BIOCHEMICAL AND GENETIC STUDIES OF ANTIBODY (IMMUNOGLOBULIN-M) PRODUCING CELLS (GLYCOPROTEINS, U-CHAIN, HYBRIDOMAS).

Abstract

We have chosen the murine immunoglobulin M (IgM) as system to study glycoprotein biosynthesis and carbohydrate processing. Secreted IgM heavy chain (m) has five glycosylation sites which location and structures have been determined. m chain variable region (VH) is involved in antigen binding, while the constant region (CH) is responsible for the effector functions in which the carbohydrate plays an important role. We have determined the carbohydrate structures present at each glycosylation site of IgM produced by a hybridoma cell line (PC 700) and its derived mutants and compared them to IgM from myeloma cell MOPC 104E. PC 700 mutants secrete altered IgM. The alterations include: deletion of one or more constant domains (mutants: 128, 313, and 562) and m chain hyperglycosylation (mutants 21 and 38). Gene analysis indicated that deletions can arise from two different mechanisms. One of these involve a major gene change (mutant 128), while others come from base point mutations (mutants 313 and 562). Cells 21 and 38 did not appear to have m gene insertions. Determination of purified single glycosylation site structures show that PC 700 m chain is processed only to biantennary. Heavy chain protein fragmentation and carbohydrate studies indicate that mutants 21 and 38 alterations are due to an increase in oligosaccharide processing and reduction of unprocessed structures. There is a trend of processing going from PC 700 < 21 < 38. In addition, our results show how growth cell conditions can affect the carbohydrate processing without altering the determinants of m chain oligosaccharide structures. Studies on the IgM molecule illustrate the need for precisely define structure-function relationships. This would allow the selection of the best antibodies for studies such as those involved in immunotherapy.