migrated with oligosaccharides of varying size from Mane-to MangGlcitolNAc.
Structural Studies Performed on Released Oligosaccharides-Oligosaccharides IB-1, IB-2, IIA, and IIB were each subjected to methylation, Smith periodate degradation, exoglycosidase degradation, and acetolysis. These techniques al- Cl, to release the oligosaccharides which were reduced with NaB3H4 and applied to a column (1.5 x 90 cm) of Bio-Gel P-4 equilibrated with 0.1 M NHdHCOa. One-milliliter fractions were collected.
An aliquot of every other fraction was assayed for radioactivity, and fractions were pooled as indicated.
by guest on July 10, 2020 http://www.jbc.org/ Downloaded from low complete elucidation of the oligosaccharide structures. Fig. 4 shows a schematic diagram for the structural studies performed using oligosaccharide IIA as an example.
Exoglycosidase Digestion of Released Oligosaccharides-In order to confirm that each of the released oligosaccharides had the expected composition (a-Man)r_7/3-ManiGlcitolNAc, samples were digested fist with a-mannosidase and then with /3-mannosidase. The products of glycosidase digestion were chromatographed in Solvent II for 19 h with appropriate standards. Results for all the oligosaccharides were identical to those shown in Fig. 5 for oligosaccharide IIA. One treatment with a-mannosidase converted the oligosaccharides to Man/31 + 4GlcitolNAc (Fig. 5A). One subsequent treatment with P-mannosidase released about 50% of the P-linked mannose, giving rise to a radioactive product that co-migrated with GlcitolNAc (Fig. 5B). When the residual disaccharide was pooled as shown and retreated with p-mancm FROM ORIGIN FIG. 3. Paper chromatography of Bio-Gel P-4 fractions IIA, IIB, IA, and IB. Each fraction was subjected to paper chromatography in Solvent I for 4% days. A small aliquot was spotted as an indicator strip and after chromatography the paper was cut into l-cm segments which were counted for radioactivity. The remainder of each sample was streaked on the paper which was scanned for radioactivity, and the areas corresponding to the bracketedpeaks indicated in the figure were eluted. Standards Mb-9 are Mannoses-[3H]GlcitolNAc. nosidase, one-half of it was cleaved and migrated with GlcitolNAc on rechromatography.
Smith Degradation-In order to determine the linkage of the outer branch point mannose to the inner one (Fig. 4), oligosaccharides were subjected to Smith degradation. The product isolated after one round of treatment in every case was a component that migrated as a trisaccharide in Solvent II (Fig. 5C). Methylation of the isolated product, in each case, gave rise to 2,3,4,6-tetramethylmannose and 2,3,4&imethylmannose in a 1:l ratio, indicating a 1 + 6 linkage between the 2 branch point mannose residues (see Table V for the methylation results).
Further Structural Studies of Oligosaccharide IIA (MansGlcitolNAc)-Methylation of IIA reveals the presence of 3 terminal nonreducing mannose residues, two 3,6-disubstituted mannose residues, and 3 mannose residues substituted only on position 2 in addition to N-acetylglucosaminitol substituted on position 4 (Table IV). Acetolysis which cleaves 1 + 6 linkages preferentially (12) was performed on the oligosaccharide (Fig. 4). Fig. 6   The major labeled species migrated as a pentasaccharide and a minor species migrated as a tetrasaccharide.
Reduction of the acetolysis products with NaB3H4 and fractionation in Solvent II (Fig. 6B) resulted in the appearance of a large disaccharide peak and a smaller mannitol peak. When the disaccharide peak was subjected to paper electrophoresis in molybdate buffer, which separates Man1 + 3 mannitol from Man1 + 2 mannitol, two peaks were observed as shown in Fig. 7. Methylation (Table V) of the material in these two peaks confirmed that disaccharide 1 was Man1 --, 3 mannitol and disaccharide 2 was Man1 --$ 2 mannitol as expected. Spectra for the two different methylated mannitol species are shown in Fig. 8, with their fragmentation patterns. Both of these methylated mannitols have the same retention time relative to tetramethylmannose (Trel = 0.45) on 1% OV-17 (160°C isothermal).
The monosaccharide peak from the reduced acetolysis mixture (Fig. 6B) is mannitol which is the expected by-product from overdegradation of the pentasaccharide fragment. The structure proposed for oligosaccharide IIA on the basis of these results is shown in Fig. 9.
