Ishmael Susan S, Ishmael Faoud T, Jones A Daniel, Bond Judith S
Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA.
J Biol Chem. 2006 Dec 8;281(49):37404-15. doi: 10.1074/jbc.M602769200. Epub 2006 Oct 13.
The meprin A homo-oligomer is a highly glycosylated, secreted zinc metalloprotease of the astacin family and metzincin superfamily. This isoform of meprin is composed of disulfide-bonded dimers of alpha subunits that further associate to form large, secreted megadalton complexes of 10 or more subunits. The aim of this study was to determine the sites of glycan attachment and to assess their ability to affect the formation and stability of the homo-oligomer. Nine of the ten potential N-linked glycosylation sites (Asn-41, Asn-152, Asn-234, Asn-270, Asn-330, Asn-426, Asn-452, Asn-546, and Asn-553) were found to be glycosylated in recombinant mouse meprin A using chemical and enzymatic deglycosylation methods and electrospray ionization mass spectrometry. Chemical cross-linking demonstrated that carbohydrates are at or near the noncovalent subunit interface. The removal of two glycans in the protease domain at Asn-234 and Asn-270, as well as one in the tumor necrosis factor receptor-associated factor domain at Asn-452, by a deglycosidase under nondenaturing conditions decreased the chemical and thermal stability of the homo-oligomer without affecting quaternary structure. Site-directed mutagenesis demonstrated that no single glycan was essential for oligomer formation; however, the combined absence of the glycans at Asn-152 and Asn-270 in the protease domain hindered intersubunit disulfide bond formation, prevented noncovalent associations, and abolished enzymatic activity. These studies provide insights into the role of glycans in the biosynthesis, activity, and stability of this extracellular protease.
甲素A同型寡聚体是一种高度糖基化的、分泌型的锌金属蛋白酶，属于虾红素家族和金属锌蛋白酶超家族。甲素的这种同工型由α亚基的二硫键连接的二聚体组成，这些二聚体进一步缔合形成由10个或更多亚基组成的大型分泌型百万道尔顿复合物。本研究的目的是确定聚糖附着位点，并评估它们影响同型寡聚体形成和稳定性的能力。使用化学和酶促去糖基化方法以及电喷雾电离质谱法，发现重组小鼠甲素A中的10个潜在N-连接糖基化位点（天冬酰胺-41、天冬酰胺-152、天冬酰胺-234、天冬酰胺-270、天冬酰胺-330、天冬酰胺-426、天冬酰胺-452、天冬酰胺-546和天冬酰胺-553）中有9个被糖基化。化学交联表明碳水化合物位于非共价亚基界面处或其附近。在非变性条件下，用去糖基酶去除蛋白酶结构域中天冬酰胺-234和天冬酰胺-270处的两个聚糖以及肿瘤坏死因子受体相关因子结构域中天冬酰胺-452处的一个聚糖，降低了同型寡聚体的化学和热稳定性，但不影响四级结构。定点诱变表明没有单个聚糖对寡聚体形成是必不可少的；然而，蛋白酶结构域中天冬酰胺-152和天冬酰胺-270处聚糖的联合缺失阻碍了亚基间二硫键的形成，阻止了非共价缔合，并消除了酶活性。这些研究为聚糖在这种细胞外蛋白酶的生物合成、活性和稳定性中的作用提供了见解。
