Co-secretion of two distinct kappa light chains by the mu-9 hybridoma.
Mu-9 is a monoclonal antibody (MAb) specific for the CSAp antigen (Ag) expressed by colorectal cancers. By using variable (V)-region-specific primers, the respective VH and VL sequences of Mu-9 were polymerase chain reaction (PCR)-amplified. However, chimeric Ab (cMu-9-1) constructed from these PCR-amplified V sequences failed to bind the CSAp Ag. Although the light chain of murine Mu-9 was not glycosylated, that of cMu-9-1 was found to be O-glycosylated, as confirmed by reducing SDS-PAGE analyses, glycoprotein blotting and O-linked specific deglycosylation studies. Removal of O-linked oligosaccharides either by enzymatic digestion or by blocking O-glycosylation with a specific inhibitor did not restore the immunoreactivity of cMu-9-1, indicating that light chain O-glycosylation was not the cause for lack of immunoreactivity. We reported earlier that screening of a Mu-9 cDNA library uncovered the presence of an additional light chain sequence that was later proven to be the authentic light chain of Mu-9. Analyses of the cDNA sequence encoding the nonimmunoreactive light chain, however, revealed no defects that would preclude the sequence from being translated and secreted by the murine hybridoma. By adapting the Mu-9 hybridoma culture to serum-free conditions, we confirmed the secretion of low levels of O-glycosylated light chain. The biological significance of the O-glycosylation as well as the cosecretion of both light chains with respect to allelic exclusion are discussed.
Krishnan
I S
IS
Immunomedics Inc., Morris Plains, NJ 07950, USA.
Hansen
H J
HJ
Gold
D V
DV
Goldenberg
D M
DM
Leung
S O
SO
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000915:Antibody Affinity; D000951:Antigens, Neoplasm / Q000276:immunology; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D015179:Colorectal Neoplasms / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D015723:Gene Library; D006031:Glycosylation; D006801:Humans; D006825:Hybridomas / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007145:Immunoglobulin kappa-Chains; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate