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  • However relatively little information is available

    2024-05-15

    However, relatively little information is available concerning the biochemical properties and other features of such enzymes in Taeniid species. In the current report, a M17 leucine aminopeptidase gene of T. pisiformis (TpLAP) was cloned and expressed, and its enzymatic activity, biochemical properties, and distribution were characterized. This study may provide valuable information on potential applications for the development of anti-cestode drugs and vaccines.
    Materials and methods
    Results
    Discussion In the present study, a full-length cDNA of the leucine aminopeptidase gene from adult T. pisiformis (TpLAP) was obtained and we performed the bioinformatic analysis, expression, enzymatic characterization and immunolocalization of the TpLAP protein. Bioinformatic analysis showed that TpLAP had characteristic domains of the M17LAP family at the C-terminus (Fig. 1), including the metal binding site 265-[VGKG]-271 and catalytic domain motif 351-[NTDAEGRL]-357 (Rawlings et al., 2006). In addition, TpLAP exhibited biological functions similar to those of the M17LAP family, such as hydrolyzing the Leu-pNA substrate and inhibition of enzymatic activity by bestatin. It has been reported that fusing LAP with GST did not affect enzymatic activity due to the GST tag not significantly altering the quaternary conformation of the enzyme (Aboge et al., 2015, Jia et al., 2009). As for the biochemical analysis of recombinant TpLAP, the soluble GST-TpLAP (≈83.5kDa) was used for assaying catalytic activity. The results showed that the optimal pH of the purified GST-TpLAP for hydrolyzing Leu-pNA was around 9.5 and the temperature for specific activity was 45 °C, at which point GST-TpLAP had a maximum activity of 46 U/ml. GST-TpLAP was relatively stable under alkaline conditions (pH 8.5–10.0), but unstable in acidic and neutral GS-9620 sale (pH 2.7–7.5) or at temperatures higher than 55 °C. Our findings revealed that the TpLAP identified in this study shared similar biochemical properties with other LAPs from known organisms (Cappiello et al., 2006, Cottrell et al., 2000, Stack et al., 2007), indicating that TpLAP is a new member of the M17LAP family. Several studies have confirmed that divalent cations are indispensable for metallopeptidase activity, and M17LAP activity is enhanced by their addition and suppressed by metal chelating agents. Biochemical analysis showed that GST-TpLAP displayed effective hydrolytic activity against a Leu-pNA substrate, which was almost completely inhibited by the known aminopeptidase inhibitor bestatin and EDTA. We further determined that GST-TpLAP activity exhibits metal-ion dependence and is enhanced by 1.0 mM divalent metal cations with in the order of Mn2+>Co2+>Mg2+>Zn2+>Ca2+>Ba2+. The known crystal structures have shown that M17LAPs contain two metal-binding sites in the active site of each subunit, site 1 (an activation site) and site 2 (a specificity site). Site 1 can be replaced by Mn2+, Co2+ and Mg2+ ions (Carpenter and Vahl, 1973, Maric et al., 2009, McGowan et al., 2010). Here, we found a significant increase in GST-TpLAP activity by the addition of Mn2+ and Co2+, which were consistent with the previous reports for M17LAPs of other parasites (Aboge et al., 2015, Acosta et al., 2008, Jia et al., 2010). Particularly, Mn2+ could be viewed as a major metal cofactor for GST-TpLAP, as it is also a potent activator typically utilized in the animal, microbial and plant LAPs (Matsui et al., 2006). These results of the enzymatic and biochemical properties of GST-TpLAP might facilitate exploration of inhibitor development. However, further investigation is warranted, especially for elucidating the key features within the TpLAP active sites, the side-chain function, and the inhibitor-binding mode to design unique and more effective TpLAP inhibitors. Interestingly, TpLAP was found to be a stage-differentially expressed protein in this study, which was confirmed by qRT-PCR, western blotting and immunolocation assay. The TpLAP transcripts were expressed in both the larval and adult stages, and the expression level gradually increased with worm maturity. The mRNA level in gravid proglottids (50.71-fold) and immature proglottids (10.36-fold) of adult worms were higher than those in larvae (P < .01), indicating that the TpLAP play a vital part in the growth and development of adult worms. Western blot revealed that the native TpLAP was confirmed as a specific constituent of ESPs from adult T. pisiformis. LAPs activity has been detected in ESPs of several helminth parasites, such as S. japonicum and F. hepatica (Acosta et al., 1998, Deng et al., 2012, Dvorak et al., 2016, Liu et al., 2009). It also exists in oncosphere ESPs from T. solium and T. saginata (Zimic et al., 2007). Helminths secrete abundant ESPs during their life in the host and ES proteomic analyses have shown that adult secretions are predominately proteases (Cao et al., 2016, Chehayeb et al., 2014, Gomez et al., 2015, Khan et al., 2017). Recently, the parasite ESPs were reported to play a major role in the host-parasite interface, which promotes worm survival and longevity in the mammalian host by modulating the host immune system and facilitates evasion from host immune attacks (Hewitson et al., 2009, Maizels and McSorley, 2016, Maizels and Yazdanbakhsh, 2003, Spolski et al., 2000). Theoretically, this modulation could be achieved by the interaction of the soluble mediators released from the parasite with the host immune cells (Lightowlers and Rickard, 1988). It has been reported that helminth ES62, a homologue of the aminopeptidases, has broad immunomodulatory activities and is a promising chemotherapeutic target (Harnett et al., 2004). Those results indicate that TpLAP is enriched at the adult stage and may have a specific function, which requires further evaluation, in modulating the immune response of host cells.