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Infection and Immunity, January 2007, p. 184-192, Vol. 75, No. 1
0019-9567/07/$08.00+0     doi:10.1128/IAI.00944-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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

Jane W. Wanyiri,1 Roberta O'Connor,1 Geneve Allison,1 Kami Kim,2 Anne Kane,3 Jiazhou Qiu,3 Andrew G. Plaut,3 and Honorine D. Ward1*

Division of Geographic Medicine and Infectious Diseases,1 GRASP Digestive Diseases Center, Tufts-New England Medical Center, 750 Washington Street, Boston, Massachusetts 02111,3 Albert Einstein College of Medicine, New York, New York2

Received 13 June 2006/ Returned for modification 19 July 2006/ Accepted 5 October 2006

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{downarrow} 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{downarrow}) 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.

* Corresponding author. Mailing address: Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, 750 Washington Street, Boston, MA 02111. Phone: (617) 636-7022. Fax: (617) 636-5292. E-mail: hward{at}tufts-nemc.org.

{triangledown} Published ahead of print on 16 October 2006.

Editor: W. A. Petri, Jr.

Infection and Immunity, January 2007, p. 184-192, Vol. 75, No. 1
0019-9567/07/$08.00+0     doi:10.1128/IAI.00944-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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