Further Structural Studies on Oligosaccharide IIB (MuneGZcitoZNAc)-Methylation of this oligosaccharide indicates that it contains 3 terminal nonreducing mannose residues, two 3,6-disubstituted mannose residues, and 1 mannose residue substituted on position 2 in addition to N-acetylglucosaminitol substituted on position 4. Acetolysis produced a The oligosaccharide fragments obtained after acetolysis were subjected to paper chromatography in Solvent II. Panels A, C, E, and G show the radioactivity profiles for the unreduced reaction products, labeled only in GlcitolNAc, from IIA, IIB, IB-1, and IB-2, respectively. Panels B, D, F, and H show the radioactivity profiles obtained when the reaction products from IIA, IIB, IB-1, and IB-2 were reduced with NaBaH to label fragments containing mannose at the reducing end. The NaB3H4 used had a higher specific activity than that initially used to reduce the intact oligosaccharides. The standards are: 1,  by guest on July 10, 2020 http://www.jbc.org/ Downloaded from tetrasaccharide labeled in N-acetylglucosaminitol (Fig. 6C) as well as a disaccharide and monosaccharide which, after NaB3H4 reduction, could be detected as a disaccharide alditol and mannitol (Fig. 6D) Fig. 6, Panel B) were eluted and subjected to electrophoresis in 0.1 M sodium molybdate buffer, pH 5.5, at 20 V/cm for 1% hours on Whatman No. 3MM paper. The paper was then scanned for radioactivity. Peaks 1 and 2 were eluted, desalted, and methylated.  (Table V) showed that it has the structure Man1 ---* 2Manl -+ 3ManI -+ 4GlcitolNAc.
Methylation of the disaccharide showed that it was Man1 --) 3 mannitol. The structure of oligosaccharide IIB is shown in Fig. 9.
Further Structural Studies of IB-1 (Man,GlcitolNAc)-Methylation analysis (Table IV) of this oligosaccharide indicates that it has 3 terminal nonreducing mannose residues, two 3,6-disubstituted mannose residues, and 1 mannose residue substituted on position 2 in addition to N-acetylglucosaminitol substituted on position 4. Acetolysis was performed and Fig. 6 (Panel E) shows that the GlcitolNAc-labeled fragment migrated as a trisaccharide. The product of a-mannosidase treatment of this trisaccharide co-migrated with Mar@1 ---* 4 GlcitolNAc.
Data from periodate oxidation and methylation of intact IB-1 showed that the linkages in the inner structure must be: Man1 + 6Manl -+ 6Manl -+ 4GlcitolNAc fL3 ?I,3 Man Man and since 1 + 6 bonds are broken in acetolysis, the GlcitolNAc containing trisaccharide from the acetolysis procedure must be Man1 -+ 3Manl + 4GlcitolNAc.
Paper chromatography of the acetolysis mixture after reduction with NaB"H4 produced the pattern seen in Fig. 6 (Panel F). The trisaccharide peak is now larger, and there is also a disaccharide peak and mannitol. Treatment of the trisaccharide material with (Ymannosidase gave rise to radioactive mannitol and Man1 ---$ 4GlcitolNAc, indicating that it was a mixture of two trisaccharides, one containing mannitol and the other GlcitolNAc. Since the two trisaccharides could not be separated adequately by chromatography in Solvent II or by borate electrophoresis, the mixed trisaccharide material was methylated directly. Methylation gave rise to tetramethylmannose, 3,4,6trimethylmannose, 2,4,6-trimethylmannose, the two methylated derivatives of 4-monosubstituted GlcitolNAc, and 1,2,4,5,6-pentamethylmannitol.
These data indicate that the trisaccharides have the following structures: Man1 -+ 3Manl +4GlcitolNAc and Man1 --, 2Manl -+ 3Mannitol. When the reduced disaccharide fragment was subjected to a-mannosidase treatment, all of the radioactivity had the mobility of mannitol. When the disaccharide was subjected to molybdate electrophoresis, two peaks were observed, which co-migrated with Man1 + 2Man and Man1 --, 3Man (varying amounts of these two components were observed in three different acetolysis experiments.) These disaccharides are presumed to arise from overdegradation of the Man1 + 2Manl --+ trisaccharide at positions 1+ 2 or 1 + 3. The large free mannose peak suggests that overdegradation is occurring. Still, the presence of a small amount of a Mans species which has a branching pattern containing the two disaccharides cannot be excluded. The amount of mannitol label in the triand disaccharide peaks is roughly equal. If there were no overdegradation in acetolysis, this would mean that two-thirds of the Mans fraction contained an oligosaccharide with trimono acetolysis fragments, and one-third contained di-di fragments. Reduction of the size of the trisaccharide peak and increase in the disaccharide peak sizes due to overdegradation in acetolysis would suggest that the trisaccharide structure accounts for more than two-thirds of the total, perhaps all. The proposed structure of this predominant oligosaccharide is shown in Fig. 9. mannose oligosaccharide chain: Manal + 6Man/31+ 4GlcNAcal-+ 3Manpl --f 4GlcNAc ---f Asn lacks the N,N'-diacetylchitobiose core present in the other high mannose oligosaccharides (8). It also contains an a-linked GlcNAc residue and displays no microheterogeneity.
