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 iii-linked carbohydrate structure attached to the C(H)2 domain of each of its heavy chains. In addition, the variable re gions of IgG often contain potential N-linked carbohydrate addition sequenc es that frequently result in the attachment of V region carbohydrate, Nonet heless, the precise role of this V region glycan remains unclear, Studies f rom our laboratory have shown that a naturally occurring somatic mutant of an anti-dextran Ab that results in a carbohydrate addition site at Asn(58) of the V-H has carbohydrate in the complementarity-determining region 2 (CD R2) of the V-H and the presence of carbohydrate leads to an increase in aff inity. However, carbohydrate attached to nearby positions within CDR2 had v ariable affects on affinity, In the present work we have extended these stu dies 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 t he novel addition sites, but the extent of glycosylation varies with the po sition of the site. In addition, we find that the position of the variable region carbohydrate influences some functional properties of the Ab, includ ing those usually associated with the V region such as affinity for Ag as w ell as other characteristics typically attributed to the Fc such as half-li fe and organ targeting, These studies suggest that modification of variable region glycosylation provides an alternate strategy for manipulating the f unctional attributes of the Ab molecule and may shed light on how changes i n carbohydrate structure affect protein conformation.