Conservation of calnexin in the early branching protozoan Tritrichomonas suis

The cloning and characterization of Ts-p66, a calcium-binding protein repre senting calnexin of the protozoan parasite Tritrichomonas suis is described . A T. suis cDNA expression library was screened with monospecific antibodi es affinity-purified on an immune-reactive 66 kDa antigen in a Triton X-114 membrane-protein fraction. The deduced amino acid sequence of the resultin g cDNA clones revealed that Ts-p66 belongs to the calreticulin protein fami ly and represents calnexin of T. suis. The key structural features and sequ ence motifs of the calnexins were all conserved. By lectin-blotting we demo nstrated that the native protein is glycosylated. Northern and Southern hyb ridizations showed that T. suis calnexin was highly expressed and encoded b y a single or low copy number gene. A cDNA encoding Ts-p66 was expressed as recombinant protein in Escherichia coli. By overlay with Ca-45 it was demo nstrated that the native and recombinant proteins bind Ca2+. Using immunofl uorescence with affinity-purified antibodies, a staining pattern was observ ed which points towards a putative localization of Ts-p66 in the nuclear me mbrane and endoplasmic reticulum. Demonstration of a structurally conserved calnexin in the amitochondriate protist T. suis indicates the very early e volutionary origin of the machinery for quality control of protein folding in the endoplasmic reticulum and the molecules involved hereby. (C) 2000 El sevier Science B.V. All rights reserved.