Structural requirements for IgM assembly and cytolytic activity. Effects of mutations in the oligosaccharide acceptor site at Asn402.
Glycosylation of IgG occurs at asparagine 297 of the gamma H chain and is necessary for the normal capacity of IgG to activate the classical pathway of complement-dependent cytolysis. IgM is glycosylated at five sites in the constant region of the mu H chain, of which glycosylation at asparagine 402 seems analogous to the glycosylation of IgG. In order to assess the importance of glycosylation at asparagine 402 for IgM cytolytic activity, we have used site-directed mutagenesis to produce IgM which is not glycosylated at this position. In particular we have tested the effects of substituting Gln for Asn 402 and Thr-Gly for Gly 403-Thr 404 in the third constant region domain. We tested the effects of these substitutions by expressing the mutant mu genes in hybridoma cells which produce the hapten-specific kappa-chain. The normal mu-chain is glycosylated at Asn 402, and, as expected, these mutations appear to abrogate glycosylation of the mutant mu-chains at position 402 and do not affect the hapten affinity of the IgM. However, both of these mutations cause the increased production of monomeric rather than polymeric IgM: the ratio of monomeric to polymeric IgM is 0.21, 3.5, and 10.3 for wild-type IgM, IgM-Gln 402, and IgM-Thr 403-Gly 404, respectively. The wild-type and mutant polymeric IgM preparations were compared for their capacity to promote complement-dependent cytolysis: IgM-Gln 402 and IgM-Thr 403-Gly 404 have approximately 31% and 4%, respectively, of the capacity of wild-type IgM.
Muraoka
S
S
Department of Immunology, University of Toronto, Canada.
Shulman
M J
MJ
D000595:Amino Acid Sequence; D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D001216:Asparagine / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003171:Complement Pathway, Classical; D004252:DNA Mutational Analysis / Q000379:methods; D006031:Glycosylation; D006825:Hybridomas / Q000378:metabolism; D007075:Immunoglobulin M / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics* / Q000502:physiology; D013329:Structure-Activity Relationship
D007075:Immunoglobulin M; D015345:Oligonucleotide Probes; D001216:Asparagine