Cysteine mutations in the MAM domain result in monomeric meprin and alter stability and activity of the proteinase
Cysteine mutations in the MAM domain result in monomeric meprin and alter stability and activity of the proteinase
复制标题
DOI：
10.1074/jbc.271.39.24236
复制
发表时间：
1996-09-27
影响因子：
4.8
通讯作者：
Bond, JS
中科院分区：
文献类型：
--
作者：
Marchand, P;Volkmann, M;Bond, JS
金属蛋白酶是寡聚的、糖基化的细胞表面或分泌的金属内肽酶，其由多结构域二硫键连接的亚基组成。为了研究亚基寡聚化是否对细胞内转运或蛋白酶的酶促和/或物理性质至关重要，对特定的半胱氨酸残基进行突变，并在293细胞中表达突变体。小鼠meprin α Cys-320在MAM结构域（在meprin中发现的胞外结构域，（A）在bar-5蛋白和受体蛋白酪氨酸磷酸酶下）突变为Ala导致meprin单体形式的表达，如SDS-聚丙烯酰胺凝胶电泳和非变性凝胶电泳所测定。与寡聚体形式的酶相比，单体亚基在体外更容易发生蛋白水解降解和热失活。使用缓激肽类似物或氨基苯甲酰基-Ala-Ala Phe-对硝基苯胺作为底物的单体meprin的蛋白水解活性与二硫键连接的低聚meprin的蛋白水解活性相似;然而，对偶氮酪蛋白的活性显著降低。MAM结构域中的另一个半胱氨酸残基（C289 A）突变，预测参与亚基内二硫键桥接，导致二硫键连接的寡聚体和单体。这些结果表明，该突变体能够形成亚基间二硫键，但效率低于野生型meprin亚基。突变体C289 A也保留了对肽的活性，但不是蛋白质底物，并且与野生型酶相比更容易发生蛋白水解降解和热失活。两种Cys突变体均以与野生型相当的水平表达并分泌到培养基中，并且具有轻微改变的糖基化。结果表明：（1）小鼠meprin α的Cys-320可能与亚基间的共价相互作用有关，（2）亚基间的共价二聚化对分泌蛋白酶的有效生物合成、运输或翻译后加工不是必需的;和3）MAM结构域中的突变影响亚基的非共价相互作用和蛋白酶结构域的稳定性和活性，表明结构域-结构域相互作用对酶的结构和功能至关重要。
Meprins are oligomeric, glycosylated cell surface or secreted metalloendopeptidases that are composed of multidomain disulfide-linked subunits. To investigate whether subunit oligomerization is critical for intracellular transport or for the enzymatic and/or physical properties of the proteinase, specific cysteine residues were mutated, and the mutants were expressed in 293 cells. Mutation of mouse meprin alpha Cys-320 to Ala in the MAM domain (an extracellular domain found in meprin, (A) under bar-5 protein, and receptor protein-tyrosine phosphatase ) resulted in expression of a monomeric form of meprin, as determined by SDS-polyacrylamide gel electrophoresis and nondenaturing gel electrophoresis. The monomeric subunits were considerably more vulnerable to proteolytic degradation and heat inactivation in vitro compared with the oligomeric form of the enzyme. Proteolytic activity of the monomeric meprin using a bradykinin analog or aminobenzoyl-Ala-Ala Phe-p-nitroanilide as substrate was similar to that of disulfide-linked oligomeric meprin; however, activity against azocasein was markedly decreased. Mutation of another cysteine residue in the MAM domain (C289A), predicted to be involved in intrasubunit disulfide bridging, resulted in disulfide-linked oligomers and monomers. These results indicated that this mutant was capable of forming intersubunit disulfide bonds but less efficiently than wild-type meprin subunits. Mutant C289A also retained activity toward peptides but not the protein substrate and was more vulnerable to proteolytic degradation and heat inactivation compared with the wild-type enzyme. Both Cys mutants were expressed and secreted into the medium at levels comparable with the wild type and had slightly altered glycosylation. This work indicates that 1) Cys-320 of mouse meprin alpha is most likely responsible for the covalent interactions of the subunits; 2) covalent dimerization of subunits is not essential for efficient biosynthesis, trafficking, or posttranslational processing of the secreted protease; and 3) mutations in the MAM domain affect noncovalent interactions of the subunits and the stability and activity of the protease domain, indicating that domain-domain interactions are critical for structure and function of the enzyme.