Position effects of variable region carbohydrate on the affinity and in vivo behavior of an anti-(1-->6) dextran antibody.
IgG is a glycoprotein with an N-linked carbohydrate structure attached to the CH2 domain of each of its heavy chains. In addition, the variable regions of IgG often contain potential N-linked carbohydrate addition sequences that frequently result in the attachment of V region carbohydrate. Nonetheless, the precise role of this V region glycan remains unclear. Studies from our laboratory have shown that a naturally occurring somatic mutant of an anti-dextran Ab that results in a carbohydrate addition site at Asn58 of the VH has carbohydrate in the complementarity-determining region 2 (CDR2) of the VH, and the presence of carbohydrate leads to an increase in affinity. However, carbohydrate attached to nearby positions within CDR2 had variable affects on affinity. In the present work we have extended these studies by adding carbohydrate addition sites close to or within all the CDRs of the same anti-dextran Ab. We find that carbohydrate is attached to all the novel addition sites, but the extent of glycosylation varies with the position of the site. In addition, we find that the position of the variable region carbohydrate influences some functional properties of the Ab, including those usually associated with the V region such as affinity for Ag as well as other characteristics typically attributed to the Fc such as half-life and organ targeting. These studies suggest that modification of variable region glycosylation provides an alternate strategy for manipulating the functional attributes of the Ab molecule and may shed light on how changes in carbohydrate structure affect protein conformation.
Coloma
M J
MJ
Department of Microbiology, Immunology, and Molecular Genetics, The Molecular Biology Institute, University of California, Los Angeles 90095, USA.
Trinh
R K
RK
Martinez
A R
AR
Morrison
S L
SL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000493:pharmacokinetics*; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D003911:Dextrans / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006031:Glycosylation; D006207:Half-Life; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007274:Injections, Intraperitoneal; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed