The Polypeptide of Immunoglobulin G Influences Its Galactosylation in Viuo*

To examine the nature of the factors influencing the galactosylation pattern of the heavy chain of murine immunoglobulin G (IgG), cell fusion was performed between a myeloma (P3x63Ag8) and a hybridoma (Sp2HL/Bu) cell line which secrete different IgGs pos- sessing structurally distinct &a-linked oligosaccha- ride moieties. The glycosylation patterns of the IgGs of the parental and fused cells were studied. Pronase digestion of the purified heavy chains and subsequent end labeling with fluorescein isothiocyanate produced fluoresceinated glycopeptides which were detected and purified by polyacrylamide gel electrophoresis. Struc- tural information was obtained by enzymatic digestion, lectin affinity chromatography, and methylation analysis. IgGs from both parental lines oligo- microheterogeneity based a common symmetrical biantennary structure terminating in

Plasma membrane and secreted glycoproteins share a common biosynthetic route involving N-glycosylation (l-3). Many of the individual steps involved in the biosynthesis and processing of the oligosaccharide chains of N-linked glycoproteins have been studied in recent years. However, little is known about the mechanisms which determine the fine structures of the completed glycan moieties. The importance of cell type, presumably related to the nature, quantity, and  (8). Influence of the acceptor polypeptide on the degree and nature of processing of the glycan unit has been suggested by a number of authors (9-11). However, inferences from these studies which indicate a role for the polypeptide chain in modulating the structure of the glycan must be tempered with the realization that different proteins, synthesized in the same cell, may enter different intracellular compartments and/or exhibit differential rates of synthesis. Presently, there has been no report of the effect of polypeptide primary structure on the fine structure of the glycan moieties of two functionally identical subunits of a single protein in which both subunits possess the same glycosylation site. Immunoglobulin G (IgG), which is a bilaterally symmetrical molecule composed of two heavy and two light polypeptide chains, is an ideal glycoprotein to examine the influence of a polypeptide backbone on the biosynthesis of N-linked glycans. All human and murine IgG heavy chains possess a conserved site of glycosylation at Asnzg7 in the CH~ domain. The Nlinked oligosaccharide moieties of all human and murine IgGs are fundamentally similar, based upon a dibranched complex structure (12). Murine IgG rarely contains terminal sialyl units and, unlike the human protein, lacks a bisecting Nacetylglucosaminyl residue (13,14). The glycan at heavy chain AsnZg7 affects a number of important biological properties of the protein. These include the ability to activate complement (15,16), to induce antibody-dependent cytotoxicity (15), and to bind to the Fc receptor of monocytes (15,16). The presence and nature of the glycan structure have been suggested to contribute to the maintenance of the spatial relationship between the two heavy chains of the holoprotein (17, 18). Clinical implications of abnormal IgG oligosaccharides have been reported. The presence of agalactosylated IgG, which possesses carbohydrates with abnormal fine structure, correlates with the appearance of symptoms of rheumatoid arthritis in humans (19,20).
To determine the effect of polypeptide structure on the fine structure of the glycan unit as AsnZg7 of murine myeloma IgG proteins, we employed cloned hybrid myeloma cell lines. These were constructed by fusion of two cell lines producing IgG of different subclasses. In this study, we demonstrate that both IgGs possess a dibranched glycan moiety, but they differ in the pattern of microheterogeneity associated with the quantity and nature of terminal galactosylation. IgGs produced by the same hybrid cell. Accordingly, comparisons can be made of the final biosynthetic product of glycosylation of two functionally identical glycoprotein subunits secreted by the same cell, but which differ in polypeptide primary structure. Results of this study demonstrate that the primary structure of the polypeptide influences the addition of terminal galactosyl residues during the biosynthesis of the glycan moiety at the conserved site of glycosylation of murine IgG heavy chains.

EXPERIMENTALPROCEDURESANDRESULTS'
Exoglycosidase Analysis of Pronase Glycopeptides-The characteristics of the myeloma proteins secreted by the parental and three hybrid clones are described in the Miniprint. When necessary, tryptic glycopeptides from each of the heterodimeric heavy chains were isolated as detailed in the Miniprint. Structural information for the glycans of each tryptic glycopeptide from the heterodimers as well as the whole molecule or isolated heavy chains of the homodimeric IgGs was obtained by Pronase digestion and end labeling of the products with fluorescein isothiocyanate. The resulting FGPs,~ isolated by Sephadex G-10 chromatography, were detected and purified by PAGE (26). Carbohydrate sequence information was obtained by the use of exo-and endoglycosidases and analysis of the products by PAGE.
