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Multiple isoforms of UDP-GalNAc:polypeptide N-acetylgalactosaminyl-
transferase (ppGaNTase) have been cloned and expressed from a variety of
organisms. In general, these isoforms display different patterns of
tissue-specific expression, but exhibit overlapping substrate
specificities, in vitro . A peptide substrate, derived from the sequence of
the V3 loop of the HIV gp120 protein (HIV peptide), has previously been
shown to be glycosylated in vitro exclusively by the ppGaNTase-T3 (Bennett
et al. , 1996). To determine if this isoform- specificity is maintained in
vivo , we have examined the glycosylation of this substrate when it is
expressed as a reporter peptide (rHIV) in a cell background (COS7 cells)
which lacks detectable levels of the ppGaNTase-T3. Glycosylation of rHIV
was greatly increased by coexpression of a recombinant ppGaNTase-T3.
Overexpression of ppGaNTase- T1 yielded only partial glycosylation of the
reporter. We have also determined that the introduction of a proline
residue at the +3 position flanking the potential glycosylation site
eliminated ppGaNTase- T3 selectivity toward rHIV observed both in vivo and
in vitro .
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Stábeli RG Sant'Ana CD Ribeiro PH Costa TR Ticli FK Pires MG Nomizo A Albuquerque S Malta-Neto NR Marins M Sampaio SV Soares AM 《International journal of biological macromolecules》2007,41(2):132-140
An L-amino acid oxidase isolated from Bothrops moojeni snake venom (BmooLAAO-I) was purified to a high degree using sequential CM-Sepharose ion-exchange and phenyl-Sepharose chromatography. When analyzed by mass spectrometry, the purified BmooLAAO-I presented a molecular weight of 64,889 and 130,779 under denaturing and nondenaturing conditions, respectively. BmooLAAO-I is a homodimeric acidic glycoprotein with a pI approximately 4.7, and the N-terminal sequence shows close structural similarity to other snake venom LAAOs. This enzyme was inactivated by freezing or low pH, and secondary structural analysis by circular dichroism revealed 48% alpha-helix, 20% beta-sheet, 12% beta-turn, and 20% random coil structures. BmooLAAO-I exhibited bactericidal, antitumoral, trypanocidal, edematogenic, and platelet-aggregating activities. All of these effects were inhibited by catalase, suggesting that these biological effects are mediated by the production of H(2)O(2). BmooLAAO-I induced typical apoptotic DNA fragmentation in HL-60 cells, which was also inhibited by catalase. These results point to the potential use of BmooLAAO-I as a therapeutic agent for treatment of diseases in which induction of H(2)O(2) production can be beneficial. 相似文献
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França SC Kashima S Roberto PG Marins M Ticli FK Pereira JO Astolfi-Filho S Stábeli RG Magro AJ Fontes MR Sampaio SV Soares AM 《Biochemical and biophysical research communications》2007,355(2):302-306
Two l-amino acid oxidases (LAAOs) were identified by random sequencing of cDNA libraries from the venom glands of Bothrops moojeni(BmooLAAO) and Bothrops jararacussu(Bjussu LAAO). Phylogenetic analysis involving other SV-LAAOs showed sequence identities within the range 83-87% being closely related to those from Agkistrodon and Trimeresurus. Molecular modeling experiments indicated the FAD-binding, substrate-binding, and helical domains of Bmoo and Bjussu LAAOs. The RMS deviations obtained by the superposition of those domains and that from Calloselasma rhodostoma LAAO crystal structure confirm the high degree of structural similarity between these enzymes. Purified BjussuLAAO-I and BmooLAAO-I exhibited antiprotozoal activities which were demonstrated to be hydrogen-peroxide mediated. This is the first report on the isolation and identification of cDNAs encoding LAAOs from Bothrops venom. The findings here reported contribute to the overall structural elucidation of SV-LAAOs and will advance the understanding on their mode of action. 相似文献
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Sant' Ana CD Ticli FK Oliveira LL Giglio JR Rechia CG Fuly AL Selistre de Araújo HS Franco JJ Stabeli RG Soares AM Sampaio SV 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2008,151(3):443-454
A thrombin-like enzyme named BjussuSP-I, isolated from B. jararacussu snake venom, is an acidic single chain glycoprotein with approximately 6% sugar, Mr=61,000 under reducing conditions and pI approximately 3.8, representing 1.09% of the chromatographic A(280) recovery. BjussuSP-I is a glycosylated serine protease containing both N-linked carbohydrates and sialic acid in its structure. BjussuSP-I showed a high clotting activity upon human plasma, which was inhibited by PMSF, leupeptin, heparin and 1,10-phenantroline. This enzyme showed high stability regarding coagulant activity when analyzed at different temperatures (-70 to 37 degrees C), pHs (4.5 to 8.0), and presence of two divalent metal ions (Ca(2+) and Mg(2+)). It also displayed TAME esterase and proteolytic activities toward natural (fibrinogen and fibrin) and synthetic (BAPNA) substrates, respectively, being also inhibited by PMSF and leupeptin. BjussuSP-I can induce production of polyclonal antibodies able to inhibit its clotting activity, but unable to inhibit its proteolytic activity on fibrinogen. The enzyme also showed crossed immunoreactivity against 11 venom samples of Bothrops, 1 of Crotalus, and 1 of Calloselasma snakes, in addition of LAAO isolated from B. moojeni venom. It displayed neither hemorrhagic, myotoxic, edema-inducing profiles nor proteolytic activity on casein. BjussuSP-I showed an N-terminal sequence (VLGGDECDINEHPFLA FLYS) similar to other thrombin-like enzymes from snake venoms. Based on its biochemical, enzymatic and pharmacological characteristics, BjussuSP-I was identified as a new thrombin-like enzyme isoform from Bothrops jararacussu snake venom. 相似文献
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