Wanyiri Jane W, O'Connor Roberta, Allison Geneve, Kim Kami, Kane Anne, Qiu Jiazhou, Plaut Andrew G, Ward Honorine D
Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, 750 Washington Street, Boston, MA 02111, USA.
Infect Immun. 2007 Jan;75(1):184-92. doi: 10.1128/IAI.00944-06. Epub 2006 Oct 16.
The apicomplexan parasite Cryptosporidium causes diarrheal disease worldwide. Proteolytic processing of proteins plays a significant role in host cell invasion by apicomplexan parasites. In previous studies, we described gp40/15, a Cryptosporidium sp. glycoprotein that is proteolytically cleaved to yield two surface glycopeptides (gp40 and gp15), which are implicated in mediating infection of host cells. In the present study, we showed that biosynthetically labeled gp40/15 is processed in Cryptosporidium parvum-infected HCT-8 cells. We identified a putative furin cleavage site RSRR downward arrow in the deduced amino acid sequence of gp40/15 from C. parvum and from all Cryptosporidium hominis subtypes except subtype 1e. Both human furin and a protease activity present in a C. parvum lysate cleaved recombinant C. parvum gp40/15 protein into 2 peptides, identified as gp40 and gp15 by size and by immunoreactivity with specific antibodies. C. hominis gp40/15 subtype 1e, in which the RSRR sequence is replaced by ISKR, has an alternative furin cleavage site (KSISKR downward arrow) and was also cleaved by both furin and the C. parvum lysate. Site-directed mutagenesis of the C. parvum RSRR sequence to ASRR resulted in inhibition of cleavage by furin and the C. parvum lysate. Cleavage of recombinant gp40/15 and a synthetic furin substrate by the C. parvum lysate was inhibited by serine protease inhibitors, by the specific furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-cmk), and by calcium chelators, suggesting that the parasite expresses a Ca2+ dependent, furin-like protease activity. The furin inhibitor Dec-RVKR-cmk decreased C. parvum infection of HCT-8 cells, suggesting that a furin-like protease activity may be involved in mediating host-parasite interactions.
顶复门寄生虫隐孢子虫在全球范围内引发腹泻疾病。蛋白质的蛋白水解加工在顶复门寄生虫入侵宿主细胞过程中发挥着重要作用。在先前的研究中，我们描述了gp40/15，一种隐孢子虫属糖蛋白，它经蛋白水解切割产生两种表面糖肽（gp40和gp15），这两种糖肽与介导宿主细胞感染有关。在本研究中，我们表明生物合成标记的gp40/15在感染微小隐孢子虫的HCT - 8细胞中被加工。我们在微小隐孢子虫以及除1e亚型外的所有人隐孢子虫亚型的gp40/15推导氨基酸序列中鉴定出一个假定的弗林蛋白酶切割位点RSRR↓。人弗林蛋白酶和微小隐孢子虫裂解物中存在的一种蛋白酶活性都将重组微小隐孢子虫gp40/15蛋白切割成两种肽段，通过大小以及与特异性抗体的免疫反应性鉴定为gp40和gp15。人隐孢子虫1e亚型的gp40/15中，RSRR序列被ISKR取代，有一个替代的弗林蛋白酶切割位点（KSISKR↓），并且也被弗林蛋白酶和微小隐孢子虫裂解物切割。将微小隐孢子虫的RSRR序列定点突变为ASRR导致弗林蛋白酶和微小隐孢子虫裂解物的切割受到抑制。微小隐孢子虫裂解物对重组gp40/15和一种合成弗林蛋白酶底物的切割受到丝氨酸蛋白酶抑制剂、特异性弗林蛋白酶抑制剂癸酰 - Arg - Val - Lys - Arg - 氯甲基酮（Dec - RVKR - cmk）以及钙螯合剂的抑制，这表明该寄生虫表达一种Ca2 + 依赖性的、类弗林蛋白酶活性。弗林蛋白酶抑制剂Dec - RVKR - cmk降低了微小隐孢子虫对HCT - 8细胞的感染，这表明类弗林蛋白酶活性可能参与介导宿主 - 寄生虫相互作用。