Enzyme Analysis of IgG2b Glycopeptides- Fig.  2 shows the PAGE fluorograms of purified and enzyme-degraded FGPs from the IgGPb protein. Pronase digestion and gel filtration of the derivatized products yielded three distinct asparaginelinked glycopeptides ( Fig. 2A, lane 4). These were purified by preparative PAGE to yield GPl (lane 1 ), GP2 (lane 2), and GP3 (lane 3). As expected for a symmetrical dibranched structure with nonreducing terminal N-acetyl$-glucosaminyl residues, GPl is not susceptible to @-galactosidase (Fig. 2B, compare lanes 7 and 9) or Lu-mannosidase (data not shown). N-Acetyl-@-glucosaminidase, however, is capable of removing two hexose units (compare lanes 7 and 8). Fig. 2C shows that after elimination of the hexosaminyl residues, the glycopeptide is susceptible to a-mannosidase, which removes two hexose moieties. The next most abundant glycopeptide, GP2, ' Portions of this paper (including "Experimental Procedures," part of "Results," part of "Discussion," Figs. 1, 3, 4, and 6, and Tables I  and II, and Footnote 2) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the Journal that is available from Waverly Press.
is susceptible to &galactosidase, which removes only 1 residue, yielding a compound of the same mobility as GPl (Fig.  2B, compare lanes 4 and 6). Treatment of GP2 with N-acetyl-P-glucosaminidase yields two fluorescent bands (lane 5). The predominant component displays an enhanced mobility corresponding to the loss of a single hexose residue from GP2. The minor component has a slower mobility. As is described in the Miniprint, the two fluorescent bands resolved by PAGE are the products of N-acetyl+glucosaminidase action on isomeric mdnogalactosylated dibranched glycopeptides differing in regard to the arm substitution of the galactose. Further exoglycosidase analysis (data not shown) of GP2 confirms that it is related to GPl except that it contains 1 P-galactosyl residue linked to an N-acetyl-@-glucosaminyl moiety equivalent to the nonreducing terminus of GPl. The least abundant glycopeptide, GP3, has a structure similar to GPl except that it possesses 2 P-galactosyl residues, each linked to an Nacetyl-&glucosaminyl residue of GPl. Thus, P-galactosidase treatment of GP3 (lane 1) results in the appearance of a glycopeptide (lane 3) with a mobility similar to GPl. Furthermore, GP3 is resistant to N-acetyl-P-glucosaminidase, indicating the absence of nonreducing terminal N-acetylglucosaminyl residues. These results are consistent with the glycans being related to a dibranched structure exhibiting microheterogeneity with 0, 1, or 2 nonreducing terminal pgalactosyl residues.
Enzyme Analysis of IgG, Glycopeptides-Results of similar studies with P3 (IgGJ show that the glycan from this protein is virtually identical to that of Sp2 except for the proportion of the two isomeric monogalactosylated glycopeptides present in the microheterogeneous mixture. This difference is most evident in Fig. 5. The action of N-acetyl+glucosaminidase on GP2 of the y, chain yields two fluorescent bands which are in approximately equal proportion (lane 2). This contrasts with GP2 from Sp2, which, when treated comparably, produces the two bands in a ratio of 4:l. The two bands are not caused by incomplete enzymatic digestion since prolonged treatment with N-acetyl-fi-glucosaminidase of GP2 from either IgG yields products similar to those seen with a 20-min exposure to the enzyme. Such results are suggestive of heterogeneity of the GP2 glycopeptides. Electrophoresis in the presence of borate is capable of resolving FGPs of the same size but of different sugar compositions (28). Borate-PAGE of GP2 from both the y1 and Y2b heavy chains shows them to be homogeneous (data not shown). Furthermore, sequential degradation of GP2 from P3 by /3-galactosidase and N-acetyl-/3-glucosaminidase results in: Step 1, the formation of a band corresponding to the loss of a single /3-galactosyl unit; and then Step 2, the formation of a single band corresponding to the loss of 2 N-acetyl-glucosaminyl residues (data not shown). GP1 from P3 contains 2 terminal N-acetyl+glucosaminyl residues (lane 5) and no nonreducing terminal P-galactosyl moieties (lane 6).

