Cloning of the immunodominant 17-kDa antigen from Cryptosporidium parvum

Priest, Jeffrey W.; Kwon, James P.; Arrowood, Michael J.; Lammie, Patrick J.

doi:10.1016/s0166-6851(99)00223-6

Cited by 70 publications

(58 citation statements)

References 33 publications

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“…Although both of these antigens include post-translational carbohydrate and/or lipid modifications, we demonstrated that much of the antibody response was directed against the protein component (19,21). While examining the IgG antibody reactivity to the subset of those antigens that were extracted into Triton X-114 detergent (the 27-and 17-kDa antigen families include both soluble and membrane-bound forms), we noted that an additional antigen having a molecular mass of Ͻ6-kDa was recognized by serum IgG antibodies from ϳ50% of the patients ( Fig.…”

Section: Serum Antibodies From Cryptosporidiosis Patients Recognize Amentioning

confidence: 94%

“…Both of these antigens are associated with the sporozoite surface, and subsets of the antigens can be partially purified from sonicated oocysts by phase partitioning into Triton X-114 detergent (19). In earlier work we used this technique to purify the 17-kDa antigen for peptide sequence analysis and to generate a native antigen fraction suitable for use in an enzyme-linked immunoassay for the detection and quantitation of serum IgG antibodies (19,21). While analyzing the antibody response to the Triton X-114 detergent extract by Western blot, we noted that a number of cryptosporidiosis patients also reacted with a novel low molecular weight antigen.…”

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confidence: 99%

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Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum

Priest

Mehlert

Arrowood

et al. 2003

Journal of Biological Chemistry

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Cryptosporidium parvum, an Apicomplexan parasite of the mammalian gut epithelium, causes a diarrheal illness in a wide range of hosts and is transmitted by contamination of food or water with oocyst-laden feces from an infected animal. We have identified a glycosylinositol phospholipid from the sporozoite stage of the parasite that is frequently recognized by serum antibodies from human cryptosporidiosis patients. The humoral immune response is dominated by IgG 1 subclass antibodies but can also include IgA and IgM antibodies. The glycosylinositol phospholipids were purified by butanol extraction of a Triton X-114-soluble fraction followed by octyl-Sepharose column chromatography and preparative high performance TLC and were shown to include at least 5 species. By using mass spectrometry and radiolabeled neutral glycan analysis, we found that the structure of the dominant glycosylinositol phospholipid antigen contained a C18:0 lyso-acylglycerol, a C16:0-acylated inositol, and an unsubstituted mannose 3 -glucosamine glycan core. Other diacyl species were also identified, most notably a series of glycosylinositol phospholipids having an acyl-linked C20:0 to C28:0 lipid on the inositol ring. Less abundant species having three acyl-linked fatty acids and species with an additional 1-3 hexoses linked to the mannose core were also observed. We are currently working to determine the role that these glycolipids may play in the development of disease and in the clearance of infection.

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Section: Serum Antibodies From Cryptosporidiosis Patients Recognize Amentioning

confidence: 94%

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confidence: 99%

Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum

Priest

Mehlert

Arrowood

et al. 2003

Journal of Biological Chemistry

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“…One research focus has been to identify the antigens on the invasive zoite stages that allow the parasite to attach to and invade the epithelial cell with the goal of preventing these parasite-host cell interactions. The best characterized of the zoite antigens is Cpgp40/15, (also called Cp17, gp15/45/60 or S60) [6][7][8][9][10], a mucin like glycoprotein antigen that is synthesized as a single precursor protein and proteolytically cleaved into the mature glycoproteins, gp40 and gp15 [11]. gp15 is anchored in the sporozoite membrane by a glycosylphosphatidyl inositol (GPI) moiety [12], while the gp40 glycoprotein does not contain any predicted transmembrane domains or GPI anchors and is predicted to be soluble [7].…”

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confidence: 99%

Cryptosporidium parvum glycoprotein gp40 localizes to the sporozoite surface by association with gp15

