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A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor.

ABSTRACT: The procyclic acidic repetitive protein is the major cell-surface glycoprotein of the insect-dwelling procyclic forms of the Trypanosoma brucei species of African trypanosomes. The glycoprotein contains an acidic Glu-Pro repeat domain, a glycosyl-phosphatidylinositol membrane anchor and a putative asparagine glycosylation site. In this paper we describe a rapid purification scheme for this glycoprotein, using solvent extraction and hydrophobic interaction chromatography, and a partial characterization of the glycosylphosphatidylinositol membrane anchor. The carbohydrate composition of the anchor is extremely unusual; it contains on average nine GlcNAc, nine Gal, and five sialic acid residues. This is the first description of such a heavily substituted and negatively charged anchor. A comparison between the trypanosome procyclic surface and the Leishmania promastigote surface is also presented.

SUBMITTER: Ferguson MA

PROVIDER: S-EPMC1132479 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Project description:Glycosylphosphatidylinositol (GPI) is a post-translational modification resulting in the attachment of modified proteins to the outer leaflet of the plasma membrane. Tissue culture experiments have shown GPI-anchored proteins (GPI-APs) to be targeted to the apical membrane of epithelial cells. However, the in vivo importance of this targeting has not been investigated since null mutations in GPI biosynthesis enzymes in mice result in very early embryonic lethality. Missense mutations in the human GPI biosynthesis enzyme pigv are associated with a multiple congenital malformation syndrome with a high frequency of Hirschsprung disease and renal anomalies. However, it is currently unknown how these phenotypes are linked to PIGV function. Here, we identify a temperature-sensitive hypomorphic allele of PIGV in Caenorhabditis elegans, pigv-1(qm34), enabling us to study the role of GPI-APs in development. At the restrictive temperature we found a 75% reduction in GPI-APs at the surface of embryonic cells. Consequently, ~80% of pigv-1(qm34) embryos arrested development during the elongation phase of morphogenesis, exhibiting internal cysts and/or surface ruptures. Closer examination of the defects revealed them all to be the result of breaches in epithelial tissues: cysts formed in the intestine and excretory canal, and ruptures occurred through epidermal cells, suggesting weakening of the epithelial membrane or membrane-cortex connection. Knockdown of piga-1, another GPI biosynthesis enzymes resulted in similar phenotypes. Importantly, fortifying the link between the apical membrane and actin cortex by overexpression of the ezrin/radixin/moesin ortholog ERM-1, significantly rescued cyst formation and ruptures in the pigv-1(qm34) mutant. In conclusion, we discovered GPI-APs play a critical role in maintaining the integrity of the epithelial tissues, allowing them to withstand the pressure and stresses of morphogenesis. Our findings may help to explain some of the phenotypes observed in human syndromes associated with pigv mutations.

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A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor.

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