McGavin M H, Krajewska-Pietrasik D, Rydén C, Höök M
Department of Biochemistry, University of Alabama, Birmingham 35294-0005.
Infect Immun. 1993 Jun;61(6):2479-85. doi: 10.1128/iai.61.6.2479-2485.1993.
A staphylococal surface protein capable of binding several extracellular matrix glycoproteins was purified as a result of our attempts to identify a receptor(s) for bone sialoprotein (BSP) on Staphylococcus aureus cells. Proteins from different staphylococcal strains were solubilized in sodium lauryl sulfate, separated by polyacrylamide gel electrophoresis, blotted onto Immobilon P membranes, and probed with 125I-BSP. Several bacterial proteins bound the radiolabeled ligand, and various strains expressed different repertoirs of BSP-binding proteins. Major BSP-binding proteins with apparent M(r)s of 72,000 or 60,000 were present on most strains, and these proteins were further studied. The 72- and 60-kDa proteins were preferentially expressed when bacteria were cultured in Luria broth compared with when they were cultured on tryptic soy broth, and the abundance of the proteins could be correlated to an increased 125I-BSP binding. Both the 72-kDa and the 60-kDa proteins were solubilized by extraction of cells with 1 M LiCl and were purified by cation-exchange chromatography. Amino acid composition analysis of the purified 72-kDa protein indicated a high content of lysine (11.9%) and hydrophobic amino acids (28.0% combined). In Western ligand blotting (immunoblotting) experiments, the 72-kDa protein bound not only BSP but also radiolabeled fibronectin, fibrinogen, vitronectin, thrombospondin, and, to some extent, collagen. Addition of the purified 60-kDa protein to S. aureus cells did not inhibit binding of the different ligands but in some cases resulted in an augmentation of the binding of 125I-ligand. Purified 60-kDa protein could hemagglutinate sheep erythrocytes at a concentration of 61 micrograms/ml. The agglutination reaction was inhibited by high concentrations of fucose, mannose, or melibiose. These data suggest that the purified proteins may serve as bacterial receptors with broad specificity for matrix glycoproteins and that the proteins may act as carbohydrate-binding proteins.
我们试图鉴定金黄色葡萄球菌细胞上骨唾液酸蛋白（BSP）的受体，在此过程中，一种能够结合多种细胞外基质糖蛋白的葡萄球菌表面蛋白被纯化出来。将来自不同葡萄球菌菌株的蛋白质用十二烷基硫酸钠溶解，通过聚丙烯酰胺凝胶电泳分离，转移到Immobilon P膜上，并用¹²⁵I-BSP进行检测。几种细菌蛋白与放射性标记的配体结合，不同菌株表达不同的BSP结合蛋白谱。大多数菌株上存在表观分子量为72,000或60,000的主要BSP结合蛋白，并对这些蛋白进行了进一步研究。与在胰蛋白胨大豆肉汤中培养相比，当细菌在Luria肉汤中培养时，72 kDa和60 kDa的蛋白优先表达，并且其丰度与¹²⁵I-BSP结合增加相关。72 kDa和60 kDa的蛋白都可以通过用1 M LiCl提取细胞而溶解，并通过阳离子交换色谱法纯化。对纯化的72 kDa蛋白的氨基酸组成分析表明，赖氨酸含量高（11.9%），疏水氨基酸含量高（合计28.0%）。在Western配体印迹（免疫印迹）实验中，72 kDa蛋白不仅结合BSP，还结合放射性标记的纤连蛋白、纤维蛋白原、玻连蛋白、血小板反应蛋白，在一定程度上还结合胶原蛋白。将纯化的60 kDa蛋白添加到金黄色葡萄球菌细胞中不会抑制不同配体的结合，但在某些情况下会导致¹²⁵I-配体结合增加。纯化的60 kDa蛋白在浓度为61微克/毫升时可使绵羊红细胞发生血凝。高浓度的岩藻糖、甘露糖或蜜二糖可抑制凝集反应。这些数据表明，纯化的蛋白可能作为对基质糖蛋白具有广泛特异性的细菌受体，并且这些蛋白可能作为碳水化合物结合蛋白发挥作用。
