Glycosylation of FcγRIII in N163 as mechanism of regulating receptor affinity
Glycosylation of FcγRIII in N163 as mechanism of regulating receptor affinity
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DOI：
10.1046/j.1365-2567.2003.01743.x
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发表时间：
2003-11-01
期刊：
影响因子：
6.4
通讯作者：
Schmidt, RE
中科院分区：
文献类型：
--
作者：
Drescher, B;Witte, T;Schmidt, RE
人FcGammaRIII(CD16)是免疫球蛋白G(Ig G)的低亲和力受体。该蛋白有两种不同的亚型：CD16a(跨膜型，表达在自然杀伤细胞和巨噬细胞上)和CD16b(糖基化磷脂酰肌醇连接，以两种等位基因NA1和Na2表达在中性粒细胞上)。这两种形式的蛋白质都有不同的糖基化模式。CD16B的NA1等位基因有4个天冬酰胺(N)连接的糖基化位点。其中一个(N163)定位于结构域II的配体结合部位，该部位由Na2等位基因和CD16A所共有。为了研究糖基化的功能作用，我们将NA1等位基因的四个糖基化位点(N39、N75、N163、N170)突变为谷氨酰胺(Q)。以野生型CD16为对照，将单个突变体稳定转染HEK293细胞。我们用抗人免疫球蛋白抗体进行免疫荧光研究，确定了人免疫球蛋白与转基因细胞的结合。单体免疫球蛋白与N163Q转染体的亲和力高于与其他转染体的亲和力，表明N163上的糖基化影响CD16与其配体的亲和力。此外，WT-CD16转基因细胞与衣霉素(N-糖基化的抑制剂)预温育后，单体免疫球蛋白结合增加，而N163Q-CD16转基因细胞未受影响。因此，N163的糖基化是调节FcGammaRIII与其配体免疫球蛋白亲和力的机制之一。
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.