immiwoloRy
and Cell Biolofty (1991) 69. 151-157
Hybrid myeloma cells which secrete hetero A model to study the /V-linked glyca S-O. LEE AND R. D. PORETZ Department of Molecular Biology and Biochemistry. Ruiger.s University. Piscataway, NJ, USA (Submitted 4 January 1991. .Accepted for publication 9 April 1991.) Fundamental questions remain unanswered regarding ihc elTec! of ihe acceptor polypeplidcslruclure on thefinestructure of the A'-linkcd glycan of glycoprotcins and conversely, the effect of the glycan structure of IgG on Ihe function and slruelure of the protein. The construction of myeloma hybrids capable of secreting multiple IgG which differ with regard to the tine structure of their /V-linked oligosaccharides would be a valuable model for studying these questions. P3X63Ag8 secretes an IgGi which possesses an oligosaccharide at Asn^'*'' that differs in fine strueture from the analogous glycan of the igGjb secreted by Sp2/HLBu. Fusion hybrids of these cells secrete parental IgGj. and to a lesser degree IgGjb. as well as a heterodimeric IgG containing both ihe 7i and Y2h chains. The oiigosaecharide of each chain is identical in structure to the appropriate parental IgG. Such cells allow for the analysis of acceptor properties that inffuence glycan fine structure, as well as the role of glycan structure on the stability of the IgG.
INTRODUCTION Somatic cell fusion involving myeloma cells, though originally performed lo explore the regulation of immunoglobulin biosythesis. has been used in the past decade primarily for the production of monoclonal antibodies (1). Nevertheless, questiotis continue to arise regarding the nature of ihe factors which influence the structure and synthesis of immunoglobulins. In analogy with other secreted giycoproteins, the heavy chain of IgG is translocated across the endoplasmic reticulum membrane and glycosylated at Asn-^^ with a mannose rich oligosaccharide (2). During migration of the Y chain from this subcellular compartment through the Golgi apparatus, the IgG is completely assembled and the glycan moiety undergoes extensive processing and restructuring. One of the final steps in oligosaccharide processing occurring in the trans-Golgi involves the addition of galactose to the termini of the glycan unit (2). The oligosaccharide at the conserved site of
glycosylation ofthey chain is a relatively simple structure, but has been implicated in numerous functions and properties of IgG. Some effector functions associated with the presence of the glycan include IgG activation of complement (3.4), antibody-dependent induction of cytotoxicity (3) and IgG binding to the Fc receptor of monocytes (3.4). The presence and specific structure of the glycan moiety maybe important in maintaining the spatial relationship of the two heavy chain subunits of the assembled immunoglobulin (5.6). Recent reports have described the high correlation between the presence of abnormally high levels of IgG lacking terminal galactose residues on the oligosaccharide at Asn-**' and the diagnosis of rheumatoid arthritis in individuals suffering from the disease (7.8). Accordingly, a better understanding of the factors that influence the biosynthesis of IgG bound glycan units may shed light on specific disease states related to IgG functions. The role of specific cell type which defines the nature, quantity and intracellular compartmentaliza-
Correspondence: Dr R. D. Poretz, Department of Molecular Biology and Biochemistry. PO Box 1059. Rutgers University, Piscataway. NJ 08855-1059. USA. Ahhrexiaiiotu used in this paper: Asn. asparagine; DMEM, Dulbecco's modified Eagle's medium; ECGS. endothelial cell growth supplement; HAT, hypoxanthino-aminopicrin-thymidine; HT. hypoxanthine-thymidine; IgG, immunoglobulin G; PAGE, polyacrylamide gel eleetrophoresis; PEG. polyethylene glycol; SDS. sodium dodeeylsulfate.
