Purification and properties of a Golgi-derived (alpha 1,2)-mannosidase-I from baculovirus-infected lepidopteran insect cells (IPLB-SF21AE) with preferential activity toward mannose6-N-acetylglucosamine2.
Purification and properties of a Golgi-derived (alpha 1,2)-mannosidase-I from baculovirus-infected lepidopteran insect cells (IPLB-SF21AE) with preferential activity toward mannose6-N-acetylglucosamine2.
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来自杆状病毒感染的鳞翅目昆虫细胞 (IPLB-SF21AE) 的高尔基衍生 (α 1,2)-甘露糖苷酶-I 的纯化和特性，对甘露糖 6-N-乙酰葡糖胺 2 具有优先活性。
DOI：
10.1021/bi00008a012
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发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Castellino,FJ
中科院分区：
文献类型：
--
作者：
Ren,J;Bretthauer,RK;Castellino,FJ
修订稿于 1994 年 11 月 21 日收到®摘要：由于细胞中加工甘露糖苷酶的可用性和亚细胞分布在确定糖缀合物的最终结构中发挥着如此强大的作用，因此我们希望鉴定、表征和研究受感染和未受感染的鳞翅目昆虫细胞中甘露糖苷酶的可能调节。由于我们之前观察到将Man6GlcNAc2转化为MansGlcNAc2的甘露糖苷酶活性在病毒感染的细胞中增强，从而为进一步加工成复合型寡糖提供了必要的中间体，因此我们尝试纯化这种酶。从重组杆状病毒感染的草地贪夜蛾 (IPLB-SF-21AE) 细胞的膜（操作上定义为高尔基体）中分离和纯化甘露糖苷酶。该蛋白质的分子量约为 63 kDa。通过测量 NaB3H4 还原的 Man6GlcNAc2-ol 到 Man5GlcNAc-[3H] GlcNAc2-ol 的转化进行的测定表明，甘露糖苷酶活性取决于二价阳离子的存在，这对于 pH 6.0 下的 Ca2+ 是最佳的。包含1-脱氧甘露尻霉素在20/iM的浓度下导致50%的抑制，而苦马豆素没有表现出这种抑制。使用对硝基苯基 aD-甘露糖苷 (4 mM) 作为底物时没有观察到活性。优选的还原寡糖底物是Man6GlcNAc2-ol，用Man9GlcNAc2-ol、MangGlcNAc2-ol和Man7GlcNAc2-ol获得的活性较低。使用Man6GlcNAc2-ol作为底物，没有观察到比还原的MansGlcNAc2-ol更小的产物。甘露糖也从糖蛋白卵清蛋白中释放出来。这些特性与 I 型 (al, 2)-Man6-甘露糖苷酶的酶分类一致。 为了进行 Asn 连接糖蛋白加工以提供复合型聚糖，从多甘醇焦磷酸转移到蛋白质中相关 Asn 残基上的 Glc3Man9GlcNAc2* 1* 必须通过葡萄糖苷酶和甘露糖苷酶进行修剪。这些步骤发生在内质网和高尔基体中。蛋白质连接的 Glc3Man9-GlcNAc2 寡糖的加工首先需要去除 Glc，这些步骤发生在粗内质网中（Shailubhai 等人，1991）。已经描述了催化修剪过程的这一阶段的两种膜结合α-葡萄糖苷酶，即葡萄糖苷酶I (Hettkamp等人，1984；Shailubhai等人，1987)和葡萄糖苷酶II (Bums & Touster，1982)。存在多种加工甘露糖苷酶。对 (a1, 2) 连接的 Man 残基具有特异性的甘露糖苷酶通常存在于 ER 以及高尔基体的顺式隔室和内侧隔室中。已经鉴定了几种这样的酶活性，并且其中几种酶已经被纯化和表征[最近的综述，参见Moremen 等人(1994)]。该酶家族的一个成员（大鼠肝 ER 甘露糖苷酶）已在体内实验中显示可催化特定 (al, 2) 连接的 Man 残基的裂解
Revised Manuscript Received November 21, 1994® abstract: Because the availability and subcellulardistribution of processing mannosidases in cells play such powerful roles in determining ultimate structures of glycoconjugates, we desired to identify, characterize, and investigate possible regulation of mannosidases in infected and noninfected lepidopteran insect cells. Since our previous observations that a mannosidase activity that converted Man6GlcNAc2 to MansGlcNAc2 was enhanced in virus-infected cells, thus providing the necessary intermediate for further processing to complex-type oligosaccharides, we attempted purification of this enzyme. A mannosidase was isolated and purified from membranes, operationally defined as Golgi, of recombinant baculovirusinfected Spodoptera frugiperda (IPLB-SF-21AE) cells. The molecular mass of this protein was approximately 63 kDa. Assays performed by measuring the conversion of NaB3H4-reduced Man6GlcNAc2-ol to Man5GlcNAc-[3H] GlcNAc2-ol demonstrated that the mannosidase activity was dependent on the presence of divalent cations, which was optimal for Ca2+ at pH 6.0. Inclusion of 1-deoxymannojirimycin resulted in 50% inhibition at a concentration of 20/iM, whereas swainsonine did not show such inhibition. No activity was observed with p-nitrophenyl aD-mannoside (4 mM) as a substrate. The preferred reduced oligosaccharide substrate was Man6GlcNAc2-ol, with lower activities obtained with Man9GlcNAc2-ol, MangGlcNAc2-ol, and Man7GlcNAc2-ol. With Man6GlcNAc2-ol as substrate, products smaller than reduced MansGlcNAc2-ol were not observed. Mannose was also liberated from the glycoprotein, ovalbumin. These properties are consistent with an enzyme classification as a type I (al, 2)-Man6-mannosidase.In order for Asn-linked glycoprotein processing to provide complex-type glycans, theGlc3Man9GlcNAc2* 1* that is trans-ferred from dolichol pyrophosphate onto relevantAsn residues in proteins must be trimmed by glucosidases and mannosidases. These steps occur in both the ER and the Golgi apparatus. Processing of protein-linked Glc3Man9-GlcNAc2 oligosaccharide first requires removal of Glc, steps that occur in the rough ER (Shailubhai et al., 1991). Two membrane-bound a-glucosidases that catalyze this phase of the trimming process, namely, glucosidase I (Hettkamp et al., 1984; Shailubhai et al., 1987) and glucosidase II (Bums & Touster, 1982), have been described. A variety of processing mannosidases exist. Mannosidases with specificity for (al, 2)-linked Man residues are normally found in the ER and the cis-and medial-compartments of the Golgi. Severalsuch enzymeactivities have been identified, and several of the enzymes have been purified and characterized [for a recent review, see Moremen et al.(1994)]. One member of this enzyme family (rat liver ER mannosidase) has been shown in in vivoexperiments to catalyze cleavage of a specific (al, 2)-linked Man residue