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 pro teins. These variant-specific surface proteins (VSPs) can be changed b y sequential expression of different VSP genes, a process for which a gradual exchange of VSP molecules appears to be required. In the prese nt 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 conv erted to a water-soluble isoform that was continuously released into t he 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 sp ecies by mass spectrometry revealed molecular mass values of 68 991 Da for mVSP4A1 and of 65 425 Da for its soluble counterpart. Amino-termi nal sequencing and metabolic labeling experiments indicated that the r elease of mVSP4A1 was associated with the cleavage of a carboxy-termin al peptide carrying the palmitic acid recently demonstrated to be atta ched to mVSP4A1. Calculations using the molecular mass and predicted a mino acid sequence data indicated that fragmentation of the protein po ssibly occurs at a site located between the lysine and serine residues of the highly conserved NKSGLS motif directly preceding the hydrophob ic sequence previously postulated to serve as a membrane-anchoring dom ain of other VSP molecules, The observed processing of the membrane-as sociated VSP to its soluble isoform is assumed to be an essential requ irement for the ability of the parasite to undergo surface antigenic v ariation and thus for its establishment and survival within the verteb rate host.