S-O. LEE A N D R. D. PORETZ
t52
tion of individual glycosyl transferases and glycosidases in determining the strueture of the glycans of giycoproteins in doeumented (9-13). Similarly there have been reports that the acceptor polyjKptideinfluenees the degree and nature of processing of the glycan unit of glycoproleins (14-16). A caveat, however, upon these latter studies is that ditferent proteins, synthesized within the same cell, may enter different intracellular compartments and/or exhibit dilferential rates of synthesis. Prior to studies (17) based upon the IgG produced by the hybrid cells reported in this paper, there had been no repoil on the effect of polypeptide primary structure on the fine structure of the glycan moieties of two functionally and structurally similar subunits of a single protein in which both subunits possess the same glycosylation site. Cloned, hybrid myeloma cell lines represent an ideal system to explore the influence of y ehain amino acid sequence on the fine stt^cture of the Asn-^^ glyean. This paper describes the construction and properties of hybrid cell lines that simultaneously secrete multiple IgG composed of either identical or different y chains. The heavy chains are of ditferent subelasses and possess dissimilar glycan units at the conserved site of glycosylation. These cell lines were constructed by fusion of a myeloma and a hybridoma. produeing IgG of different subclasses, each with a di-branched glycan moiety, differing in the quantity and nature of terminal galactosylation (17). Sueh hybrid cell lines and their 'parental' lines may serve as a valuable model to explore the influence of fine structure of the earbohydrate at .\sn-''^ on the strueture and function of IgG.
MATERIALS AND M E T H O D S Cells and media Fusions were performed with the myeloma cell line P3X63Ag8. an azaguanine resistant P3 clone derived from MOPC-21 which secretes an IgG 1. and a hybridoma which secretes an IgGib. Sp2/HLBu. obtained from a fusion of P3X63Ag8 and a murine splenocyte and selected for resistance to 5-bromouridine. Both cell lines were developed in the laboratory of Dr C. Milstein (18) and were a gift from Dr N. Cowan. These cells and their hybrids were cultured In vitro using Dulbecco's modified Eagle's medium (DMEM) (Gibco. Grand Island. NY. USA) supplemented with 10% fetal calf serum (Gibco,
Grand Island. NY. USA), non-essetitial amino acids (Gibco. Grand Island, NY. USA), glutamine. penicillin-streptomycin, under an atmosphere of 5% CO;. Cell fusion and detection of clones The cell fusion was performed aecording to the method of Pearson et al. (19). Cells (I X 10*') of each cell line were combined and mixed with 0.3 mL of 35% polyethylene glycol (PEG 1000. Sigma Chemical Co.. St Louis. MO. USA) and centrifuged. After 8 min at 37°, the eell mixture was diluted with 5 mL of DMEM. centrifuged. washed with DMEM and suspended in 200 mL of HAT medium eontaining 100 ^ig/mL of endothelial cell-growth supplement (ECGS. Collaborative Research Inc.. Bedford, MA. USA) (20). One hundred microlitres of appropriately diluted cell suspension were placed into each well of 96-well plates. Three to four days later, cells were fed with HT medium and allowed to grow for an additional 2 weeks. The supernatants were removed from eaeh well and subjected to immuno-dot blot analysis using anti-IgG serum (Miles Laboratories Inc.. Kankakee. IL. USA). Cells of randomly selected wells, positive for IgG. were subeloned on plates containing l%Sea Plaque agarose (FMC Bioproducts. Rockland, ME. USA) and ECGS in HT medium as described by others (21.22). After the clones reached a visible growth, multiple nitrocellulose membrane biois were obtained and probed with rabbit subclass specific antiserum (Litton Bionetics. Charleston. SC. USA); one membrane with anti-lgG| and another with antl-IgG2b serum. Positive reactions were detected with horse-radish peroxidase-conjugated anti-rabbit IgG and 4-chloro-l-naphthol. Comparisons of matching membranes developed with the different subclass specific antibody allowed selection of clones secreting both IgG subclasses. Following expansion of the selected clones in suspension culture, the cells (2-4 X lO**) were injected intraperitoneally into pristane-primed Balb/c female mice resulting in the production of ascitic Huids.
