Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis
Recognition of saccharides by the OpcA, OpaD, and OpaB outer membrane proteins from Neisseria meningitidis
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DOI：
10.1074/jbc.m506354200
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发表时间：
2005-09-09
影响因子：
4.8
通讯作者：
Derrick, JP
中科院分区：
文献类型：
--
作者：
Moore, J;Bailey, SES;Derrick, JP
脑膜炎奈瑟菌与宿主细胞表面蛋白多糖的黏附由外膜蛋白OPCA和OpA介导，在细菌的侵袭和侵袭过程中起着重要作用。然而，OPCA和OpA蛋白的确切特异性尚不清楚。在这里，我们使用基于荧光的结合分析来表明这两种蛋白质都以高亲和力与单糖和双糖结合。糖的结合导致两种蛋白质的内源荧光发射减弱，而外环区域内选定的Tyr残基的突变导致荧光显著下降。我们认为本征荧光来自于结构的β-桶部分从Tyr到Trp残基的共振能量转移。OPCA结合唾液酸的K-d为0.31µM，并被证明是吡喃糖的专一性。比较了两种不同的OpA蛋白的结合特性；与OPCA不同，两种蛋白都不与单糖结合，但都与麦芽糖、乳糖和含唾液酸的寡糖结合，K-d值在微摩尔范围内。由于突变Y165V，OpaB对含唾液酸配体的亲和力是OPAD的10倍，这表明OpaB恢复了OPAD的这种特异性。最后，外源添加唾液酸和麦芽糖可部分抑制OPCA和OpA依赖的脑膜炎双球菌与上皮细胞的黏附。结果表明，OPCA和OpA蛋白可以被认为是外膜凝集素，简单的糖可以调节它们对复杂的蛋白多糖受体的识别。
The adhesion of the pathogen Neisseria meningitidis to host cell surface proteoglycan, mediated by the integral outer membrane proteins OpcA and Opa, plays an important part in the processes of colonization and invasion by the bacterium. The precise specificities of the OpcA and Opa proteins are, however, unknown. Here we use a fluorescence-based binding assay to show that both proteins bind to mono- and disaccharides with high affinity. Binding of saccharides caused a quench in the intrinsic fluorescence emission of both proteins, and mutation of selected Tyr residues within the external loop regions caused a substantial decrease in fluorescence. We suggest that the intrinsic fluorescence arises from resonance energy transfer from Tyr to Trp residues in the beta-barrel portion of the structure. OpcA bound sialic acid with a K-d of 0.31 mu M and was shown to be specific for pyranose saccharides. The binding specificities of two different Opa proteins were compared; unlike OpcA, neither protein bound to monosaccharides, but both bound to maltose, lactose, and sialic acid-containing oligosaccharides, with K-d values in the micromolar range. OpaB had a 10-fold higher affinity for sialic acid-containing ligands than OpaD as a result of the mutation Y165V, which was shown to restore this specificity to OpaD. Finally, the OpcA- and Opa-dependent adhesion of meningococci to epithelial cells was shown to be partially inhibited by exogenously added sialic acid and maltose. The results show that OpcA and the Opa proteins can be thought of as outer membrane lectins and that simple saccharides can modulate their recognition of complex proteoglycan receptors.