Peptide : N-glycanase-catalyzed De-N-glycosylation of Glycoproteins in Mammalian Cells

最近，我们提出了这样的假设，即肽：N-聚糖酶（PNGase）催化的糖蛋白去N-糖基化是一个翻译后修饰过程，以调节某些功能蛋白的理化和生理特性。为了验证我们的假设，我们已经预料到在更广泛的活细胞中普遍存在PNGase和PNGase催化的反应，并且我们已经开始研究PNGase在哺乳动物器官和组织中的分布。本研究项目对哺乳动物PNGases的分布和性质进行了如下研究.从小鼠成纤维细胞L-929中纯化出PNGase（命名为L-929 PNGase），并发现其不同于植物和细菌PNGase的独特性质：（1）需要-SH基团;（2）中性和碱性pH最适;（3）受游离N-连接聚糖链的抑制. L-929 PNGase具有糖基化活性， ...更多信息 糖结合活性，表明L-929 PNGase可能具有脱糖基酶和糖结合蛋白的双重作用.建立了以去唾液<14>酸胎球蛋白糖肽I为底物的PNGase活性的简易测定方法，并对去糖肽产物进行了纸层析和纸理论分析.已证实小鼠器官中广泛存在PNGase。可溶性和膜性组分中均检测到PNGase活性，但不同器官的活性水平不同.从猪脾中部分纯化了可溶性PNGases。鉴定了至少四种PNGase，它们在物理化学性质（疏水性和分子量）上彼此不同，表明可溶性PNGase可能构成“PNGase家族”。这些可溶性酶与L-929 PNGase具有某些酶特性（见上文），并且这些可溶性和中性PNGase可能参与某些细胞内非溶酶体事件中的基本生物学过程。少

Recently, we have proposed the hypothesis that peptide : N-glycanase (PNGase) -catalyzed de-N-glycosylation of glycoproteins is a post-translational remodification process to modulate physicochemical and physiological properties of certain functional proteins. In order to validate our hypothesis, we have anticipated the general occurrence of PNGase and PNGase-catalyzed reaction in a wider variety of living cells, and we have initiated to examine the distribution of PNGase among mammalian organs and tissues. In this reserch project, we have carried out the following studies on distribution and properties of mammalian PNGases.1. PNGase was purified to homogeneity from mouse fibroblast L-929 cells (designated as L-929 PNGase) , and its unique properties, which were different from those of plant and bacterial PNGases, were revealed : (1) requirement of-SH group (s) ; (2) neutral and alkaline pH optima ; (3) inhibition by free N-linked glycan chains.2. L-929 PNGase was shown to have carbohy … More drate-binding activity, suggesting that L-929 PNGase may play dual roles as a deglycosylating enzyme and as a carbohydrate-binding protein.3. The simple assay method for PNGase activity using ^<14>C-labeled asialo-fetuin glycopeptide I as a substrate was developed, which was based on the paper chromatographical and paper electrophoretical analyzes of the deglycosylated peptide product.4. A wide occurrence of PNGase in mouse organs was demonstrated. PNGase activities were detected in both soluble and membranous fractions, although the levels of the activities were different from organ to organ.5. Soluble PNGases were partially purified from pig spleen. At least four PNGases were identified, which differed in physicoshemical properties (hydrophobicity and molecular weight) from each other, suggesting soluble PNGases may possibly constitute a "PNGase family" . These soluble enzymes shared some enzymatic properties (see above) with L-929 PNGase, and these soluble and neutral PNGases may be involved in basic biological processes in certain intracellular non-lysosomal events. Less