The purification of/gG To isolate individual subclasses of IgG, ascites fluid was clarified by centrifugation at 1000 .i? for 10 min, mixed with an equal volume of I.5mol/L glycine buffer (pH 8.9) containing 3 mol/L NaC'l and 0.01% NaN^ and applied to a protein A-Sepharose column (0.5 cm X 5 cm)
HETERODIMERIC IgG SECRETING CELLS
(Pharmacia. Piscataway. NJ, USA). The column was washed with 40 mL of the same buffer and eluted sequentially with O.I mol/L citrate buffers of pH 6.0, 5.5.4.5 and 3.5 containing 0.01% NaN.i as described by Ey et al. (23). The pH 4.5 and 3.5 eluants of the eolumn were colleeted in tubes (2 mL) containing I mL of 0.1 mol/L borate buffer. pH 9.0. Following optical density measurements at 280 nm. the appropriate fractions were combined, dialysed against H2O,and lyophilized. Immuno-eleclrophoresis Agarose eleetrophoresis was conducted as described elsewhere (24) using O.Oi mol/L barbital buffer (pH 8.6). Upon completion of eleetrophoresis. the agarose plates were fixed with 10% trichloroacetic acid solution containing 3.5% sulfosalicylic aeid and stained with Coomassie Blue R. Immunodijfusion tests Double immunodifTusion was conducted in agarose as described by Ouchterlony (25). SDS-page Gels and samples were prepared, and the eleetrophoresis was conducted according to the conditions of Laemmli and Favre (26). The separating gel contained 9% acrylamide and 0.25% jV.A'-methylenebisacrylamide. and the stacking gel contained 4% acrylamide and 0.1% A'.iV'methylenebisacrylamide. The electrophoretograms werefixedand stained as described above for immuno-electrophoresis. Gelfiltrationchromatography One-half millilitre of sample or standards (1 mg/mL) was applied in 0.05 mol/L potassium phosphate buffer (pH 6.8) containing 0.1 mol/L NaCI and 0.02% NaN3 to a column (0.9 cm X 150 cm) of Sephadex G-200 (Pharmacia. Piscataway, NJ, USA) and eluted with the same buffer at a flow rate of 4 mL/h. The optical density of individual fractions (1.5 mL) was determined at 280 nm. The standards employed were: ferritin (450 000) (27): rabbit IgG (155 000) (28): and bovine serum albumin (68 000) (29), as well as Blue Dextran (Pharmacia, Piscataway, NJ, USA).
153
RESULTS AND DISCUSSION Fusion hybrids of P3X63Ag8 and Sp2/HLBu were constructed and seeded at a limiting dilution of 0.5 ceils/well in 96-well plates. Following subeloning in soft agar of IgG producing eells. 36 colonies showed secretion of reactive material to both anti-lgGi and antiIgGab sera and were selected and expanded. These colonies were each on plates containing fewer than 20 colonies per 48 mm diameter plate, were physically separated from neighbouring colonies and appeared to have circular symmetry of growth. These characteristics were taken as additional assurance of the monoclonal nature of the selected eolonies. Though many of the colonies exhibited an unequal reactivity with the two subclass specific antisera. three hybrid subelones, with apparent equal reactivity with the antisera and originating from different wells of the initial plating, were chosen for further analysis. Each clone was grown as an ascitic form in BALB/c mice and the IgG from the fluid so developed was isolated by protein A-Sepharose affinity adsorption. It is evident from the elution profiles (Fig. 1) obtained with the fluid produeed by clone 27-1 and both parental cell lines that the hybrid cell secretes both lgG|- and IgG2b-like proteins. Fluid from all tfie fused constructs produced similar profiles with the protein A affinity eolumn. The purity of each IgG pool separated by protein A adsorption is shown by agarose eleetrophoresis (Fig. 2). A single protein-staining region with a mobility corresponding to that exhibited by the appropriate immunoglobulin present in the ascitic