PRIMARY STRUCTURE AND BIOCHEMICAL-PROPERTIES OF A VARIANT-SPECIFIC SURFACE PROTEIN OF GIARDIA

Trophozoites of Giardia duodenalis express at their cell surface varia nt-specific proteins (VSPs) that are believed to contribute to the pro tection of the parasite from immunological and other host defense mech anisms. In the present study, we have cloned and characterized the gen e encoding a VSP (VSP4A1, originally designated CRISP-90) that is expr essed by the sheep-derived Giardia variant clone 02-4A1. The gene was isolated by probing a genomic library with a near-full-length gene-spe cific polymerase chain reaction (PCR) product. The VSP4A1 gene specifi es a 70729 Da protein with features common to all previously reported VSPs, including a high cysteine and threonine content, a highly conser ved hydrophobic carboxy-terminal domain and little similarity in the r emaining polypeptide sequence. Comparison of the predicted sequence wi th the amino-terminal sequence of purified VSP4A1 revealed the absence of an amino-terminal hydrophobic extension from the mature protein. V SP4A1 purified from the O2-4A1 variant clone was found to undergo conf ormational changes resulting in the formation of two additional electr ophoretic species. Free thiol groups were not detected in purified VSP 4A1, indicating that all cysteine residues may be involved in disulphi de crosslinking. Possibly as a consequence of this, VSP4A1 was found t o be fairly resistant to proteolytic digestion. Although VSP4A1 is abl e to bind zinc following blotting to a nitrocellulose membrane, other analyses with both the purified and cell associated VSP have failed to confirm significant zinc ion binding to this protein. The latter resu lt questions the assumption previously made by other authors that zinc binding to VSPs constitutes an important structural and functional as pect of these proteins. (C) 1997 Elsevier Science B.V.