Influence of asparagine-linked oligosaccharides on antigenicity, processing, and cell surface expression of herpes simplex virus type 1 glycoprotein D.
Glycoprotein D (gD) is an envelope component of herpes simplex virus types 1 and 2. gD-1 contains three sites for the addition of N-linked carbohydrate (N-CHO), all of which are used. Three mutants were constructed by site-directed mutagenesis, each of which altered one N-CHO addition site from Asn-X-Thr/Ser to Asn-X-Ala. A fourth mutant was altered at all three sites. The mutant genes were inserted into an expression vector, and the expressed protein was analyzed in transiently transfected COS-1 cells. The mutant protein lacking N-CHO at site 1 (Asn-94) had a reduced affinity for monoclonal antibodies (MAbs) to discontinuous epitopes, suggesting that the conformation of the protein had been altered. However, the protein was processed and transported to the cell surface. The absence of N-CHO at site 2 (Asn-121) had no apparent effect on processing or transport of gD-1 but resulted in reduced binding of two MAbs previously shown to be in group VI. Binding of other MAbs to discontinuous epitopes (including other group VI MAbs) was not affected. The absence of N-CHO at site 3 (Asn-262) had no effect on processing, transport, or conformation of the gD-1 protein. The absence of N-CHO from site 1 or from all three sites resulted in the formation of high-molecular-weight aggregates or complexes and a reduction in MAb binding. However, these proteins were modified by the addition of O-glycans and transported to the cell surface. We conclude that the absence of the first or all N-linked carbohydrates alters the native conformation of gD-1 but does not prevent its transport to the cell surface.
Sodora
D L
DL
Department of Microbiology, University of Pennsylvania, Philadelphia 19104-6003.
Cohen
G H
GH
Eisenberg
R J
RJ
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface / Q000032:analysis / Q000235:genetics; D000956:Antigens, Viral / Q000032:analysis / Q000235:genetics*; D001216:Asparagine; D015153:Blotting, Western; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D009154:Mutation; D009844:Oligosaccharides / Q000235:genetics* / Q000276:immunology; D018139:Simplexvirus / Q000235:genetics* / Q000276:immunology; D014162:Transfection; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000956:Antigens, Viral; D009844:Oligosaccharides; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1; D001216:Asparagine