fluid from the parental cells is obtained from the pH 6 and 3.5 eluates. In all cases the protein band of the purified IgG from the fused cells was more diffuse and exhibited a decreased mobility as compared with tbe IgG of the parental ceils. Presumably, this is due to the random association of the light chains derived from each parent with the heavy chains of both subclasses (1). This possibility is made more evident from the SDS-PAGE of the purified IgGs. Figure 3 demonstrates that the light chains of both parents are found associated with both the 71 and yih ehains of the fused cell products. Furthermore the pH 3.5 eluate contains both yi andyibchains. Similar results were obtained with the pH 3.5 pool from the protein A-Sepharose affinity adsorption of the ascites fluid produeed by all three fused eell lines studied. Re-adsorption of the protein in the pH 3.5 pool to a protein A-Sepharose eolumn resulted in protein being eluted only with the pH 3.5 buf-
S-O. LEE AND R. D. PORETZ
154
.£)
' ostcoarthritis with changes in the glycosylation pattern of total serum IgG. NatureiW. 452-457. 8. Parekh, R. B.. Dwek, R. A. and Rademacher, T. W. 1988, Rheumatoid arthritis as a glycosylation disorder. Br. J. Rheum. 27 (Suppl. 11): 162169. 9. Hsieh. P., Rosner. M. R. and Robbins, P, W. 1983. Host-dependent variation of asparaginelinked oligosaccharides at individual glycosylation sites of Sindbis virus giycoproteins. J. Biol, Chem. 258: 2548-2554. 10. Williams. D.B. and Lennarz.W. 1984. Control of asparaginc-linked oligosaccharide chain processing: studies on bovine pancreatic ribonuclease B. J. Biol. Chem. 259: 5105-5114. 11. Taylor, A.K. and Wall. R. 1988. Selective removal of heavy-chain glycosylation sites causes
HETERODIMERIC IgG SECRETING CELLS
12.
13.
14.
15.
16. 17.
18.
19.
20.
21.
22.
immunoglobulin A degradation and reduced secretion. Mot. Cell. Biol. 8: 4197-4203. Sheares, B.T. and Robbins, P. W. 1986. Glycosylation of ovalbuniin in a heterologous cell: analysis of oligosaccharide chains of the cloned giycoprotein in mouse L cells. Proc. NatlAcad Sci. USAS3: 1993-1997, Parekh, R. B.. Tse. A. G. D., Dwek, R.A., Williams, A. F. and Rademacher. T. W. 1987. Tissuespecific /V-glycosylation. site-specific oligosaccharide patterns and lentil Icctin recognition of rat Thy-I. EMBOJ. 6: 1233-1244. Anderson. D. R. and Grimes. W. J. 1982. Heterogeneity of asparagine-linked oligosaccharides of five glycosylation sites on immunoglobulin M heavy chain from mineral oil plasmacytoma 104E. / Biol. Chem. 257: 14858-14864. Green. M. 1982. Incorporation of amino acid analog interferes with the processing of the asparagine-l inked oligosaccharide ofthe MOPC46B tight chain. / Biot. Chem. ISl: 9039-9042. Dahms. N. M. and Hart. G. W. 1986. Influence of quaternary structure on glycosylation. / Biot. Chem- 261: 13186-13196. Lee. S-O. Connolly, J. M.. Ramirez-Soto. D. and Poretz. R. D. 1990. The polypcptide of immunoglobulin G influences its galactosylation in vivo. J. Biot. Chem. 265: 5833-5839. Kohler. C. and Milstein. C. 1976. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. / Immunol. 6; 511519. Pearson, T. W.. Pinder. M., Poclanls. G. E. el at. 1980. Methods for derivation and detection of anti-parasile monoclonal antibodies. J. Immunot. Melt}. 34: 141-154. Maciag. T.. Hoover, G. A., Stemerman. M. B. and Weinstein. R. 1981. Serial propagation of human endothelial cells in vilro. J. Cetl. Biot. 91: 420426. Sharon. J.. Morrison. S. L. and Rabat, E. A. 1979. Detection of specific hybridoma clones by replica immunoadsorption of their secreted antibodies. Froc. NaltAcaci Sci. USA 76: 1420-1424. Coffino, P., Baumal, R.. Laskov, R. and Scharff, M. D. 1972. Cloning of myeloma cells and detection of rare variants. / Cett. Physiot. 79: 429440.
