Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii

Roberta M. O'Connor, Jane W. Wanyiri, Boguslaw S. Wojczyk, Kami Kim, Honorine Ward

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Cryptosporidium is a cause of diarrheal disease worldwide. Parasite glycoproteins involved in invasion of Cryptosporidium into host cells have been investigated as possible targets for effective interventions against this parasite. One of these, Cpgp40/15, is expressed as a precursor protein that is cleaved by a parasite-derived furin-like protease activity into gp15, a glycophosphatidyl inositol anchored surface protein, and gp40, that associates with gp15 and binds to host cells. Investigation of the functions of these glycoproteins requires an expression system that can produce similar glycosylation patterns to the native antigens. Previous work demonstrated that Cpgp40/15 transiently expressed in Toxoplasma gondii was appropriately localized and glycosylated. In this study, T. gondii stable transfectants expressing gp40/15, gp15, gp40 and hemagglutinin (HA) tagged gp40 were generated. T. gondii recombinant gp40HA and gp40/15 (recTggp40HA and recTggp40/15) were isolated from infected cells by HA affinity chromatography and Helix pomatia lectin affinity chromatography, respectively. Mass spectrometry confirmed that recTggp40-HA and native Cpgp40 were similarly glycosylated. Like native Cpgp40/15, recTggp40/15 could be cleaved into the gp40 and gp15 products by human furin or by a furin-like protease activity in T. gondii tachyzoite lysates. However, processing was inefficient in intact tachyzoites. Unlike the N-terminus of native Cpgp40/15, which appears to be processed following signal peptide cleavage, the N-terminus of recTggp40/15 began at the predicted signal sequence cleavage site, 11 amino acids upstream of the N-terminus of native Cpgp40. The ability to express and isolate appropriately glycosylated Cryptosporidium glycoproteins will enable further investigations into host-parasite interactions of this important pathogen.

Original languageEnglish (US)
Pages (from-to)149-158
Number of pages10
JournalMolecular and Biochemical Parasitology
Volume152
Issue number2
DOIs
StatePublished - Apr 2007

Fingerprint

Cryptosporidium parvum
Toxoplasma
Furin
Cryptosporidium
Glycoproteins
Hemagglutinins
Parasites
Protein Sorting Signals
Affinity Chromatography
Peptide Hydrolases
Host-Parasite Interactions
Protein Precursors
Inositol
Glycosylation
Mass Spectrometry
Membrane Proteins
Antigens
Amino Acids

Keywords

  • Cryptosporidium
  • Mucin-like glycoproteins
  • Proteolytic processing
  • Toxoplasma

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii. / O'Connor, Roberta M.; Wanyiri, Jane W.; Wojczyk, Boguslaw S.; Kim, Kami; Ward, Honorine.

In: Molecular and Biochemical Parasitology, Vol. 152, No. 2, 04.2007, p. 149-158.

Research output: Contribution to journalArticle

O'Connor, Roberta M. ; Wanyiri, Jane W. ; Wojczyk, Boguslaw S. ; Kim, Kami ; Ward, Honorine. / Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii. In: Molecular and Biochemical Parasitology. 2007 ; Vol. 152, No. 2. pp. 149-158.
@article{49ee0458f2354cd781bb14836c24b2a0,
title = "Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii",
abstract = "Cryptosporidium is a cause of diarrheal disease worldwide. Parasite glycoproteins involved in invasion of Cryptosporidium into host cells have been investigated as possible targets for effective interventions against this parasite. One of these, Cpgp40/15, is expressed as a precursor protein that is cleaved by a parasite-derived furin-like protease activity into gp15, a glycophosphatidyl inositol anchored surface protein, and gp40, that associates with gp15 and binds to host cells. Investigation of the functions of these glycoproteins requires an expression system that can produce similar glycosylation patterns to the native antigens. Previous work demonstrated that Cpgp40/15 transiently expressed in Toxoplasma gondii was appropriately localized and glycosylated. In this study, T. gondii stable transfectants expressing gp40/15, gp15, gp40 and hemagglutinin (HA) tagged gp40 were generated. T. gondii recombinant gp40HA and gp40/15 (recTggp40HA and recTggp40/15) were isolated from infected cells by HA affinity chromatography and Helix pomatia lectin affinity chromatography, respectively. Mass spectrometry confirmed that recTggp40-HA and native Cpgp40 were similarly glycosylated. Like native Cpgp40/15, recTggp40/15 could be cleaved into the gp40 and gp15 products by human furin or by a furin-like protease activity in T. gondii tachyzoite lysates. However, processing was inefficient in intact tachyzoites. Unlike the N-terminus of native Cpgp40/15, which appears to be processed following signal peptide cleavage, the N-terminus of recTggp40/15 began at the predicted signal sequence cleavage site, 11 amino acids upstream of the N-terminus of native Cpgp40. The ability to express and isolate appropriately glycosylated Cryptosporidium glycoproteins will enable further investigations into host-parasite interactions of this important pathogen.",
keywords = "Cryptosporidium, Mucin-like glycoproteins, Proteolytic processing, Toxoplasma",
author = "O'Connor, {Roberta M.} and Wanyiri, {Jane W.} and Wojczyk, {Boguslaw S.} and Kami Kim and Honorine Ward",
year = "2007",
month = "4",
doi = "10.1016/j.molbiopara.2007.01.003",
language = "English (US)",
volume = "152",
pages = "149--158",
journal = "Molecular and Biochemical Parasitology",
issn = "0166-6851",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Stable expression of Cryptosporidium parvum glycoprotein gp40/15 in Toxoplasma gondii

AU - O'Connor, Roberta M.

