Marchand P, Volkmann M, Bond J S
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033-0850, USA.
J Biol Chem. 1996 Sep 27;271(39):24236-41. doi: 10.1074/jbc.271.39.24236.
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-5 protein, and receptor protein-tyrosine phosphatase mu) 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.
膜蛋白水解酶是一种寡聚的、糖基化的细胞表面或分泌型金属内肽酶，由多结构域二硫键连接的亚基组成。为了研究亚基寡聚化对于细胞内运输或蛋白酶的酶学和/或物理性质是否至关重要，对特定的半胱氨酸残基进行了突变，并在293细胞中表达了这些突变体。通过SDS-聚丙烯酰胺凝胶电泳和非变性凝胶电泳测定，将小鼠膜蛋白水解酶α的MAM结构域（在膜蛋白水解酶、A-5蛋白和受体蛋白酪氨酸磷酸酶μ中发现的一种细胞外结构域）中的半胱氨酸-320突变为丙氨酸，导致了单体形式的膜蛋白水解酶的表达。与该酶的寡聚形式相比，单体亚基在体外对蛋白水解降解和热失活更为敏感。以缓激肽类似物或氨基苯甲酰-Ala-Ala-Phe-对硝基苯胺为底物时，单体膜蛋白水解酶的蛋白水解活性与二硫键连接的寡聚膜蛋白水解酶相似；然而，其对偶氮酪蛋白的活性明显降低。MAM结构域中另一个预测参与亚基内二硫键桥接的半胱氨酸残基（C289A）发生突变，导致了二硫键连接的寡聚体和单体的产生。这些结果表明，该突变体能够形成亚基间二硫键，但效率低于野生型膜蛋白水解酶亚基。突变体C289A对肽仍具有活性，但对蛋白质底物则无活性，并且与野生型酶相比，其对蛋白水解降解和热失活更为敏感。两个半胱氨酸突变体均得以表达并分泌到培养基中，其水平与野生型相当，且糖基化略有改变。这项研究表明：1）小鼠膜蛋白水解酶α的半胱氨酸-320最有可能负责亚基的共价相互作用；2）亚基的共价二聚化对于分泌型蛋白酶的高效生物合成、运输或翻译后加工并非必不可少；3）MAM结构域中的突变影响亚基的非共价相互作用以及蛋白酶结构域的稳定性和活性，表明结构域间相互作用对于该酶的结构和功能至关重要。