157
23. Ey, P. L.. Prowse, S.J. and Jenkin. C. R. 1978. Isolation of pure IgGj, lgG2a and lgG2b immunoglobulins from mouse serum using protein ASepharose. Immutwchemisiry 15: 429-436. 24. Elevitch. F. R. 1966. Thin gel electrophoresis in agarose. J. Ctin. Path. 46: 692-697. 25. Ouchterlony. O. 1948. Antigen-antibody reactions in gels. Ada Path. Microhiot.. Scand. 25: 186. 26. Laemmli, U. K. and Favre, M, 1974. Maturation ofthe head of bacteriophage T4. / Mot. Biol. 80: 575-599. 27. Rothen. A. 1944. Ferritin and apoferritin in the ultracentrifuge: studies on the relationship of ferritin and apoferritin. J. Biot. Chem. 152: 679693. 28. Cammack.K. A. 1962. Molecular weight of rabbit gamma-globulin. Nature 194: 745-747. 29. DanhofT, M. O.. Perlmann. G. E. and Maclnnes. D. A. 1952. The partial specific volumes in aqueous solution, of three proteins. J. Amer. Chem. Soc. 74: 2515-2517. 30. Eden. A., Lammn, M. E. and Nussenzweig. V. 1972. Complemenlarily of H and L chains from antihapten antibodies of different classes. J. Immunot. 108: 1605-1608. 31. Margulies, D. H., Cieplinski. W.. Dharmgrongatama. B. etai 1976. Regulation of immunogiobulin expression in mouse myeloma cells. Cold Spring Hart). Symp. Quani- Bioi 41: 781-791. 32. Rademacher. T. W.. Homans, S. W.. Fernandes. D. L. and Dwek. R. A. 1987. Immunoglobulin G as a giycoprotein. Biochem. Soc. Symp. 51: 131148. 33. Mizuochi, T., Hamako. J. and Titani, K. 1987. Structures of the sugar chains of mouse immunoglobulin G. Arch. Biochem. Biophys. 257: 387394. 34. Rothman, R. J., Warren. L.. Vliengenthart. F. G. and Hard, K. J, 1987. Clonal analysis of the glycosylation of immunoglobulin G secreted by murine hybdridomas. Bioctwmi.slry 26: 13771384. 35. Bole. D. G.. Hendershot, L. M. and Kearney. J. F. 1986, Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas. J. Cetl. Biot. 102: 1558-1566.
and Cell Biolofty (1991) 69. 151-157
Hybrid myeloma cells which secrete hetero A model to study the /V-linked glyca S-O. LEE AND R. D. PORETZ Department of Molecular Biology and Biochemistry. Ruiger.s University. Piscataway, NJ, USA (Submitted 4 January 1991. .Accepted for publication 9 April 1991.) Fundamental questions remain unanswered regarding ihc elTec! of ihe acceptor polypeplidcslruclure on thefinestructure of the A'-linkcd glycan of glycoprotcins and conversely, the effect of the glycan structure of IgG on Ihe function and slruelure of the protein. The construction of myeloma hybrids capable of secreting multiple IgG which differ with regard to the tine structure of their /V-linked oligosaccharides would be a valuable model for studying these questions. P3X63Ag8 secretes an IgGi which possesses an oligosaccharide at Asn^'*'' that differs in fine strueture from the analogous glycan of the igGjb secreted by Sp2/HLBu. Fusion hybrids of these cells secrete parental IgGj. and to a lesser degree IgGjb. as well as a heterodimeric IgG containing both ihe 7i and Y2h chains. The oiigosaecharide of each chain is identical in structure to the appropriate parental IgG. Such cells allow for the analysis of acceptor properties that inffuence glycan fine structure, as well as the role of glycan structure on the stability of the IgG.