O’Connor

Wanyiri

Cevallos

et al. 2007

Molecular and Biochemical Parasitology

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Cryptosporidium spp. are ubiquitous waterborne parasites responsible for outbreaks of diarrheal disease worldwide [1]. The infection is self-limiting in immunocompetent individuals, but in immunocompromised individuals the infection can develop into a chronic, debilitating, and sometimes life-threatening disease [2]. In malnourished children, cryptosporidiosis is associated with growth and developmental delays [1]. Currently, there are no vaccines to prevent cryptosporidiosis, and nitazoxanide, the only approved drug in the US [3] is not effective in AIDS patients [4]. Because the parasite cannot be propagated in vitro or genetically manipulated, data that would permit targeted drug design or vaccine development are sorely lacking.Ingested Cryptosporidium oocysts excyst in the intestine, releasing sporozoites that attach to and invade intestinal epithelial cells, initiating the lifecycle [5]. The parasite undergoes two rounds of asexual reproduction, in each cycle producing merozoites that invade new epithelial cells, before entering the sexual cycle and producing oocysts. One research focus has been to identify the antigens on the invasive zoite stages that allow the parasite to attach to and invade the epithelial cell with the goal of preventing these parasite-host cell interactions. The best characterized of the zoite antigens is Cpgp40/15, (also called Cp17, gp15/45/60 or S60) [6][7][8][9][10], a mucin like glycoprotein antigen that is synthesized as a single precursor protein and proteolytically cleaved into the mature glycoproteins, gp40 and gp15 [11]. gp15 is anchored in the sporozoite membrane by a glycosylphosphatidyl inositol (GPI) moiety [12], while the gp40 glycoprotein does not contain any predicted transmembrane domains or GPI anchors and is predicted to be soluble [7]. However, gp40 has been shown to bind intestinal epithelial cells in a dose dependent and saturable manner [7] suggesting that this antigen recognizes a host cell receptor. If this interaction is important for zoite attachment and invasion, then gp40 must have some mechanism of associating with the parasite membrane. In these studies we explore the possibility that gp40 and gp15 associate to form a protein complex capable of linking zoite and host cell surfaces.* Corresponding Authors NEMC Box 041, 750 Washington St, Boston, MA 02111, e-mail: roconnor@tufts-nemc.org; hward@tufts-nemc.org, tel ROC: 617 636 2684, HW: 617 636 7022, fax: 617 636 5292. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To generate gp40 specific antiserum, recombinant (r)gp40 fusion protein [7] was purified via the His...

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“…These proteins, as well as proteins on the surface of the zoite, facilitate attachment to the cell surface, gliding motility, and subsequent invasion and intracellular development of the parasite. However, unlike those of other apicomplexans, most of the Cryptosporidium surface and apical complex antigens that have been characterized are glycoproteins [9][10][11][12][13][14]. Two of these, gp900 [10] and gp40/15 [11,13,14], are mucin-like glycoproteins, proteins with high percentages of serine and threonine residues that are sites for O-linked glycosylation.…”

Section: Introductionmentioning

confidence: 99%

“…However, unlike those of other apicomplexans, most of the Cryptosporidium surface and apical complex antigens that have been characterized are glycoproteins [9][10][11][12][13][14]. Two of these, gp900 [10] and gp40/15 [11,13,14], are mucin-like glycoproteins, proteins with high percentages of serine and threonine residues that are sites for O-linked glycosylation. Recently, the Cryptosporidium genome projects identified 31 putative mucin genes [15,16], suggestive of an essential role for this class of glycoprotein in Cryptosporidium host-parasite interactions.…”

Section: Introductionmentioning

confidence: 99%

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

O’Connor

Wanyiri

Wójczyk

et al. 2007

Molecular and Biochemical Parasitology

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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 T. 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.

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Cloning of the immunodominant 17-kDa antigen from Cryptosporidium parvum

Cited by 70 publications

References 33 publications

Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum

Characterization of a Low Molecular Weight Glycolipid Antigen from Cryptosporidium parvum

Cryptosporidium parvum glycoprotein gp40 localizes to the sporozoite surface by association with gp15

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

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