AU - Wanyiri, Jane W.

AU - Wojczyk, Boguslaw S.

AU - Kim, Kami

AU - Ward, Honorine

PY - 2007/4

Y1 - 2007/4

N2 - Cryptosporidium is a cause of diarrheal disease worldwide. Parasite glycoproteins involved in invasion of Cryptosporidium into host cells have been investigated as possible targets for effective interventions against this parasite. One of these, Cpgp40/15, is expressed as a precursor protein that is cleaved by a parasite-derived furin-like protease activity into gp15, a glycophosphatidyl inositol anchored surface protein, and gp40, that associates with gp15 and binds to host cells. Investigation of the functions of these glycoproteins requires an expression system that can produce similar glycosylation patterns to the native antigens. Previous work demonstrated that Cpgp40/15 transiently expressed in Toxoplasma gondii was appropriately localized and glycosylated. In this study, T. gondii stable transfectants expressing gp40/15, gp15, gp40 and hemagglutinin (HA) tagged gp40 were generated. T. gondii recombinant gp40HA and gp40/15 (recTggp40HA and recTggp40/15) were isolated from infected cells by HA affinity chromatography and Helix pomatia lectin affinity chromatography, respectively. Mass spectrometry confirmed that recTggp40-HA and native Cpgp40 were similarly glycosylated. Like native Cpgp40/15, recTggp40/15 could be cleaved into the gp40 and gp15 products by human furin or by a furin-like protease activity in T. gondii tachyzoite lysates. However, processing was inefficient in intact tachyzoites. Unlike the N-terminus of native Cpgp40/15, which appears to be processed following signal peptide cleavage, the N-terminus of recTggp40/15 began at the predicted signal sequence cleavage site, 11 amino acids upstream of the N-terminus of native Cpgp40. The ability to express and isolate appropriately glycosylated Cryptosporidium glycoproteins will enable further investigations into host-parasite interactions of this important pathogen.

AB - Cryptosporidium is a cause of diarrheal disease worldwide. Parasite glycoproteins involved in invasion of Cryptosporidium into host cells have been investigated as possible targets for effective interventions against this parasite. One of these, Cpgp40/15, is expressed as a precursor protein that is cleaved by a parasite-derived furin-like protease activity into gp15, a glycophosphatidyl inositol anchored surface protein, and gp40, that associates with gp15 and binds to host cells. Investigation of the functions of these glycoproteins requires an expression system that can produce similar glycosylation patterns to the native antigens. Previous work demonstrated that Cpgp40/15 transiently expressed in Toxoplasma gondii was appropriately localized and glycosylated. In this study, T. gondii stable transfectants expressing gp40/15, gp15, gp40 and hemagglutinin (HA) tagged gp40 were generated. T. gondii recombinant gp40HA and gp40/15 (recTggp40HA and recTggp40/15) were isolated from infected cells by HA affinity chromatography and Helix pomatia lectin affinity chromatography, respectively. Mass spectrometry confirmed that recTggp40-HA and native Cpgp40 were similarly glycosylated. Like native Cpgp40/15, recTggp40/15 could be cleaved into the gp40 and gp15 products by human furin or by a furin-like protease activity in T. gondii tachyzoite lysates. However, processing was inefficient in intact tachyzoites. Unlike the N-terminus of native Cpgp40/15, which appears to be processed following signal peptide cleavage, the N-terminus of recTggp40/15 began at the predicted signal sequence cleavage site, 11 amino acids upstream of the N-terminus of native Cpgp40. The ability to express and isolate appropriately glycosylated Cryptosporidium glycoproteins will enable further investigations into host-parasite interactions of this important pathogen.

KW - Cryptosporidium

KW - Mucin-like glycoproteins

KW - Proteolytic processing

KW - Toxoplasma

UR - http://www.scopus.com/inward/record.url?scp=33847266927&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33847266927&partnerID=8YFLogxK

U2 - 10.1016/j.molbiopara.2007.01.003

DO - 10.1016/j.molbiopara.2007.01.003

M3 - Article

C2 - 17275106

AN - SCOPUS:33847266927

VL - 152

SP - 149

EP - 158

JO - Molecular and Biochemical Parasitology

JF - Molecular and Biochemical Parasitology

SN - 0166-6851

IS - 2

ER -