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

肽：N-聚糖酶催化哺乳动物细胞中糖蛋白的去-N-糖基化

• 批准号: 05808053
• 负责人: KITAJIMA Ken
• 金额: $ 1.22万
• 依托单位: THE UNIVERSITY OF TOKTO
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1994
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05808053/
• 关键词: PNGase (Peptide : N-glycanase); Post-translational Modification Of Proteins; Mammalian PNGase; L-929 PNGase; De-N-glycosylation Of Glycoproteins; Biological Function Of PNGase; Carbohydrate-Binding Activity; PNGase Family; PNGase(ペプチド:N-ブリカナーゼ)

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

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

项目成果

SUZUKI TADASHI: "Purification and Enzymatic Properties of Peptide:N-Glycanase (PNGase) from C3H Mouse-derived L-929 Fibroblast Cells." The Journal of Biological Chemistry. 269. 17611-17618 (1994)

SUZUKI TADASHI：“来自 C3H 小鼠 L-929 成纤维细胞的肽：N-聚糖酶 (PNGase) 的纯化和酶学特性。”

SUZUKI TADASHI: "N-Glycosylation/deglycosylation as a mechanism for the post-translational modification/remodification of proteins" Glycoconjugate Journal. 12(印刷中). (1995)

SUZUKI TADASHI：“N-糖基化/去糖基化作为蛋白质翻译后修饰/再修饰的机制”，Glycoconjugate Journal 12（出版中）。

SUZUKI TADASHI: "Purification and enzymatic properties of peptide N-glycanase from C3H mouse-derived L-929 fibroblast cells. Possible widespread occurrence of posttranslational remodification of proteins by N-deglycosylation" J.Biol. Chem.269-26. 17611-17

SUZUKI TADASHI：“来自 C3H 小鼠来源的 L-929 成纤维细胞的肽 N-聚糖酶的纯化和酶学特性。N-去糖基化对蛋白质的翻译后修饰可能广泛发生”J.Biol。

SUZUKI TADASHI: "N-Glycosylation/deglycosylation as a mechanism for the post-translational modification/remodification of proteins" Glycoconjugate Journal. 12(発表予定). (1995)

SUZUKI TADASHI：“N-糖基化/去糖基化作为蛋白质翻译后修饰/再修饰的机制”，Glycoconjugate Journal 12（待出版）。

KITAJIMA KEN: "Identification and distribution of peptide:N-glycanase (PNGase) in mouse organs:Demonstration of ubiquitous occurrence of PNGase activities in animal cells" Archives of Biochemistry and Biophysics. (発表予定). (1995)

KITAJIMA KEN：“小鼠器官中肽：N-聚糖酶（PNGase）的识别和分布：动物细胞中普遍存在的 PNGase 活性的证明”生物化学和生物物理学档案（即将出版）。