INTRODUCTION Somatic cell fusion involving myeloma cells, though originally performed lo explore the regulation of immunoglobulin biosythesis. has been used in the past decade primarily for the production of monoclonal antibodies (1). Nevertheless, questiotis continue to arise regarding the nature of ihe factors which influence the structure and synthesis of immunoglobulins. In analogy with other secreted giycoproteins, the heavy chain of IgG is translocated across the endoplasmic reticulum membrane and glycosylated at Asn-^^ with a mannose rich oligosaccharide (2). During migration of the Y chain from this subcellular compartment through the Golgi apparatus, the IgG is completely assembled and the glycan moiety undergoes extensive processing and restructuring. One of the final steps in oligosaccharide processing occurring in the trans-Golgi involves the addition of galactose to the termini of the glycan unit (2). The oligosaccharide at the conserved site of
glycosylation ofthey chain is a relatively simple structure, but has been implicated in numerous functions and properties of IgG. Some effector functions associated with the presence of the glycan include IgG activation of complement (3.4), antibody-dependent induction of cytotoxicity (3) and IgG binding to the Fc receptor of monocytes (3.4). The presence and specific structure of the glycan moiety maybe important in maintaining the spatial relationship of the two heavy chain subunits of the assembled immunoglobulin (5.6). Recent reports have described the high correlation between the presence of abnormally high levels of IgG lacking terminal galactose residues on the oligosaccharide at Asn-**' and the diagnosis of rheumatoid arthritis in individuals suffering from the disease (7.8). Accordingly, a better understanding of the factors that influence the biosynthesis of IgG bound glycan units may shed light on specific disease states related to IgG functions. The role of specific cell type which defines the nature, quantity and intracellular compartmentaliza-
Correspondence: Dr R. D. Poretz, Department of Molecular Biology and Biochemistry. PO Box 1059. Rutgers University, Piscataway. NJ 08855-1059. USA. Ahhrexiaiiotu used in this paper: Asn. asparagine; DMEM, Dulbecco's modified Eagle's medium; ECGS. endothelial cell growth supplement; HAT, hypoxanthino-aminopicrin-thymidine; HT. hypoxanthine-thymidine; IgG, immunoglobulin G; PAGE, polyacrylamide gel eleetrophoresis; PEG. polyethylene glycol; SDS. sodium dodeeylsulfate.
S-O. LEE A N D R. D. PORETZ
t52
tion of individual glycosyl transferases and glycosidases in determining the strueture of the glycans of giycoproteins in doeumented (9-13). Similarly there have been reports that the acceptor polyjKptideinfluenees the degree and nature of processing of the glycan unit of glycoproleins (14-16). A caveat, however, upon these latter studies is that ditferent proteins, synthesized within the same cell, may enter different intracellular compartments and/or exhibit dilferential rates of synthesis. Prior to studies (17) based upon the IgG produced by the hybrid cells reported in this paper, there had been no repoil on the effect of polypeptide primary structure on the fine structure of the glycan moieties of two functionally and structurally similar subunits of a single protein in which both subunits possess the same glycosylation site. Cloned, hybrid myeloma cell lines represent an ideal system to explore the influence of y ehain amino acid sequence on the fine stt^cture of the Asn-^^ glyean. This paper describes the construction and properties of hybrid cell lines that simultaneously secrete multiple IgG composed of either identical or different y chains. The heavy chains are of ditferent subelasses and possess dissimilar glycan units at the conserved site of glycosylation. These cell lines were constructed by fusion of a myeloma and a hybridoma. produeing IgG of different subclasses, each with a di-branched glycan moiety, differing in the quantity and nature of terminal galactosylation (17). Sueh hybrid cell lines and their 'parental' lines may serve as a valuable model to explore the influence of fine structure of the earbohydrate at .\sn-''^ on the strueture and function of IgG.