Structures of the IgG Glycans from Parental and Hybrid Cell
Lines-The ability to separate di-, mono-, and agalactosylated FGPs by PAGE and the resolution of the isomeric monogalactosylated compounds by concanavalin A-Sepharose chromatography allow for determination of the structures of the glycan units isolated from the conserved site of glycosylation. Table III records  isolated from the IgGi chains is identical. This microheterogeneity is reflected in the di-, mono (isomers A and B)-, and agalactosylated structures in a proportion of approximately 5:20:20:55, respectively. This composition differs from the proportion of the structures associated with the -j'2b chains, which is 5:38:9:48 in di-, mono (isomers A and B)-, and agalactosylated glycopeptides, respectively. Most noteworthy, in all the cases studied (regardless of cell type or dimeric form of the IgG), the proportion of each structure within the microheterogeneous mixture isolated from either the y1 or T$& chains is virtually identical to the composition determined for the corresponding parental homodimeric y chains. Interestingly, the proportion of the digalactosylated species is significantly greater in the glycopeptides isolated from the heterodimeric IgG than in structures obtained from the corresponding homodimeric proteins. This increase occurs at the expense of the quantity of agalactosylated glycopeptide in the IgGi series, but of the monogalactosylated structures of the IgGZb glycopeptides. Nevertheless, the ratio of isomer A to B of the monogalactosylated glycopeptides is not significantly different from that obtained from the appropriate heavy chain in either the homodimeric or heterodimeric protein.

Sp2HLIBu
and P3x63Ag8 Produce IgGs Which Exhibit Differences in Glycan Microheterogeneity-Both IgGs from P3 and Sp2 cell lines possess a non-bisected dibranched oligosaccharide, although the proportions of the 1,6-and 1,3-arm monogalactosylated isomers differ in the two proteins. The ratios of the monogalactosylated isomers of IgG from the P3 and Sp2 cells are 1:l and 4.2:1, respectively. Our results with the myeloma protein from clone P3x63AgB are similar to those reported by Rademacher et al. (12) for the protein  The overall microheterogeneous mixture from the P3K protein appears qualitatively similar to P3x63Ag8, although significant amounts of nonfucosylated oligosaccharides were obtained by Rademacher et al. (12). As with the P3 and Sp2 IgGs, however, the glycans from pooled mouse serum IgG (13) and a number of mouse monoclonal antibodies (14) are greater than 94% fucosylated.
In contrast to what may be expected from the proportions of monogalactosylated isomers observed for IgG, the /34-galactosyltransferase isolated from a number of sources (31,33,34) preferentially transfers galactose to the 1,3-arm of symmetrically branched acetylated glycopeptide and oligosaccharide acceptors terminating in N-acetyl-@-glucosaminyl residues. Apparently, either cellular factors or polypeptide structure influences the branch specificity of the reaction catalyzed by the transferase. The ability to obtain fusion constructs of myeloma cells which secrete heterodimeric IgG composed of a heavy chain derived from each parental cell (P3 and Sp2) allows for the determination of the nature of the factors that influence the galactosylation process. Regardless of subclass, IgG is assembled completely with two heavy and two light chains prior to transfer to the Golgi apparatus (35), where giycan processing and galactosylation occur (3). Accordingly, the finding that the glycan moiety from each of the heavy chains of the heterodimeric IgG possesses similar levels of microheterogeneity but differs in the proportion of each monogalactosylated species demonstrates that the polypeptide chain is capable of influencing, in viva, the branch specificity of the P-galactosyltransferase.
There is no need to invoke the presence of a @-galactosyltransferase different from that reported previously to explain the nature of the glycan microheterogeneity found in IgG. Furthermore, the structure of the heterodimeric IgG glycan reflects properties representative of the parental cells. The microheterogeneous mixture of the glycopeptides from the yi chain of the heterodimeric protein secreted by the three fused cell lines examined is very similar to that found with the IgG secreted by P3 cells and the homodimeric IgG, obtained from the fused constructs. Similarly, the glycopeptide mixture obtained from the IgG of Sp2 cells is virtually identical to the composition of the glycopeptides obtained from the -yzb chain of the heterodimeric protein produced by the fused cell lines. The possible basis for the observed polypeptide influence on galactosylation is presented in the Miniprint.
