The metalloproteinases/disintegrins in the snake venom act as platelet aggregation inhibitor by an antagonism against integrin on platelet through its RGD sequence and may play other important role in cell-cell fusion, cell matrix interaction and other cellular function. Ussurin is a new metalloproteinase/disintegrin that was cloned from Gloydius ussuriensis. Poly (A+) RNA was purified from the total RNA preparation from venom gland of a single G. ussuriensis using the poly (A+) tract-mRNA isolation system. A cDNA library was constructed with the SMART PCR cDNA library construction kit. The cDNA library was screened and the positive clones were selected. The full-length cDNA of Ussurin was obtained. The cDNA encoding the Ussurin precursor has a 51bp 5'-UTR, the open reading frame of Ussurin and a 490 bp 3'-UTR, the open reading frame of Ussurin cDNA nucleotide sequence is 1434 bp and codes for 478 amino acids with a predicted molecular mass of 53.2 kD and an isoelectric point of 5.37. There is no potential N-glycosylation site in the deduced sequence region. Its deduced amino acid sequence consists of four region, a signal sequence of 18 amino acid residues, a zymogen pro-peptide of 171 amino acid residues with a cysteine switch motif (PK-MCGVT) in it, a central metalloproteinase domain of 201 amino acid residues containing a conserved zinc-chelating sequence (HEXXHXXGXXH) and a methionine-turn CIM involving zinc banding also, a space sequence between metalloproteinase domain and disintegrin domain of 15 amino acid residues with a conserved T392, T397, S400, which is specific residues of the P-II snake venom metalloproteinases, a disintegrin domain of 73 amino acid residues with a characteristic RGD region and six-disulfide bonds. Ussurin belongs to P-II class. The cDNA sequence and deduced amino acid sequence of Ussurin precursor were compared with homologous sequence in the GenBank database, the result reveals high degree of homology in sequence and organization pattern of domain with metalloproteinase/disintegrin gene family of other snake species. Compared with the alignment of amino acid sequence of metalloproteinase/disintegrin member, hypervariable regions of this member were revealed, besides they share higher homologous in the zymogen domain. It suggests that the hypervariable regions are the counterparts directly suitable for interacting with different domain of receptors, different receptors or substrates.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; Disintegrins ; chemistry ; genetics ; metabolism ; Metalloproteases ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Sequence Alignment ; Viper Venoms ; enzymology ; genetics ; Viperidae ; genetics ; metabolism

Trichomonas vaginalis secretes a number of proteases which are suspected to be the cause of pathogenesis; however, little is understood how they manipulate host cells. The mammalian target of rapamycin (mTOR) regulates cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription. We detected various types of metalloproteinases including GP63 protein from T. vaginalis trophozoites, and T. vaginalis GP63 metalloproteinase was confirmed by sequencing and western blot. When SiHa cells were stimulated with live T. vaginalis, T. vaginalis excretory-secretory products (ESP) or T. vaginalis lysate, live T. vaginalis and T. vaginalis ESP induced the mTOR cleavage in both time- and parasite load-dependent manner, but T. vaginalis lysate did not. Pretreatment of T. vaginalis with a metalloproteinase inhibitor, 1,10-phenanthroline, completely disappeared the mTOR cleavage in SiHa cells. Collectively, T. vaginalis metallopeptidase induces host cell mTOR cleavage, which may be related to survival of the parasite.
Blotting, Western ; Cell Line, Tumor ; Epithelial Cells/metabolism/parasitology ; Humans ; Metalloproteases/genetics/*metabolism ; Proteolysis ; Sequence Analysis, DNA ; TOR Serine-Threonine Kinases/*metabolism ; Trichomonas vaginalis/*enzymology/genetics

This study is to investigate the effect of Euphorbia humifusa effective fraction (EHEF) on the CYP51 enzyme activity, the lanosterol content and the MEP, SUB gene expression of Trichophyton rubrum. Trichophyton rubrum was treated by EHEF for 7 days at 26 degrees C. The activity of CYP51 enzyme of Trichophyton rubrum in the cell membrane was determined by using ELISA kit, and the lanosterol content was investigated by using high performance liquid chromatography (HPLC), and the MEP, SUB gene expression of Trichophyton rubrum was detected with the reverse transcription polymerase chain reaction (RT-PCR) method. Results showed that EHEF can decrease the membrane CYP51 enzyme activity, and it also can accumulate the fungal lanosterol in a dose-dependent manner, and it also can decrease the gene expression of MEP and SUB. The antifungal mechanism of EHEF may be related to the inhibition on CYP51 enzyme activity, and to the effects on fungal cell membrane ergosterol biosynthesis. It may also play an antifungal effect by inhibiting the MEP, SUB gene expression of fungal proteases.
Antifungal Agents ; isolation & purification ; pharmacology ; Cell Membrane ; drug effects ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Enzyme Activation ; drug effects ; Euphorbia ; chemistry ; Gene Expression Regulation, Fungal ; Lanosterol ; metabolism ; Metalloproteases ; metabolism ; Plants, Medicinal ; chemistry ; Sterol 14-Demethylase ; metabolism ; Subtilisins ; metabolism ; Trichophyton ; drug effects ; genetics ; metabolism