MATERIALS AND M E T H O D S Cells and media Fusions were performed with the myeloma cell line P3X63Ag8. an azaguanine resistant P3 clone derived from MOPC-21 which secretes an IgG 1. and a hybridoma which secretes an IgGib. Sp2/HLBu. obtained from a fusion of P3X63Ag8 and a murine splenocyte and selected for resistance to 5-bromouridine. Both cell lines were developed in the laboratory of Dr C. Milstein (18) and were a gift from Dr N. Cowan. These cells and their hybrids were cultured In vitro using Dulbecco's modified Eagle's medium (DMEM) (Gibco. Grand Island. NY. USA) supplemented with 10% fetal calf serum (Gibco,
Grand Island. NY. USA), non-essetitial amino acids (Gibco. Grand Island, NY. USA), glutamine. penicillin-streptomycin, under an atmosphere of 5% CO;. Cell fusion and detection of clones The cell fusion was performed aecording to the method of Pearson et al. (19). Cells (I X 10*') of each cell line were combined and mixed with 0.3 mL of 35% polyethylene glycol (PEG 1000. Sigma Chemical Co.. St Louis. MO. USA) and centrifuged. After 8 min at 37°, the eell mixture was diluted with 5 mL of DMEM. centrifuged. washed with DMEM and suspended in 200 mL of HAT medium eontaining 100 ^ig/mL of endothelial cell-growth supplement (ECGS. Collaborative Research Inc.. Bedford, MA. USA) (20). One hundred microlitres of appropriately diluted cell suspension were placed into each well of 96-well plates. Three to four days later, cells were fed with HT medium and allowed to grow for an additional 2 weeks. The supernatants were removed from eaeh well and subjected to immuno-dot blot analysis using anti-IgG serum (Miles Laboratories Inc.. Kankakee. IL. USA). Cells of randomly selected wells, positive for IgG. were subeloned on plates containing l%Sea Plaque agarose (FMC Bioproducts. Rockland, ME. USA) and ECGS in HT medium as described by others (21.22). After the clones reached a visible growth, multiple nitrocellulose membrane biois were obtained and probed with rabbit subclass specific antiserum (Litton Bionetics. Charleston. SC. USA); one membrane with anti-lgG| and another with antl-IgG2b serum. Positive reactions were detected with horse-radish peroxidase-conjugated anti-rabbit IgG and 4-chloro-l-naphthol. Comparisons of matching membranes developed with the different subclass specific antibody allowed selection of clones secreting both IgG subclasses. Following expansion of the selected clones in suspension culture, the cells (2-4 X lO**) were injected intraperitoneally into pristane-primed Balb/c female mice resulting in the production of ascitic Huids.
The purification of/gG To isolate individual subclasses of IgG, ascites fluid was clarified by centrifugation at 1000 .i? for 10 min, mixed with an equal volume of I.5mol/L glycine buffer (pH 8.9) containing 3 mol/L NaC'l and 0.01% NaN^ and applied to a protein A-Sepharose column (0.5 cm X 5 cm)
HETERODIMERIC IgG SECRETING CELLS
(Pharmacia. Piscataway. NJ, USA). The column was washed with 40 mL of the same buffer and eluted sequentially with O.I mol/L citrate buffers of pH 6.0, 5.5.4.5 and 3.5 containing 0.01% NaN.i as described by Ey et al. (23). The pH 4.5 and 3.5 eluants of the eolumn were colleeted in tubes (2 mL) containing I mL of 0.1 mol/L borate buffer. pH 9.0. Following optical density measurements at 280 nm. the appropriate fractions were combined, dialysed against H2O,and lyophilized. Immuno-eleclrophoresis Agarose eleetrophoresis was conducted as described elsewhere (24) using O.Oi mol/L barbital buffer (pH 8.6). Upon completion of eleetrophoresis. the agarose plates were fixed with 10% trichloroacetic acid solution containing 3.5% sulfosalicylic aeid and stained with Coomassie Blue R. Immunodijfusion tests Double immunodifTusion was conducted in agarose as described by Ouchterlony (25). SDS-page Gels and samples were prepared, and the eleetrophoresis was conducted according to the conditions of Laemmli and Favre (26). The separating gel contained 9% acrylamide and 0.25% jV.A'-methylenebisacrylamide. and the stacking gel contained 4% acrylamide and 0.1% A'.iV'methylenebisacrylamide. The electrophoretograms werefixedand stained as described above for immuno-electrophoresis. Gelfiltrationchromatography One-half millilitre of sample or standards (1 mg/mL) was applied in 0.05 mol/L potassium phosphate buffer (pH 6.8) containing 0.1 mol/L NaCI and 0.02% NaN3 to a column (0.9 cm X 150 cm) of Sephadex G-200 (Pharmacia. Piscataway, NJ, USA) and eluted with the same buffer at a flow rate of 4 mL/h. The optical density of individual fractions (1.5 mL) was determined at 280 nm. The standards employed were: ferritin (450 000) (27): rabbit IgG (155 000) (28): and bovine serum albumin (68 000) (29), as well as Blue Dextran (Pharmacia, Piscataway, NJ, USA).
