Mechanism of Ca2+ and monosaccharide binding to a C-type carbohydrate-recognition domain of the macrophage mannose receptor
Mechanism of Ca2+ and monosaccharide binding to a C-type carbohydrate-recognition domain of the macrophage mannose receptor
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DOI：
10.1074/jbc.272.9.5668
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发表时间：
1997-02-28
影响因子：
4.8
通讯作者：
Taylor, ME
中科院分区：
文献类型：
--
作者：
Mullin, NP;Hitchen, PG;Taylor, ME
定点诱变已被用来确定残基连接钙和糖的第四个C型碳水化合物识别结构域（CRD）的巨噬细胞甘露糖受体。CRD-4是甘露糖受体的八个CRD中唯一一个在分离表达时表现出可检测的单糖结合的CRD，并且它是受体结合配体的核心。CRD-4需要两个Ca 2+来结合糖，就像大鼠血清甘露糖结合蛋白（MBP-A）的CRD一样。两个CRD之间的序列比较表明，直接连接到MBP-A中的结合糖的一个Ca 2+的结合位点在CRD-4中是保守的，但辅助Ca 2+结合位点不是。CRD-4中与MBP-A中辅助Ca ~（2+）结合位点相当的四个残基的突变表明，只有Asn（728）参与Ca ~（2+）的连接。丙氨酸扫描突变用于鉴定可能参与辅助Ca ~（2+）与CRD-4连接的另外两个天冬酰胺残基和一个谷氨酸残基。与其他C型CRD的序列比较表明，所提出的甘露糖受体的CRD-4中辅助Ca 2+的结合位点是独特的。CRD-4中保守的Ca 2+以类似于MBP-A的方式在蛋白质和结合糖之间桥接的证据是通过在该位点的氨基酸侧链之一的突变获得的。在CRD-4和定点诱变存在下，甘露糖、GlcNAc和岩藻糖的甲基糖苷的H-1 NMR光谱中观察到的环电流位移表明，与Tyr（729）的堆积相互作用也参与了糖与CRD-4的结合。这种相互作用贡献了与甘露糖结合的总自由能的约25%。甘露糖的C-5和C-6与Tyr（729）相互作用，而GlcNAc的C-2最接近该残基，表明这两种糖以相反的方向与CRD-4结合。与其他甘露糖/GlcNAc特异性C型CRD的序列比较表明，在这些糖的结合中使用堆积相互作用可能是甘露糖受体的CRD-4所独有的。
Site-directed mutagenesis has been used to identify residues that ligate Ca2+ and sugar to the fourth C-type carbohydrate-recognition domain (CRD) of the macrophage mannose receptor. CRD-4 is the only one of the eight CRDs of the mannose receptor to exhibit detectable monosaccharide binding when expressed in isolation, and it is central to ligand binding by the receptor. CRD-4 requires two Ca2+ for sugar binding, like the CRD of rat serum mannose-binding protein (MBP-A). Sequence comparisons between the two CRDs suggest that the binding site for one Ca2+, which ligates directly to the bound sugar in MBP-A, is conserved in CRD-4 but that the auxiliary Ca2+ binding site is not. Mutation of the four residues at positions in CRD-4 equivalent to the auxiliary Ca2+ binding site in MBP-A indicates that only one, Asn(728), is involved in ligation of Ca2+, Alanine-scanning mutagenesis was used to identify two other asparagine residues and one glutamic acid residue that are probably involved in ligation of the auxiliary Ca2+ to CRD-4. Sequence comparisons with other C-type CRDs suggest that the proposed, binding site for the auxiliary Ca2+ in CRD-4 of the mannose receptor is unique. Evidence that the conserved Ca2+ in CRD-4 bridges between the protein and bound sugar in a manner analogous to MBP-A was obtained by mutation of one of the amino acid side chains at this site. Ring current shifts seen in the H-1 NMR spectra of methyl glycosides of mannose, GlcNAc, and fucose in the presence of CRD-4 and site-directed mutagenesis indicate that a stacking interaction with Tyr(729) is also involved in binding of sugars to CRD-4. This interaction contributes about 25% of the total free energy of binding to mannose. C-5 and C-6 of mannose interact with Tyr(729), whereas C-2 of GlcNAc is closest to this residue, indicating that these two sugars bind to CRD-4 in opposite orientations. Sequence comparisons with other mannose/GlcNAc-specific C-type CRDs suggest that use of a stacking interaction in the binding of these sugars is probably unique to CRD-4 of the mannose receptor.