14.  Williams, D. B., and Lennarz, W. J. (1984) J. Biol. Chem. 259, 5105-5114 Preparation of Fluoresceinated PronaSe Glycopeptides -Whole IgG, ~&at& heavy chains, or tryptic glyc&t?pti&* were digested exhaustively with pronase, and fluoresceinated with FIX as described by Poretz and Pieczenik (26). The fluorescehated glycopzptide.5 EGPsl were isolated by gel fdtration through Sephadex G-10. Glycosidase treatment of the FGPs and electrophoretic separation of individual FGs or glycosidase treated EP* was perfond as describe-3 elSewhere @6). Previously (26, 271, we *onstat& that the logarithm of the relative difference in ability of two FWs, which have identical amino acid structures, is proportional to the difference in the number of neutral hexo*e units present. The** sizing gels were calibrated periodically employing FGPs of known stmcture*. Borate-PAGE of the FGps was conducted to separate FGP* of the sane size but which complex with different annunts of borate @8).

Methylaticm
Analysis of the FGP Glycan -Each FGP (2-10 nmol) was penrethylated in dimethylsulfoxide using N&H and methyl ia,i& as describe.2 by Ciucanu and Kerek (30). Sonicatian was snployed to enhance mixing. Upon termination of the reaction, the extracted methylated material was hydrolyzed.
The product* were partially purified by ion exchange chromatography, reduced and acetylated @I. CC-N5 analysis of the partially "?ethylat& alditol acetate* was performed with a Hewlett-Packard m&l 5890 GC and model 5970 Mass Selective Detector using a 30 m ultra I column. Temperature was programed to increase from 90°C to 200°C at a rate of ZO"C/min and maintained at 200°C for 5 min. Partially methylated hexosaminitol acetate* were eluted by increasing the temperature to 240°C at a rate of POWmin.
Chdracteri'i~tlc.7 of IgG from paiencal and hybrid cell line* -The IqG secreied by cloned parental and fused cell lines were isolated from ascites fluid and fractionated into abclasse* by Protein A-Sepharose affinity adsorption.
The properties of the IgG fran the parental cell line, P3X63.W G21) and Sp2HL/Bu (21) as well a* the cloned fusion products3 have been reported elsewhere. Table I shows the characteristics of fhe poly!xpti&s of the I@ produced by each cell line.
Parental cell, P3, produces a myelcana protein composed of two identxal 7, subclass heavy chains and c light chains; Sp2, a hybridoma which was used a* a parental cell line, produces a smgle homodimeric molecule composed of two ';a heavy chains and K light chains.
Each of the three cloned fusion CDN~N~'E produced three individual IgG molecules.
In each case, one IgG is a homodimer of 7, heavy chain*; another IgG contains two identical T,, chains and the third is a heterodimric molecule containing one Y1 and one Ya heavj chain. R11 of the secreted 1gG.s are associated with the TV, possible light chains &rived from the parental cells.
SDS-P?GE of the I$*, secreted fron each fused cell line and separated by Protein A-Sepharose affinity chrowtography, dMiOnst~ate* that one contains only the IgGl homodimer and and the other is a mixture containing the IgGm honodimer and the hetercdineric I&.
The proportion of heterodimeric to homodimeric Iq& in the mixture varies from 4:1 to *:1 depending upon the cell line.
Separation of glycopeptides frm individual glycosylation sites -Each Iq2 pool (10-E mg) was isolated from the appropriate ascites fluid and the nature of the carbohydrate unit at As" 297 of the different heavy chains was analyzed.
Tmtic mapping demonstrated that the IgG secreted by each parental cell line possesses only one glyco*ylation site, presumed to be at the conserwd site of qlycosylation.
The heteradimeric molecule*, produced by the fused cells, contain two tryptic glycopeptl&?*. Each is characteristic of the tryptic glycopeptide from the heavy chain derived from the corresponding parental cell.
To accarpllish the tr-,ptic peptide analysis, the appropriate cells were grown in vitro in the presence of 2-Xkwannose. I"di"i*al, radiolabeled IgGS, cmtaining appropriate *on-radioacti"e lrwlOm3 protein as a carrier, were purified from the culture medium by affimty adsorption onto Protein A-Sepharose. Following digestion with 'TM-trypsin, reverse phase HPX was errplayed to resolve the peptide mixture. Elution profiles of the peptides relatng radioactivity and absorption at 215 nm with resect to time showed that each parental IqG possesses only one detectable radioactive glyccpptide.
