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

最近，我们提出了一种假说，即PNGase催化的糖蛋白去N-糖基化是一种翻译后再修饰过程，调节某些功能蛋白的物理化学和生理性质。为了验证我们的假设，我们预测了PNGase和PNGase催化的反应在更广泛的活细胞中的普遍发生，并开始研究PNGase在哺乳动物器官和组织中的分布。在本研究项目中，我们对哺乳动物PNGase的分布和性质进行了以下研究。从小鼠成纤维细胞L-929细胞中纯化了PNGase(命名为L-929PNGase)，并揭示了其不同于植物和细菌PNGase的独特性质：(1)需要-SH基团(S)；(2)中性和碱性pH最适；(3)游离N-连接的糖链抑制。L-929PNG酶具有羧基…更强的糖酸结合活性，提示L-929PNGase可能具有脱糖酶和糖结合蛋白的双重作用。在对脱糖肽产物进行纸层析和纸电泳法分析的基础上，建立了以~&lt；14&gt；C标记去唾液酸胎蛋白糖肽I为底物的PNGase活性简易测定方法。PNGase在小鼠各器官中广泛存在。PNGase活性在可溶性组分和膜组分中均有检测到，但不同器官的PNGase活性水平不同。从猪脾中分离纯化了部分可溶性PNGase。至少鉴定出四种PNGase，它们的理化性质(疏水性和相对分子质量)各不相同，这表明可溶性PNGase可能构成一个“PNGase家族”。这些可溶性酶与L-929PNGase有一些相同的酶性质(见上文)，这些可溶性和中性的PNGase可能参与了某些细胞内非溶酶体事件的基本生物学过程。较少

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