| Protein tyrosine phosphatases (PTPase) are a family of Anportan enzymes inthe cells. They have been implicated as key players in signaiing pathway controllinggrowh, differeniation, metabolism, cell cycle regulation and cytoskeletal function.They catalyze specifically phosphate hydrlysis of phosphotyrosine and actstogether with protein kinases to control the extent of protein tyrosinephosphorylation. Some baceria (such as Yersinia genus) and viruses (such as poxviruses) express PTPases, which are related to their infectivity and pathogenesis. Ofwhich, the PTPase encoded by vaccinia virus is dual specific in substrae, i.e.catalyzes the dephosphorylation of both tyrosine- and serine/thronine-phosphorylated proteins.Some baculoviruses posses open reading frames encoding protein tyrosinephosphatases in their genomes. of which, the PTPase gene of AcMNPV has beencloned and sequenced, and its encoded protein contains the conserved "HC" activesite motif, (I/V) HCXAGXXR(S/T)G, Which is a distinctive featUr of the PTPasefamily, and is dual specific in catalysis activity hydrolyzing substrates containing notonly phosphotyrosine but also phosphoserine/threonine. In this paper, a PTPase geneframent was generated using the polymerase chain reaction (PCR) from thegenome of BombN mori nuclear polyhedrosis virus Zhenjiang staln (BmNPV-ZJ).Oligonucleotide primers for PCR were based on the nucleotide sequences of thegenomes of AcMNPV and BmNPV-T3 strain. The nucleotide sequence of theamplified fragment was determined, and compared with its homologues of otherbaculoviruses. Then the gene was inserted into an expression plasmid and expressedin bacterium Escherichia coli.The coding region of the BANPV-ZJ PTPase gene consists of 507 nucleotideswith the base count of 163 A, 99 C, 113 G, 132 T and a G+C content of 42%, andencodes a protein of 168 amino acid residues. The amino acid -sequence deducedfrom the nucleotide sequence coniains the conserved 11 amno acid "HC" motif(residues 117-127) characterizing PTPase family Wth nucleotide sequencecomparison, BmNPV-ZJ PTPase gene shows 96.8% identity with its AcMNPVhomologue and 98.2% identity with BInNPV-T3. The newly determined sequencehas been submitted to GenBank and the accession number is AF3 16871.The identity of BmNPV-ZJ PTPase to the PTPases of AcMNPV BmNPV-T3,AWPV (Anagrapha falcifera multicapsid nuclear polyhedrosis virus) and PTPase-1 of OpMNPV (Orgyia Pseudotsugata multicapsid nuclear polyhedrosis virus) is97%, 97.6%, 96% and 60% respectively, and that to OpMNPV-2 only 20%.By searching homologous sequences from the 599,38l amino acid sequences inNCBI data base, at least l4 mRNA caPping enZymes were found to contain thePTPase active site "HC" motif at the region of their N-tCrminals, such as humanmRNA caPping enZyIne l- HCAPl, HOmo SaPiens mRNA caPPing enZyIne lA -HCAPlA, mouse InRNA caPping enZyme-MCE, Caenorhabdnd eleganS InRNAcaPping enZyme- CeCAP etc. Howevr, their identity in complete arnino acidsequence is only 3l%-32%.BmNPV-ZJ PTPase gene was inserted into the EcoR I- and BamH I-digestedexpression plasmid pBV22l and the resulting p1asmid was transformed into E cQlistrain JMl09 for exprestion of the PTPase under control of the temperAn sensitivePRPL promoter A l9 ku protein, identical tO the predicted molecular mass, wasexpressed and the SDS-PAGE analysis demonStrates that the protein is produced toan amoUn aboat 2.2% of the total bacterial cellular proteins. ExPressed productsdephosphorylated PNPP, a substrae utilized by most phoSPhataSes, in a reaction thatwas linear with respect to time and the amowt of enzyme added. The activity wasalmost comPletely inhibited by 200 ll moim vanadate, an effective iambitor ofPTPases, and by 1 nunol/L ZnCl,, Which inhibitS some but not all euknyoticPTPases.(II )Interleukin-ll (IL-1l) is a pleiotropic cytokine with importan biologicalactivities in the hematopoietic microenvirotunent... |
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