In an earlier study of another human IgM myeloma protein, Hickman et al. (3) partially characterized a high mannose glycopeptide with the structure:

Further
Structural Studies of IB-2 ( Man5GlcitolNAc)-Methylation analysis of oligosaccharide IB-2 (Table IV) shows that it has 3 terminal mannose residues and two 3,6-disubstituted mannose residues, in addition to N-acetylglucosaminitol substituted on position 4.
Acetolysis of the oligosaccharide shows that the glucosaminitol labeled fragment (Fig. 6G) moves as a trisaccharide. After reduction of the acetolysis products, paper chromatography (Fig. 6H) shows two additional peaks, a disaccharide, and mannitol. Methylation of the trisaccharide from acetolysis (Table V) shows that it has the structure Man1 + 3Manl+ 4GlcitolNAc.
Methylation of the disaccharide shows that it has the structure Man1 + 3mannitol. The only possible structure for oligosaccharide IB-2 is shown in Fig. 9.
This oligosaccharide displays heterogeneity in the number of mannose residues but differs from the structures found in IgM (Wa) in having an additional mannose attached to N-acetylglucosamine, probably in p linkage.

DISCUSSION
The amino acids present in glycopeptide I (Asn, Pro, Ala, Thr, and His) and glycopeptide II (Asn, Ser, and Val), correspond to those found by Putnam et al. (1) in the sequences around Asn 402 and Asn 563 to which high mannose oligosaccharides are attached in the human IgM (0~). The amino acid sequence of the Fc regions of five pathological human p chains have now been compared and shown to be the same and to be substituted with oligosaccharide on the same asparagine residues (21). Therefore, we conclude that the oligosaccharides of glycopeptide I are attached to Asn 402 and the oligosaccharides of glycopeptide II are attached to Asn 563 in the p chain of IgM (Wa). Studies on the carbohydrate of two of these IgM molecules by the same authors (21) and on a third pathological IgM by Jonneau and Bourrillon (22) show a high mannosetype glycopeptide appears at Asn 563 in these proteins as well as in those studied by Shimizu and co-workers earlier (2).
All of these oligosaccharides have a common structure of: Although all four of the IgM (Wa) high mannose chains contain the same structure for the 5 innermost mannose residues, oligosaccharide IB-1 has its sixth mannose attached to position b of the common structure, whereas IIB has its sixth mannose attached to position c. Clearly, the biosynthesis of these oligosaccharides proceeds with specificity and yet heterogeneity is observed in the size of the chains attached to a particular asparagine residue. Since myeloma proteins such as IgM (Wa) are products of a single clone of cells, this heterogeneity cannot be attributed to diversity in the cells of origin. Since plasma contains a-mannosidase activity, this heterogeneity could possibly arise from degradation of the oligosaccharides as the IgM circulates. However the amount of a-mannosidase activity at the physiologic pH of 7.4 is very low (26). The other explanation for the heterogeneity is that it arises during the biosynthesis of the molecules. We now know that the biosynthesis of high mannose oligosaccharides occurs by transfer of a large lipid-linked high mannose oligosaccharide to asparagine followed by "processing" of the sugar chain by removal of mannose residues (27-29). An interruption of this sequence of events could result in a spectrum of oligosaccharide chain lengths. In fact glycopeptide I contained, in addition to the two major high mannose oligosaccharide chains described here, small amounts of four other oligosaccharides whose structures have been analyzed. These results and their possible relevance to oligosaccharide processing are reported in the following paper.
Mann1 + 6Manal + 6Manpl + 4GlcNAcPl+ 4GlcNAc a fal,3 ?a133 Man Man b c with additional mannose residues linked u1,2 at positions n, 6, or c. High mannose oligosaccharide structures have been studied in detail in two other human myeloma immunoglobulins, with results suggesting that the oligosaccharides in these 1.
cases are somewhat different. The structure for the IgE high 11.