大鼠肝脏胞质α-D-甘露糖苷酶的底物特异性。寡甘露糖型聚糖的新型降解途径。

Substrate specificity of rat liver cytosolic alpha-D-mannosidase. Novel degradative pathway for oligomannosidic type glycans.

作者信息

Haeuw J F, Strecker G, Wieruszeski J M, Montreuil J, Michalski J C

机构信息

Laboratoire de Chimie Biologique, UMR du CNRSn. 111, Université des Sciences et Techniques de Lille Flandres-Artois, Villeneuve d'Ascq, France.

出版信息

Eur J Biochem. 1991 Dec 18;202(3):1257-68. doi: 10.1111/j.1432-1033.1991.tb16498.x.

DOI:10.1111/j.1432-1033.1991.tb16498.x

PMID:1837268

Abstract

The substrate specificity of rat liver cytosolic neutral alpha-D-mannosidase was investigated by in vitro incubation with a crude cytosolic fraction of oligomannosyl oligosaccharides Man9GlcNAc, Man7GlcNAc, Man5GlcNAc I and II isomers and Man4GlcNAc having the following structures: Man9GlcNAc, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-2)Man(alpha 1-6)]Man(alpha 1-6) [Man(alpha 1-2)Man(alpha 1-3)]Man(beta 1-4)GlcNAc; Man5GlcNAc I, Man(alpha 1-3)[Man(alpha 1-6)]-Man(alpha 1-6)Man(alpha 1-3)] Man(beta 1-4)GlcNAc; Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3) [Man(alpha 1-6)]Man(beta 1-4)GlcNAc; Man4GlcNAc, Man(alpha 1-2)Man(alpha 1-2)Man(alpha 1-3)Man(beta 1-4)GlcNAc. The different oligosaccharide isomers resulting from alpha-D-mannosidase hydrolysis were analyzed by 1H-NMR spectroscopy after HPLC separation. The cytosolic alpha-D-mannosidase activity is able to hydrolyse all types of alpha-mannosidic linkages found in the glycans of the oligomannosidic type, i.e. alpha-1,2, alpha-1,3 and alpha-1,6. Nevertheless the enzyme is highly active on branched Man9GlcNAc or Man5GlcNAc I oligosaccharides and rather inactive towards the linear Man4GlcNAc oligosaccharide. Structural analysis of the reaction products of the soluble alpha-D-mannosidase acting on Man5-GlcNAc I and Man9GlcNAc gives Man3GlcNAc, Man(alpha 1-6)[Man(alpha 1-3)]Man(beta 1-4)GlcNAc, and Man5GlcNAc II oligosaccharides, respectively. This Man5GlcNAc II, Man(alpha 1-2)Man(alpha 1-3)[Man(alpha 1-6)]Man(beta 1-4)GlcNAc, represents the 'construction' Man5 oligosaccharide chain of the dolichol pathway formed in the cytosolic compartment during the biosynthesis of N-glycosylprotein glycans. The cytosolic alpha-D-mannosidase is activated by Co2+, insensitive to 1-deoxymannojirimycin but strongly inhibited by swainsonine in the presence of Co2+ ions. The enzyme shows a highly specific action different from that previously described for the lysosomal alpha-D-mannosidases [Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]. A possible complementarity between cytosolic and lysosomal alpha-D-mannosidase activities in the catabolism of N-glycosylprotein is proposed.

摘要

通过将大鼠肝脏胞质中性α-D-甘露糖苷酶与低聚甘露糖基寡糖Man9GlcNAc、Man7GlcNAc、Man5GlcNAc I和II异构体以及具有以下结构的Man4GlcNAc的粗胞质级分进行体外孵育，研究了其底物特异性：Man9GlcNAc，Man(α1-2)Man(α1-3)[Man(α1-2)Man(α1-6)]Man(α1-6)[Man(α1-2)Man(α1-3)]Man(β1-4)GlcNAc；Man5GlcNAc I，Man(α1-3)[Man(α1-6)]-Man(α1-6)Man(α1-3)]Man(β1-4)GlcNAc；Man5GlcNAc II，Man(α1-2)Man(α1-2)Man(α1-3)[Man(α1-6)]Man(β1-4)GlcNAc；Man4GlcNAc，Man(α1-2)Man(α1-2)Man(α1-3)Man(β1-4)GlcNAc。在HPLC分离后，通过1H-NMR光谱分析α-D-甘露糖苷酶水解产生的不同寡糖异构体。胞质α-D-甘露糖苷酶活性能够水解低聚甘露糖型聚糖中发现的所有类型的α-甘露糖苷键，即α-1,2、α-1,3和α-1,6。然而，该酶对分支的Man9GlcNAc或Man5GlcNAc I寡糖具有高活性，而对线性的Man4GlcNAc寡糖活性较低。可溶性α-D-甘露糖苷酶作用于Man5-GlcNAc I和Man9GlcNAc的反应产物的结构分析分别得到Man3GlcNAc、Man(α1-6)[Man(α1-3)]Man(β1-4)GlcNAc和Man5GlcNAc II寡糖。这种Man5GlcNAc II，Man(α1-2)Man(α1-3)[Man(α1-6)]Man(β1-4)GlcNAc，代表了在N-糖基化蛋白聚糖生物合成过程中在胞质区室形成的多萜醇途径的“构建”Man5寡糖链。胞质α-D-甘露糖苷酶被Co2+激活，对1-脱氧甘露基野尻霉素不敏感，但在Co2+离子存在下被苦马豆素强烈抑制。该酶显示出与先前描述的溶酶体α-D-甘露糖苷酶[Michalski, J.C., Haeuw, J.F., Wieruszeski, J.M., Montreuil, J. and Strecker, G. (1990) Eur. J. Biochem. 189, 369-379]不同的高度特异性作用。提出了在N-糖基化蛋白分解代谢中胞质和溶酶体α-D-甘露糖苷酶活性之间可能的互补性。