Mapping rheumatoid factor binding sites using genetically engineered, chimeric IgG antibodies.
We are using chimeric IgG antibodies consisting of murine variable regions joined to human constant regions as rheumatoid factor (RF) binding substrates to localize and map IgM RF binding sites on IgG. Using chimeric antibodies in a modified RF ELISA, we showed that RFs from rheumatoid arthritis (RA) and Waldenstrom's macroglobulinemia (WMac) patients differ in their binding specificities for IgG3, although some of these RFs share common specificity for IgG1, IgG2, and IgG4. By shuffling constant region domains between IgG3 and IgG4, we showed that sequence variation in the CH3 domain is responsible for WMac-derived RF differentiation of IgG3 and IgG4. By making site-directed mutations in the wild-type IgG3 or IgG4 human gamma constant genes, we showed that His-435 is an essential residue in RF binding to IgG for most WMac RFs. The allotypic polymorphism in IgG3 at 436 is not responsible for differences in previous reports of high-frequency IgG3 binding by WMac RFs. A amino acid loop in the CH2 domain of IgG4 proximal to the CH2-CH3 interface is important in WMac RF binding to IgG; a more distal CH2 loop in CH2 has a more variable effect on WMac RF binding. To evaluate the contribution of the N-linked carbohydrate moiety at Asn-297 to RF binding sites on IgG, we measured RF binding to aglycosylated IgG antibodies produced by mutating the glycosylation signal Asn-297 to another amino acid. Of all four IgG subclasses, only aglycosylated IgG3 was a better RF binding substrate than its glycosylated subclass counterpart.(ABSTRACT TRUNCATED AT 250 WORDS)
Bonagura
V R
VR
Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Artandi
S E
SE
Agostino
N
N
Tao
M H
MH
Morrison
S L
SL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002678:Chimera; D005818:Genetic Engineering; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000378:metabolism*; D007075:Immunoglobulin M / Q000235:genetics / Q000378:metabolism; D051379:Mice; D009154:Mutation; D012217:Rheumatoid Factor / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D012217:Rheumatoid Factor