title: Increased avidity of mutant IgM antibodies caused by the absence of COOH-terminal glycosylation of the mu H chain.
abstract: We have previously described the isolation of two hybridoma variants secreting higher avidity IgM (D5 and 7F5), starting from the E11 hybridoma cell line, which produces an antibody specific for the A Ag of the ABO blood group system. In order to explain at the molecular level this increased reactivity, cDNA encoding the H and L chains of the E11, D5, and 7F5 mAb were cloned and sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAb produced by the hybridoma variants. The mutations were both located in the H chain C region and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at position 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence (Asn-X-Ser/Thr) located in the tail piece of the secretory mu-chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the [14C]mannose-labeled mAb. The two single point mutations were solely responsible for the increased avidity of the antibodies, as confirmed by site-directed mutagenesis of the E11 mu-chain and serologic analysis of the mutated E11 antibodies. We conclude that the absence of glycosylation at Asn 563 is responsible for the increased avidity of the mutant, possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the H chain C region can influence the reactivity of IgM mAb.
authors.affiliation: Canadian Red Cross Society, Blood Services, Quebec City, P. Qc.
authors.forename: R
authors.initials: R
authors.lastname: Bazin
authors.forename: A
authors.initials: A
authors.lastname: Darveau
authors.forename: F
authors.initials: F
authors.lastname: Martel
authors.forename: A
authors.initials: A
authors.lastname: Pelletier
authors.forename: L
authors.initials: L
authors.lastname: Piché
authors.forename: M
authors.initials: M
authors.lastname: St-Laurent
authors.forename: L
authors.initials: L
authors.lastname: Thibault
authors.forename: A
authors.initials: A
authors.lastname: Demers
authors.forename: L
authors.initials: L
authors.lastname: Boyer
authors.forename: G
authors.initials: G
authors.lastname: Lemieux
mesh_terms: D000595:Amino Acid Sequence; D000915:Antibody Affinity; D000917:Antibody Formation; D001483:Base Sequence; D003001:Cloning, Molecular; D006031:Glycosylation; D006801:Humans; D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M / Q000276:immunology*; D007148:Immunoglobulin mu-Chains / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid
chemical_list: D007147:Immunoglobulin Light Chains; D007075:Immunoglobulin M; D007148:Immunoglobulin mu-Chains