Suzuki T, Kitajima K, Inoue S, Inoue Y
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, Japan.
Glycoconj J. 1994 Oct;11(5):469-76. doi: 10.1007/BF00731283.
Recently, we have reported purification and characterization of a de-N-glycosylating enzyme, peptide: N-glycanase (PNGase) found in C3H mouse fibroblast L-929 cells, and designated L-929 PNGase [Suzuki T, Seko A, Kitajima K, Inoue Y, Inoue S (1994) J Biol Chem 269, 17611-18]. The unique properties of L-929 PNGase are that the enzyme had a high affinity to the substrate glycopeptide (e.g. Km = 114 microM for fetuin derived glycopentapeptide) and that the PNGase-catalysed reaction is strongly inhibited by the released free oligosaccharides but not by the free peptides formed, suggesting that L-929 PNGase is able to bind to a certain type of carbohydrate chain. In this study, we report the new findings of the mannan-binding property of L-929 PNGase: the de-N-glycosylating enzyme activity of L-929 PNGase was inhibited by yeast mannan and triomannose, Man alpha 1-->3(Man alpha 1-->6)Man, but not by mannose and alpha-methyl-D-mannoside. Furthermore, L-929 PNGase was revealed to bind to the glycan moiety of yeast mannan by using mannan-conjugated Sepharose 4B gel as a ligand, suggesting that L-929 PNGase could serve not only as an enzyme but also as a carbohydrate recognition protein in vivo. Such 'dual' properties found for animal-derived L-929 PNGase are unique and are not shared with other previously characterized plant- and bacterial-origin PNGases--PNGase A and PNGase F, respectively.
最近，我们报道了在C3H小鼠成纤维细胞L-929中发现的一种去N-糖基化酶——肽：N-聚糖酶（PNGase）的纯化和特性，并将其命名为L-929 PNGase [铃木T，世耕A，北岛K，井上Y，井上S（1994）《生物化学杂志》269，17611 - 18]。L-929 PNGase的独特性质在于，该酶对底物糖肽具有高亲和力（例如，对胎球蛋白衍生的糖五肽的Km = 114 microM），并且PNGase催化的反应受到释放的游离寡糖的强烈抑制，但不受形成的游离肽的抑制，这表明L-929 PNGase能够与某种类型的碳水化合物链结合。在本研究中，我们报道了L-929 PNGase甘露聚糖结合特性的新发现：L-929 PNGase的去N-糖基化酶活性受到酵母甘露聚糖和三甘露糖（Manα1→3(Manα1→6)Man）的抑制，但不受甘露糖和α-甲基-D-甘露糖苷的抑制。此外，通过使用甘露聚糖偶联的琼脂糖4B凝胶作为配体，发现L-929 PNGase与酵母甘露聚糖的聚糖部分结合，这表明L-929 PNGase在体内不仅可以作为一种酶，还可以作为一种碳水化合物识别蛋白。动物来源的L-929 PNGase所具有的这种“双重”性质是独特的，与之前分别鉴定的植物和细菌来源的PNGase——PNGase A和PNGase F不同。
