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    • The functional significance of the cap residues with

    2023-05-15

    The functional significance of the cap residues with regard to the established role of PfA-M1 in hemoglobin catabolism in the food vacuole is a key question that is difficult to address with in vitro studies. We note that three of the dipeptide substrates used in our study are found in the sequences of human α- and β-globin: Leu-Ala (5 occurrences), Lys-Ala (two occurrences) and Ala-Leu (7 occurrences). However, we do not know whether these dipeptides are generated in the food vacuole. Perhaps the best way to evaluate the biological significance of the cap residues will be to attempt to introduce mutations in the parasite’s genomic copy of PfA-M1. Previously, we have shown that mutation of a weakly conserved residue that contributes to the formation of the S1 cylinder (V459 in PfA-M1) can dramatically alter the S1 subsite preferences of the enzyme [23]. Here, we have identified a second, independent determinant of S1 subsite specificity. To explore the level of conservation in cap residues, we aligned ten homologous M1-family aminopeptidases from eight genera of apicomplexan parasites. The residue corresponding to E572 was conserved either as an acidic residue (Glu or Asp) or a polar residue (Asn). In contrast, the residue corresponding to M1034 was highly variable, with Val, Ile, Leu, Gln, His and Arg found in this position. Although the functional implications of this natural variation are unknown, it is conceivable that variation of the two cap residues is a means by which the S1 subsite preferences can be modulated in response to selective pressures, allowing the rapid pitavastatin of novel specificities. Finally, we note that there appears to be a context dependence to the specificity imparted through the cap residues. In previous work, we found that the catalytic efficiencies of EcPepN with Arg-Ala and Lys-Ala matched or exceeded those of PfA-M1, even when all four cylinder “wall” residues were identical [23]. However, when we substituted PfA-M1 cap residues with those of EcPepN (i.e., in the E572N/M1034Q mutant), the catalytic efficiencies of the E572N/M1034Q mutant were 5- to 10-fold lower than those of wild-type PfA-M1 (Fig. 3). Clearly, there are factors beyond the identities of the six S1 subsite residues contributing to overall catalytic efficiency.
    Acknowledgments This work was funded by the National Institutes of Health (grant AI077638) and by the Virginia Agricultural Experiment Station and the Hatch Program of the National Institute of Food and Agriculture, U.S. Department of Agriculture (project VA-139761). We appreciate the support of M.R. through a Summer Undergraduate Research Fellowship from Fralin Life Science Institute, Virginia Tech. We are grateful to Feng Li for contributing to the initiation of this project.
    Introduction Porcine epidemic diarrhea virus (PEDV) is a positive single-strand RNA virus in the family Coronaviridae and order Nidovirales. PEDV is the causative agent of porcine epidemic diarrhea (PED), which is characterized by acute watery diarrhea, vomiting, dehydration and high mortality in pigs of all ages, especially in neonatal piglets (Li et al., 2012). PED was first reported in England in 1971 (Wood, 1977), and subsequently spread to other swine production countries. Since 2010, PED outbreaks have swept throughout East Asia and North America and dramatically damaged pig industry (Ding et al., 2014, Kim et al., 2015, Li et al., 2012, Liu et al., 2015, Mole, 2013). Porcine aminopeptidase N (APN), an important entry factor on cell surface, plays a crucial role in PEDV infection (Li et al., 2007, Oh et al., 2003). It is well known that Vero E6 cell is widely used for the isolation and passage of PEDV as well as the model for studies on virus pathogenesis in vitro. In addition, PEDV has also been confirmed to infect other kinds of cell lines, including PK15 cell (pig kidney), huh-7 cell (human liver), Tb1-Lu cell (bat lung) and so on (Liu et al., 2015). However, Vero cell line is recognized to be unable to secrete type I interferon when infected with viruses, due to loss of the type I interferon gene cluster (Ding et al., 2014, Le Bon and Tough, 2002). Therefore, Vero cells are not recommended as the ideal model to investigate viral infection-driven innate immune responses. Although porcine intestine epithelial cells are the target cells for PEDV infection in vivo, the immortalized porcine intestinal epithelial cell clone J2 (IPEC-J2) has a low susceptibility to PEDV infection in vitro (Guo et al., 2016, Luo et al., 2017). Our previous research has demonstrated that HEK293 cells can be efficiently infected by PEDV and APN is involved in the virus infection, which might provide a useful tool for understanding the fundamental mechanisms involved in PEDV infection in vitro (Zhang et al., 2017). Moreover, HEK293 cells have been widely used in cell biology research for many years because of their reliable growth and propensity for transfection. Thus, the HEK293 cell as a new cell model is essential to gain a better understanding of the mechanisms of PEDV infection in vitro.