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MOLECULAR MECHANISMS OF OPSONIZATION

调理作用的分子机制

基本信息

批准号：
3445513
负责人：
MARGARET K HOSTETTER
金额：
$ 4.9万
依托单位：
UNIVERSITY OF MINNESOTA
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-01-01 至 1987-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/3445513
关键词：
Streptococcus pneumoniae acyl group bacterial polysaccharides bactericidal immunity complement complement pathway covalent bond host organism interaction human tissue neutrophil newborn human (0-6 weeks) opsonin phagocytosis radiotracer sickle cell anemia thiols virulence

项目摘要

The binding of C3b, the opsonic fragment of the third component of complement (C3) to bacterial surface structures prepares the microorganisms for ingestion by phagocytic cells. We propose to examine, at the molecular level, this interaction between C3 and pathogenic bacteria. We contend that a particular structure of C3--the reactive thiolester--mediates the attachment of the protein to the organism by means of a transacylation reaction. The structure of the organism, in turn, provides specific sites of acylation for the protein. The thiolester of C3 resides within a transiently generated binding site on the Alpha' chain of the C3 protein and is formed by the linking of a cysteinyl residue with a glutamyl residue. In our published studies of the covalent reaction of C3 with small carbohydrates and amines, we have shown that the glutamyl component of the thiolester is the site of transacylation reactions, donating its acyl group to form covalent ester or amide bonds with appropriate carbohydrates or amines. We propose that opsonization of virulent bacteria proceeds by the identical biochemical reaction. First, using radiolabeled, purified complement components, C4-deficient human serum, and polymorphonuclear leukocytes from normal adults, we shall analyze differences in the opsonization of virulent serotypes of Streptococcus pneumoniae (types 3, 4, 6, 8, 14, and 18) as a function of number of covalent binding sites for C3b per organism. Contributions of type-specific anticapsular antibody to the binding of C3b, as well as binding to polymorphonuclear leukocyte receptors for complement, will also be examined with these methods. Secondly, employing the methods which we used for the characterization of the transacylation reaction with small carbohydrates, we shall identify the constituents of the covalent bond formed in opsonic interactions. C3 will be covalently attached to pneumococcal polysaccharide, the peptide containing the binding site will be isolated, and the reactive species from both C3 and polysaccharide will be analyzed. Thirdly, we shall relate the reactivity of the thiolester to opsonic function in two human models of defective opsonization: neonates and patients homozygous for hemoglobin-SS.

C3 b的结合，C3 b的第三组分的调理素片段，细菌表面结构的补体（C3）使微生物供吞噬细胞摄取。我们建议，在分子水平上，水平，这种C3和致病菌之间的相互作用。我们主张 C3的一种特殊结构--反应性硫酯--介导了通过转酰作用将蛋白质附着到生物体上反应有机体的结构反过来提供了特定的位点蛋白质的酰化反应。 C3的硫酯存在于一个瞬时产生的结合位点上， C3蛋白的α '链，并通过连接半胱氨酰残基与谷氨酰残基。在我们发表的研究中， C3与小分子碳水化合物和胺的共价反应，我们已经证明硫酯的谷氨酰成分是转酰作用的位点反应，使其酰基形成共价酯键或酰胺键与适当的碳水化合物或胺。我们建议，调理作用的毒性细菌通过相同的生化反应进行。首先，使用放射性标记的纯化的补体成分，C4-缺陷型人血清和正常成人的多形核白细胞，我们将分析在调理作用的毒性血清型的差异，肺炎链球菌（3、4、6、8、14和18型）作为每个生物体C3 b的共价结合位点数。贡献型特异性抗囊抗体结合C3 b，以及与多形核白细胞补体受体结合，也将用这些方法检查。第二，采用我们用于表征转酰化反应，碳水化合物，我们将确定共价键的成分在调理素的相互作用中形成。 C3将共价连接到肺炎球菌多糖，含有结合位点的肽将被分离，并且来自C3和多糖两者的反应性物种将被分离。被分析。第三，我们将把硫酯的反应性与两种缺陷性调理作用人类模型的调理功能：新生儿和血红蛋白-SS纯合子患者。