Primary structure and biochemical properties of a variant-specific surface protein of Giardia.
Trophozoites of Giardia duodenalis express at their cell surface variant-specific proteins (VSPs) that are believed to contribute to the protection of the parasite from immunological and other host defense mechanisms. In the present study, we have cloned and characterized the gene encoding a VSP (VSP4A1, originally designated CRISP-90) that is expressed by the sheep-derived Giardia variant clone O2-4A1. The gene was isolated by probing a genomic library with a near-full-length gene-specific polymerase chain reaction (PCR) product. The VSP4A1 gene specifies a 70729 Da protein with features common to all previously reported VSPs, including a high cysteine and threonine content, a highly conserved hydrophobic carboxy-terminal domain and little similarity in the remaining polypeptide sequence. Comparison of the predicted sequence with the amino-terminal sequence of purified VSP4A1 revealed the absence of an amino-terminal hydrophobic extension from the mature protein. VSP4A1 purified from the O2-4A1 variant clone was found to undergo conformational changes resulting in the formation of two additional electrophoretic species. Free thiol groups were not detected in purified VSP4A1, indicating that all cysteine residues may be involved in disulphide crosslinking. Possibly as a consequence of this VSP4A1 was found to be fairly resistant to proteolytic digestion. Although VSP4A1 is able 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 result questions the assumption previously made by other authors that zinc binding to VSPs constitutes an important structural and functional aspect of these proteins.
Papanastasiou
P
P
Institute of Parasitology, University of Zürich, Switzerland.
Bruderer
T
T
Li
Y
Y
Bommeli
C
C
Köhler
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000954:Antigens, Surface / Q000096:biosynthesis / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D005872:Giardia / Q000737:chemistry / Q000502:physiology*; D006867:Hydrolases; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry; D015800:Protozoan Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D015032:Zinc / Q000378:metabolism
D000954:Antigens, Surface; D017931:DNA Primers; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D006867:Hydrolases; C100894:atrazine chlorohydrolase; D015032:Zinc