Glycosylation of the variable region of immunoglobulin G--site specific maturation of the sugar chains.
The structure of the N-linked sugar chains attached to three IgG antibodies, identical in amino acid sequence except for the changes required to introduce the carbohydrate addition sites, has been determined. All three antibodies are specific for dextran but differ in their ability to bind antigen. The heavy chains with a murine variable region (V region) attached to the human gamma 4 constant region were expressed in a murine hybridoma synthesizing the specific light chain. In addition to the glycosylation site in the Fc portion, each antibody has a different glycosylation site in the second complementarity determining region (CDR2) of the heavy chain (Asn54, Asn58, or Asn60). The sugar chains were released from purified Fab and Fc fragments by hydrazinolysis and converted to radioactive oligosaccharides by reduction with sodium borotritide. The structures of these radioactive oligosaccharides were determined by a combination of sequential exoglycosidase digestion and Bio-Gel P-4 and lectin column chromatography. For all three antibodies, the carbohydrate attached to the Fc portion was a mixture of complex-type biantennary sugar chains. The variable region carbohydrate structures attached at Asn54 and Asn58 were also complex-type but more highly sialylated than were the Fc-associated sugars. Moreover, unlike the Fc-associated sugars, a significant population of Fab-associated sugars contained a Gal alpha 1-->3 residue as a non-reducing terminus. In contrast, the carbohydrate attached at Asn60 was a high mannose structure. These results demonstrate that slight changes in the position of carbohydrate attachment within CDR2 of the variable region of the heavy chain can substantially alter carbohydrate processing and that complex-type carbohydrates contained within the same polypeptide chain can have different structures.
D000595:Amino Acid Sequence; D000818:Animals; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D000939:Epitopes; D006031:Glycosylation; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data
D002241:Carbohydrates; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D007135:Immunoglobulin Variable Region