A binding site for highly sulfated heparan sulfate is identified in the N terminus of the circumsporozoite protein - Significance for malarial sporozoite attachment to hepatocytes
A binding site for highly sulfated heparan sulfate is identified in the N terminus of the circumsporozoite protein - Significance for malarial sporozoite attachment to hepatocytes
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DOI：
10.1074/jbc.m401979200
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发表时间：
2004-05-21
影响因子：
4.8
通讯作者：
Kisilevsky, R
中科院分区：
文献类型：
--
作者：
Ancsin, JB;Kisilevsky, R
环子孢子蛋白（Circumsporozoite protein，CSP）是包裹在疟原虫子孢子表面的一种蛋白质，其功能是靶向肝脏进行感染，这是疟疾发展的第一步。CSP的一种重要组织配体是在肝细胞表面和Disse间隙基底膜中发现的硫酸乙酰肝素（HS）。为了更好地理解这种有效的靶向过程，我们着手鉴定和表征CSP的HS结合位点。我们合成了一系列的肽对应于5个区域的恶性疟原虫CSP含有碱性残基，HS结合位点的共同要求，并筛选它们的肝素和HS结合活性。其中只有一种肽（Pf 2）含有我们命名为区域I-plus的基序，它既表现出高亲和力肝素/ HS结合活性，又表现出阻断重组CSP与肝素- Sepharose 4 B结合的能力。通过等温滴定量热法分析表明，区域I-加具有K-d = 5.0 μ m M的结合常数和n = 7.8结合位点/肝素链的化学计量。肝素结合依赖于区域I-plus的氨基酸序列，并且肝素/ HS上的结合位点包含在十糖内。此外，高效结合需要富含硫酸盐和艾杜糖醛酸含量（肝素样）的HS寡糖。由于肝脏HS在这两种组分中相对于其他器官的HS特别高，因此有效区域I-plus/ HS结合的HS结构要求与该肽序列一致，该肽序列起作用以将子孢子靶向肝脏以附着于肝细胞。最后，在感染人类的另外两个物种三日疟原虫和间日疟原虫中也发现了I区+肝素/ HS结合位点，这进一步支持了CSP的N-末端部分存在HS结合结构域.
Circumsporozoite protein ( CSP) coats the malarial sporozoite and functions to target the liver for infection, which is the first step to developing malaria. An important tissue ligand for CSP is the glycosaminoglycan heparan sulfate ( HS) found on the surface of hepatocytes and in the basement membrane of the space of Disse. To better understand this efficient targeting process, we set out to identify and characterize the HS binding site( s) of CSP. We synthesized a series of peptides corresponding to five regions of Plasmodium falciparum CSP containing basic residues, a common requirement of HS binding sites, and screened them for heparin and HS binding activity. Only one of these peptides ( Pf 2), which contains a motif we have named region I- plus, demonstrated both high affinity heparin/ HS binding activity and the ability to block the binding of recombinant CSP to heparin- Sepharose 4B. Analysis by isothermal titration calorimetry revealed that region I- plus has a binding constant of K-d = 5.0 mum M and a stoichiometry of n = 7.8 binding sites/ heparin chain. Heparin binding was dependent on the amino acid sequence of region I- plus, and the binding sites on heparin/ HS are contained within a decasaccharide. Furthermore, HS oligosaccharides rich in sulfate and iduronic acid content ( heparin- like) are required for efficient binding. Because liver HS is exceptionally high in both these components relative to the HS of other organs, the HS structural requirements for efficient region I- plus/ HS binding are consistent with this peptide sequence functioning to target sporozoites to the liver for attachment to hepatocytes. Finally, the region I- plus heparin/ HS binding site was also discovered for two other species that infect humans, Plasmodium malariae and Plasmodium vivax, further supporting the existence of a HS binding domain in the N- terminal portion of CSP.