The patterx for the heteradimric IqC appeared to be the sum of the correspon&nq parental protein profile*.
The peptide map for the heterodimeric IgG obtained from the cloned fused cell line 27-I is shown in Fig. 1. The glycapeticks corresponding to the I', and Y, chains, as detemdned from analyses perfomd on each *ubclas* protein, are indicawd. Preparative HPLC , using a more shallow gradient of eluant than shown in Fig. 1, allowed for cmplete preparative separation of the individual tryptic glycopeptides from the Y1 and T',, chains of the heierodimer. The YSb qlycopeptide is derived from 1& chains of both the heterodimer and hamodimeric Iq&.
However, the & glycopeprIde from the heterodimer represents from 50-70% of the mixture, depending upon rhe cell line from which they are obtained.
AU of the YI q1ycc+zeptx& from the pool containing the heteradirer is derived from the hetercdirrer.
Separation of ismric mnogalactosylated qlycopepcides -Since each 82 from either the 7, or 7% chain: 1) when treated wirh @-qalactosidase results in a product indistinguishable from its corresponding GP1, 2) appear* homogeneous on borate-PAGE, and 3) yields two electrophoretically distinct comnents, when treated with N-acetyl-P-glucosaminidase. the possibility exists that Gp2 is a mixture of two monogalactose substitution isomers such as *hoar in Fig. 6. Paquet et al.011 have denon*trated that concanavalin A-Sepharose is capable of discriminating di-branched complex type glycopeptide* which differ I" *ZT sub*titution of the terminal qalactosyl unit.
They reported that thl* affinity matrix retards the elution of oligosaccharides with structures such a* Homer A, but bind tightly glycopeptides resembling isomer 8. Accordingly, when GP2 frcan the U, heavy chain is passed through a concanavalin A-Sepharose column, the elutian profile as shown in Fig. 3 is obtained. A portion of the fluorescence is unretained by the colwul.
Elution of the remainder, however, requires 0.1 M methyl or-mannoside for &sorption.
The FGPs were pooled separately, and aliquots were analyzed by PAGE prior to and following exposure to WacetylQ glucosminidase.
Consistent with the observation that the original GP2 appears hmcqeneaus an PAGE, the fluoroqraph deprcted m Fig. 4 dennnstrates that the unbound (lane l)and bound (lane 4) FGPs exhibit the **me electrophoretic mobility. N-Acetyl-p-glucosaminidase treatment of the individual FGPs shows that the unbound fraction of Gp2 corresponds to the isaner yielding the fa*ter wing product. The praduct of *1ower nobility is derived frm the GP2 isomer which binds to concanavalin A-Sepharase.
The assignment of the I,6 arm substituted isomer (isomer Al as the unbound fraction of GP2, and the bound fraction as the 1,3 arm *ti*tituted isomer Umrer Bl is based upon the conclusions of Paquet et al. (31). We find that the behavior of the EWs differs from those of the acetylated glycopeptides, as repOrted by otter.5 (29, 31). me Km, though separable by concanavalin A-Sepharose chromatography, bind 1855 strongly than do acetylated glycopeptides.
The fluore*cei"ated peptide moiety may be responsible for this decreased interaction since we observed also that GE'2 and @3 bind less well to Ricinus cmunis dgglutinin ItO-Spharose than reported (32) for the corresponding idnat& glycopeptides.
;he transit tire* through rhe tran* Golgi of be hcmo-and hetercdimeric proteins may accaunt, in pazI, for tIhi5 effect.
Accordingly, the increase in the prcportion of digalaccosylated glycan of Y, is associated with a decrease in the quantity of the agalactosylaced spxies.
.Sesulcs observed for the Ya chain however, suggests that other factor* al*0 may influence Che distribution.
The apparent decrease in the qua*ciLy of the 1.3~branched moncqalactasylaa2d ismer, and not the agalac:osylated species Of Che hereradirreric I@*, may be accounted for, in part, by a" increase in the ability of the galactosy?tra"*ferase to act o" the 1,6-branched mor.ogalacm*ylated isomer at Lhe expense of Lhe production of the 1,3-branch& iscmr.
Though Lhis propas. is concmry co the result* described for the action of the enzyme on glycopeptides (31, 331 and olicjosaccharides (34,. the reL3cive effecx Of the different polypepcide matrice* in rhe homodi~~eric and hecercdineric proteins on mmir.al galactosylation must caPsidx&.

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