Proteolytic Processing of the Cryptosporidium Glycoprotein gp40/15 by Human Furin and by a Parasite-Derived Furin-Like Protease Activity

Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, 750 Washington Street, Boston, MA 02111, USA.
Infection and Immunity (Impact Factor: 3.73). 02/2007; 75(1):184-92. DOI: 10.1128/IAI.00944-06
Source: PubMed


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.

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Available from: Anne V Kane, Oct 07, 2015
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    • "hominis), II (C. parvum); GalNAc-specific lectins such as HPA, AIA, and MPA prevent cell attachment and invasion indicating GPs might bind via GalNAc to cells; moreover Nac could reduce binding of the mAb against GP15 but not glucose, mannose or galactose; immunization of mice with mAb against GP15 could reduce oocyst shedding by 67.5%; GP15/GP40 are shed during gliding GP40: predicted N-terminal signal peptide, a polyserine domain, multiple predicted glycosylation sites, a single potential N-glycosylation site GP15: glycosylated, GPI anchor GP15 is localized on the surface of sporozoites and merozoites; GP40 is localized in the apical region whereas GP15 is found on the complete surface; since GP40 does not have a transmembrane domain or GPI anchor it is proposed that GP15 and GP40 form a complex (Tilley et al. 1991; Jenkins et al. 1993; Cevallos et al. 2000a, b; Strong et al. 2000; Sestak et al. 2002; O'Connor et al. 2003, 2007a, b; Wanyiri et al. 2009, 2007) GP900 Ab against GP900 or subdomains reduced infection of cells in a concentration-dependent manner; GP900 mRNA levels peak at 14–26 h p.i. of cells; comparison of Ab reactivity between C. parvum and C. hominis against GP900 revealed that Ab did not cross-react between the species, indicating that morphological differences of surface molecules might account for species-specific infectivity Mucin-like glycoprotein composed of distal cysteine-rich domains separated by polythreonine domains and a large membrane proximal N-glycosylated core region; deglycosylated protein app. 190 kDa GP900 is stored in micronemes prior to appearance on the surface of invasive forms (Petersen et al. 1992; Barnes et al. 1998; Sturbaum et al. 2003, 2008; Jakobi and Petry, 2006) "
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    • "subtilisin-like serine protease activity cleaves gp40/15 for the production of gp40 and gp15 implicated in infection of HCT-8 cells (Wanyiri et al., 2007). The C. parvum gene encoding CpSUB1, a subtilisin-like serine protease, is overexpressed during infection of HCT-8 cells and localizes within the apical region of infecting sporozoites (Wanyiri et al., 2009). "
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    • "This might be one of the reasons why HA7 transcript remained high in the AEBSF treated mice. Another possible explanation could be that the activity of the subtilisin-related furin is efficiently abolished with polybasic peptide inhibitors fused to cholromethylketone but only partially inhibited by the AEBSF inhibitor (45%; [57,58]). Thus, it has to be taken in consideration that the efficacy of the protease inhibitors to block infection might vary among various influenza subtypes depending on the susceptibility of their HAs to be cleaved by host proteases based on their cleavage site. "
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