Poulsen K, Reinholdt J, Kilian M
Department of Medical Microbiology and Immunology, University of Aarhus, Denmark.
Infect Immun. 1996 Oct;64(10):3957-66. doi: 10.1128/iai.64.10.3957-3966.1996.
Bacterial immunoglobulin A1 (IgA1) proteases constitute a very heterogenous group of extracellular endopeptidases which specifically cleave human IgA1 in the hinge region. Here we report that the IgA1 protease gene, iga, of Streptococcus pneumoniae is homologous to that of Streptococcus sanguis. By using the S. sanguis iga gene as hybridization probe, the corresponding gene from a clinical isolate of S. pneumoniae was isolated in an Escherichia coli lambda phage library. A lysate of E. coli infected with hybridization-positive recombinant phages possessed IgA1-cleaving activity. The complete sequence of the S. pneumoniae iga gene was determined. An open reading frame with a strongly biased codon usage and having the potential of encoding a protein of 1,927 amino acids with a molecular mass of 215,023 Da was preceded by a potential -10 promoter sequence and a putative Shine-Dalgarno sequence. A putative signal peptide was found in the N-terminal end of the protein. The amino acid sequence similarity to the S. sanguis IgA1 protease indicated that the pneumococcal IgA1 protease is a Zn-metalloproteinase. The primary structures of the two streptococcal IgA1 proteases were quite different in the N-terminal parts, and both proteins contained repeat structures in this region. Using a novel assay for IgA1 protease activity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we demonstrated that the secreted IgA1 protease was present in several different molecular forms ranging in size from approximately 135 to 220 kDa. In addition, interstrain differences in the sizes of the pneumococcal IgA1 proteases were detected. Southern blot analyses suggested that the S. pneumoniae iga gene is highly heterogenous within the species.
细菌免疫球蛋白A1（IgA1）蛋白酶构成了一组非常异质的细胞外内肽酶，它们特异性地切割铰链区的人IgA1。在此我们报道，肺炎链球菌的IgA1蛋白酶基因iga与血链球菌的该基因同源。通过使用血链球菌iga基因作为杂交探针，在大肠杆菌λ噬菌体文库中分离出了一株肺炎链球菌临床分离株的相应基因。感染杂交阳性重组噬菌体的大肠杆菌裂解物具有切割IgA1的活性。测定了肺炎链球菌iga基因的完整序列。一个开放阅读框，其密码子使用存在强烈偏好，有可能编码一个由1927个氨基酸组成、分子量为215,023 Da的蛋白质，其前面有一个潜在的-10启动子序列和一个推定的Shine-Dalgarno序列。在该蛋白质的N末端发现了一个推定的信号肽。与血链球菌IgA1蛋白酶的氨基酸序列相似性表明，肺炎球菌IgA1蛋白酶是一种锌金属蛋白酶。两种链球菌IgA1蛋白酶的一级结构在N末端部分有很大差异，并且这两种蛋白质在该区域都含有重复结构。使用一种在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上检测IgA1蛋白酶活性的新方法，我们证明分泌的IgA1蛋白酶以几种不同的分子形式存在，大小范围约为135至220 kDa。此外，还检测到肺炎球菌IgA1蛋白酶大小的菌株间差异。Southern印迹分析表明，肺炎链球菌iga基因在该物种内高度异质。
