Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.
A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.
Lustigman
S
S
Laboratory of Virology and Parasitology, Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021.
Brotman
B
B
Huima
T
T
Prince
A M
AM
McKerrow
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D015853:Cysteine Proteinase Inhibitors / Q000032:analysis / Q000235:genetics* / Q000494:pharmacology; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005982:Glutathione Transferase / Q000235:genetics / Q000494:pharmacology; D015801:Helminth Proteins; D006801:Humans; D007814:Larva; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D005810:Multigene Family; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D009854:Onchocerca / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000378:metabolism; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D015801:Helminth Proteins; D009838:Oligodeoxyribonucleotides; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; C076278:Onchocystatin protein, Onchocerca volvulus; D004247:DNA; D005982:Glutathione Transferase; D012697:Serine Endopeptidases