Primary structure and biochemical properties of a variant-specific surface protein of Giardia
Primary structure and biochemical properties of a variant-specific surface protein of Giardia
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DOI：
10.1016/s0166-6851(97)02836-3
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发表时间：
1997-05-01
影响因子：
1.5
通讯作者：
Kohler, P
中科院分区：
文献类型：
--
作者：
Papanastasiou, P;Bruderer, T;Kohler, P
十二指肠贾第鞭毛虫滋养体在其细胞表面表达变异特异性蛋白(VSP)，被认为有助于保护寄生虫免受免疫和其他宿主防御机制的影响。在本研究中，我们克隆并鉴定了由绵羊贾第鞭毛虫变异克隆02-4A1表达的VSP(VSP4A1，最初命名为CRISP-90)基因。该基因是通过用接近全长的基因特异性聚合酶链式反应(PCR)产物探测基因组文库来分离的。VSP4A1基因编码一个70729 Da的蛋白，具有所有已报道的VSP的共同特征，包括高半胱氨酸和苏氨酸含量，高度保守的疏水羧基末端结构域，以及与其余多肽序列几乎没有相似性。将预测的序列与纯化的VSP4A1的氨基末端序列进行比较，发现成熟蛋白没有氨基末端的疏水延伸。从O2-4A1变异克隆中纯化的VSP4A1发生了构象变化，导致了另外两个电泳种的形成。在纯化的VSP4A1中没有检测到游离的硫醇基团，这表明所有的半胱氨酸残基都可能参与了二硫键的交联。可能正因为如此，VSP4A1被发现对蛋白水解性消化具有相当的抵抗力。虽然VSP4A1能够在印迹后与硝酸纤维素膜结合锌，但其他对纯化的和细胞相关的VSP的分析都未能证实锌离子与该蛋白的显著结合。后者的结果质疑了其他作者之前的假设，即锌与VSP结合构成了这些蛋白质的重要结构和功能方面。(C)1997年爱思唯尔科学公司。
Trophozoites of Giardia duodenalis express at their cell surface variant-specific proteins (VSPs) that are believed to contribute to the protection of the parasite from immunological and other host defense mechanisms. In the present study, we have cloned and characterized the gene encoding a VSP (VSP4A1, originally designated CRISP-90) that is expressed by the sheep-derived Giardia variant clone 02-4A1. The gene was isolated by probing a genomic library with a near-full-length gene-specific polymerase chain reaction (PCR) product. The VSP4A1 gene specifies a 70729 Da protein with features common to all previously reported VSPs, including a high cysteine and threonine content, a highly conserved hydrophobic carboxy-terminal domain and little similarity in the remaining polypeptide sequence. Comparison of the predicted sequence with the amino-terminal sequence of purified VSP4A1 revealed the absence of an amino-terminal hydrophobic extension from the mature protein. VSP4A1 purified from the O2-4A1 variant clone was found to undergo conformational changes resulting in the formation of two additional electrophoretic species. Free thiol groups were not detected in purified VSP4A1, indicating that all cysteine residues may be involved in disulphide crosslinking. Possibly as a consequence of this, VSP4A1 was found to be fairly resistant to proteolytic digestion. Although VSP4A1 is able to bind zinc following blotting to a nitrocellulose membrane, other analyses with both the purified and cell associated VSP have failed to confirm significant zinc ion binding to this protein. The latter result questions the assumption previously made by other authors that zinc binding to VSPs constitutes an important structural and functional aspect of these proteins. (C) 1997 Elsevier Science B.V.