Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis

Two different types of peptide:N-glycanase (PNGase) were identified in deve loping embryos of medaka fish (Oryzias latipes). Because the optimum pH val ues for their activities were acidic and neutral, they were designated as a cid PNGase RI and neutral PNGase M, respectively, The acid PNGase M corresp onded to the enzyme that had been partially purified from medaka embryos (S eko,A., Kitajima,K., Inoue,Y, and Inoue,S (1991) J. Biol. Chem., 266, 22110 -22114). The apparent molecular weight of this enzyme was 150 K, and the op timal pH was 3.5-4.0, and the K-m for L-hyosophorin was 44 mu M. L-Hyosopho rin is a cortical alveolus-derived glycononapeptide with a large N-linked g lycan chain present in the perivitelline space of the developing embryo. Ac id PNGase M was competitively inhibited by a free de-N-glycosylated nonapep tide derived from L-hyosophorin. This enzyme was expressed in ovaries and e mbryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gast rula. Several independent lines of evidence suggested that acid PNGase M ma y be responsible for the unusual accumulation of free N-glycans derived fro m yolk glycoproteins (Imasaki,M., Seko,A,, Kitajima,K., Inoue,Y, and Inoue, S, (1992) J, Biol, Chem., 267, 24287-24296). In contrast, the neutral PNGas e RI was expressed in blastoderms from the 4-8 cell stage and in cells up t o early gastrula. The general significance of these findings is that they s how a developmental stage-dependent expression of the two PNGase activities , and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-s tage-related de-N-glycosylation of the L-hyosophorin.