Glycosylation of FcgammaRIII in N163 as mechanism of regulating receptor affinity.
Human FcgammaRIII (CD16) is a low-affinity receptor for immunoglobulin G (IgG). There are two different isoforms of this protein: CD16a (transmembranous, expressed on natural killer cells and on macrophages) and CD16b (glycosylphosphatidylinositol-linked, expressed on neutrophilic granulocytes in two allelic forms NA1 and NA2). Both forms of the protein have a variable glycosylation pattern. The NA1 allele of CD16B has four asparagine (N)-linked glycosylation sites. One of them (N163) is localized in the ligand-binding site of domain II. This site is shared by the NA2 allele and CD16A. To examine the functional role of the glycosylation we mutated the four glycosylation sites of the NA1 allele (N39, N75, N163, N170) into glutamine (Q). HEK293 cells were stably transfected with the single mutants and wild-type CD16 as control. We determined binding of human IgG to transfected cells using immunofluorescence studies with anti-human IgG antibody. Monomeric IgG bound to N163Q transfectants with higher affinity than to other transfectants, showing that glycosylation in N163 influences the affinity of CD16 to its ligand. In addition, preincubation of WT-CD16-transfected cells with Tunicamycin (an inhibitor of N-glycosylation) resulted in an increased binding of monomeric IgG whereas N163Q-CD16-transfected cells remained unaffected. Therefore, glycosylation in N163 is a mechanism of regulating affinity of FcgammaRIII to its ligand IgG.
Drescher
Bettina
B
Department of Clinical Immunology, Hannover Medical School, Carl-Neuberg-Str 1, D-30625 Hannover, Germany.
Witte
Torsten
T
Schmidt
Reinhold E
RE
D000915:Antibody Affinity / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D018604:Epitope Mapping; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D017452:Receptors, IgG / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D007074:Immunoglobulin G; D017452:Receptors, IgG; D014415:Tunicamycin