The release of the variant surface protein of Giardia to its soluble isoform is mediated by the selective cleavage of the conserved carboxy-terminal domain.

The trophozoites of Giardia duodenalis are covered by a coat composed of an apparently single species of a group of novel, cysteine-rich proteins. These variant-specific surface proteins (VSPs) can be changed by sequential expression of different VSP genes, a process for which a gradual exchange of VSP molecules appears to be required. In the present study, we have examined the in vitro release of one of these VSPs (VSP4A1, formerly named CRISP-90) expressed by a sheep-derived variant Giardia clone. During in vitro incubation of the cloned trophozoites, the membrane-associated form of VSP4A1 (mVSP4A1) was specifically converted to a water-soluble isoform that was continuously released into the culture medium. The time required for mVSP4A1 to decline to half of the initial amount was 7.8 h. Analysis of the two purified protein species by mass spectrometry revealed molecular mass values of 68,991 Da for mVSP4A1 and of 65,425 Da for its soluble counterpart. Amino-terminal sequencing and metabolic labeling experiments indicated that the release of mVSP4A1 was associated with the cleavage of a carboxy-terminal peptide carrying the palmitic acid recently demonstrated to be attached to mVSP4A1. Calculations using the molecular mass and predicted amino acid sequence data indicated that fragmentation of the protein possibly occurs at a site located between the lysine and serine residues of the highly conserved NKSGLS motif directly preceding the hydrophobic sequence previously postulated to serve as a membrane anchoring domain of other VSP molecules. The observed processing of the membrane-associated VSP to its soluble isoform is assumed to be an essential requirement for the ability of the parasite to undergo surface antigenic variation and thus for its establishment and survival within the vertebrate host.