153
RESULTS AND DISCUSSION Fusion hybrids of P3X63Ag8 and Sp2/HLBu were constructed and seeded at a limiting dilution of 0.5 ceils/well in 96-well plates. Following subeloning in soft agar of IgG producing eells. 36 colonies showed secretion of reactive material to both anti-lgGi and antiIgGab sera and were selected and expanded. These colonies were each on plates containing fewer than 20 colonies per 48 mm diameter plate, were physically separated from neighbouring colonies and appeared to have circular symmetry of growth. These characteristics were taken as additional assurance of the monoclonal nature of the selected eolonies. Though many of the colonies exhibited an unequal reactivity with the two subclass specific antisera. three hybrid subelones, with apparent equal reactivity with the antisera and originating from different wells of the initial plating, were chosen for further analysis. Each clone was grown as an ascitic form in BALB/c mice and the IgG from the fluid so developed was isolated by protein A-Sepharose affinity adsorption. It is evident from the elution profiles (Fig. 1) obtained with the fluid produeed by clone 27-1 and both parental cell lines that the hybrid cell secretes both lgG|- and IgG2b-like proteins. Fluid from all tfie fused constructs produced similar profiles with the protein A affinity eolumn. The purity of each IgG pool separated by protein A adsorption is shown by agarose eleetrophoresis (Fig. 2). A single protein-staining region with a mobility corresponding to that exhibited by the appropriate immunoglobulin present in the ascitic fluid from the parental cells is obtained from the pH 6 and 3.5 eluates. In all cases the protein band of the purified IgG from the fused cells was more diffuse and exhibited a decreased mobility as compared with tbe IgG of the parental ceils. Presumably, this is due to the random association of the light chains derived from each parent with the heavy chains of both subclasses (1). This possibility is made more evident from the SDS-PAGE of the purified IgGs. Figure 3 demonstrates that the light chains of both parents are found associated with both the 71 and yih ehains of the fused cell products. Furthermore the pH 3.5 eluate contains both yi andyibchains. Similar results were obtained with the pH 3.5 pool from the protein A-Sepharose affinity adsorption of the ascites fluid produeed by all three fused eell lines studied. Re-adsorption of the protein in the pH 3.5 pool to a protein A-Sepharose eolumn resulted in protein being eluted only with the pH 3.5 buf-
S-O. LEE AND R. D. PORETZ
154
.£)
' ostcoarthritis with changes in the glycosylation pattern of total serum IgG. NatureiW. 452-457. 8. Parekh, R. B.. Dwek, R. A. and Rademacher, T. W. 1988, Rheumatoid arthritis as a glycosylation disorder. Br. J. Rheum. 27 (Suppl. 11): 162169. 9. Hsieh. P., Rosner. M. R. and Robbins, P, W. 1983. Host-dependent variation of asparaginelinked oligosaccharides at individual glycosylation sites of Sindbis virus giycoproteins. J. Biol, Chem. 258: 2548-2554. 10. Williams. D.B. and Lennarz.W. 1984. Control of asparaginc-linked oligosaccharide chain processing: studies on bovine pancreatic ribonuclease B. J. Biol. Chem. 259: 5105-5114. 11. Taylor, A.K. and Wall. R. 1988. Selective removal of heavy-chain glycosylation sites causes
HETERODIMERIC IgG SECRETING CELLS
12.
13.
14.
15.
16. 17.
18.
19.
20.
21.
22.
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