Conservation of calnexin in the early branching protozoan Tritrichomonas suis

Abstract

The cloning and characterization of Ts-p66, a calcium-binding protein representing calnexin of the protozoan parasite Tritrichomonas suis is described. A T. suis cDNA expression library was screened with monospecific antibodies affinity-purified on an immuno-reactive 66 kDa antigen in a Triton X-114 membrane-protein fraction. The deduced amino acid sequence of the resulting cDNA clones revealed that Ts-p66 belongs to the calreticulin protein family and represents calnexin of T. suis. The key structural features and sequence motifs of the calnexins were all conserved. By lectin-blotting we demonstrated that the native protein is glycosylated. Northern and Southern hybridizations showed that T. suis calnexin was highly expressed and encoded by a single or low copy number gene. A cDNA encoding Ts-p66 was expressed as recombinant protein in Escherichia coli. By overlay with 45Ca it was demonstrated that the native and recombinant proteins bind Ca 2+. Using immunofluorescence with affinity-purified antibodies, a staining pattern was observed which points towards a putative localization of Ts-p66 in the nuclear membrane and endoplasmic reticulum. Demonstration of a structurally conserved calnexin in the amitochondriate protist T. suis indicates the very early evolutionary origin of the machinery for quality control of protein folding in the endoplasmic reticulum and the molecules involved hereby.