Sperm Proteases that May Be Involved in the Initiation of Sperm Motility in the Newt,Cynops pyrrhogaster

A protease of sperm in the newt Cynops pyrrhogaster that is released after the acrosome reaction (AR) is proposed to lyse the sheet structure on the outer surface of egg jelly and release sperm motility-initiating substance (SMIS). Here, we found that protease activity in the sperm head was potent to widely digest substrates beneath the sperm. The protease activity measured by fluorescein thiocarbamoyl-casein digestion was detected in the supernatant of the sperm after the AR and the activity was inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), an inhibitor for serine or cysteine protease, suggesting the release of serine and/or cysteine proteases by AR. In an in silico analysis of the testes, acrosins and 20S proteasome were identified as possible candidates of the acrosomal proteases. We also detected another AEBSF-sensitive protease activity on the sperm surface. Fluorescence staining with AlexaFluor 488-labeled AEBSF revealed a cysteine protease in the principal piece; it is localized in the joint region between the axial rod and undulating membrane, which includes an axoneme and produces powerful undulation of the membrane for forward sperm motility. These results indicate that AEBSF-sensitive proteases in the acrosome and principal piece may participate in the initiation of sperm motility on the surface of egg jelly.     

Crystal Structures of BapA Complexes with β‐Lactam‐Derived Inhibitors Illustrate Substrate Specificity and Enantioselectivity of β‐Aminopeptidases

β‐Aminopeptidases have exclusive biocatalytic potential because they react with peptides composed of β‐amino acids, which serve as building blocks for the design of non‐natural peptidomimetics. We have identified the β‐lactam antibiotic ampicillin and the ampicillin‐derived penicilloic acid as novel inhibitors of the β‐aminopeptidase BapA from Sphingosinicella xenopeptidilytica (K_i values of 0.69 and 0.74 mM , respectively). We report high‐resolution crystal structures of BapA in noncovalent complexes with these inhibitors and with the serine protease inhibitor 4‐(2‐aminoethyl)benzenesulfonyl fluoride. All three inhibitors showed similar binding characteristics; the aromatic moiety extended into a hydrophobic binding pocket of the active site, and the free amino group formed a salt bridge with Glu133 of BapA. The exact position of the inhibitors and structural details of the ligand binding pocket illustrate the specificity and the enantioselectivity of BapA‐catalyzed reactions with β‐peptide substrates.