In the present study AP

In the present study, AP31-B preferentially released hydrophobic 3,4-DAA such as leucine and alanine from N-terminal of peptide (Fig. 4). The debittering ability of AP31-B seemed to depend on such substrate specificity, but additional tests against other hydrophobic amino acids are needed to confirm this. Gordon and Speck (1965) reported bitter peptides isolated from milk culture of Streptococcus cremoris, used as yogurt starter, and Barry et al. (2000) reported the debittering of casein tryptically digested with pig kidney AP. The debittering ability of AP31-B suggests its potential utility for dairy food processing such as yogurt production or cheese ripening. In conclusion, in this study we found that P. hubeiensis 31-B, a newly isolated yeast, produced extracellular AP, but we did not detect proteinase activities in the culture filtrate. The AP31-B purified was likely a Leucyl aminopeptidase (EC 3.4.11.1) with properties similar to C. albicans metallopeptidase and aminopeptidase Y from S. cerevisiae. Given the debittering ability and extracellular enzymes observed, this AP may be useful as food additive enzyme for processing of dairy foods such as yogurt or cheese.
Disclosure We have no conflicts of interest. This research was supported by a donation from Amano Enzyme Inc. (Nagoya, Japan). The funding source had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the article for publication. All the experiments undertaken in this study comply with current Japanese law.
Acknowledgments We thank the members of the Higashiyama Botanical Garden, Nagoya, for providing the Nepenthes digestive juices.
MHCI peptide binding and presentation The MHCI antigen processing and presentation pathway samples cellular proteins and presents peptide fragments bound to MHCI molecules for scrutiny by CD8+ T cells (Rock et al., 2016). Many of these peptides are generated after protein degradation by the proteasome, a multi-subunit enzyme that degrades proteins to peptides 4–20 amino acids in length (Cascio et al., 2001). The majority of these peptides are rapidly destroyed by cytosolic aminopeptidases, but a small fraction escapes to the endoplasmic reticulum (ER) through the action of the transporter associated with antigen processing, TAP (Saunders and van Endert, 2011). Most peptides translocated into the ER contain the same C-terminus as the antigenic epitopes finally presented by MHCI, due to similarities of proteasome cleavage sites and MHC peptide binding preferences, but many contain N-terminal extensions that have to be trimmed by ER resident aminopeptidases (ERAPs), before loading onto MHCI molecules (Blanchard et al., 2010). MHCI molecules assemble in the ER with a polymorphic type I integral membrane glycoprotein heavy chain and an invariant soluble light chain (β2 microglobulin, β2m). The heavy chain forms the peptide binding site, a groove on the upper surface of the MHCI molecule (Hewitt, 2003). MHCI residues within the binding groove interact via hydrogen bonds and salt bridges with the free N- and C-termini of peptides, thus limiting binding to (mostly) peptides of 8–9 residues. Side-chain binding pockets within the peptide binding site accommodate two or more residues (anchor residues) of the bound peptide (Neefjes and Ovaa, 2013; Blum et al., 2013). MHCI molecules are highly polymorphic with variation focused in side-chain binding pockets, so that a large variety of different peptides can be bound by various MHCI allelic variants. The loading of the peptide results in stabilization of MHCI-peptide complex, dissociation from the ER peptide loading machinery, and transit through Golgi to the cell surface to become available for immunosurveillance by receptors on CD8+ T cells (Yewdell et al., 2003; Neefjes et al., 2011).
Biological function of ERAP1