O-glycan analysis of natural human neutrophil gelatinase B using a combination of normal phase- HPLC and online tandem mass spectrometry: Implications for the domain organization of the enzyme
O-glycan analysis of natural human neutrophil gelatinase B using a combination of normal phase- HPLC and online tandem mass spectrometry: Implications for the domain organization of the enzyme
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DOI：
10.1021/bi001367j
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发表时间：
2000-12-26
期刊：
影响因子：
2.9
通讯作者：
Rudd, PM
中科院分区：
文献类型：
--
作者：
Mattu, TS;Royle, L;Rudd, PM
明胶酶B是一种基质金属蛋白酶（MMP-9），在严格控制下由许多细胞类型（包括中性粒细胞、单核细胞、巨噬细胞和肿瘤细胞）表达。MMP-9是在组织重塑和发育中细胞外基质的生理维持中的关键介质，而不受控制的酶活性有助于诸如癌症和炎症的病理。中性粒细胞响应于IL-8刺激从颗粒释放MMP-9。人MMP-9具有三个潜在的N-连接的糖基化位点，并含有富含Ser/Pro/Thr的结构域，称为V型胶原样结构域，预期其是高度O-糖基化的。事实上，人嗜中性粒细胞MMP-9上约85%的总糖是O-连接的。本文介绍了皮摩尔量的这些O-聚糖的详细分析，使用一种新的基于HPLC的策略，O-聚糖分析，提供连接和臂的具体信息，除了单糖序列。使用HPLC和在线MS/MS裂解分析确认初始结构归属。12种糖被鉴定为含有2至9个单糖残基。其中大多数含有2型核心结构，具有Gal β 1 -4GlcNAc（N-乙酰乳糖胺）延伸，含或不含唾液酸或岩藻糖。使用寡糖结构数据库对O-聚糖进行建模。基于明胶酶A（MMP-2）的结构，MMP-9的模型表明明胶酶B中的V型胶原样结构域位于远离活性位点的环上。十四个潜在的O-糖基化位点多重呈现在这个52个氨基酸的环上。许多鉴定的O-聚糖含有末端半乳糖残基，可提供识别表位。重要的是，这个环区域的重糖基化，在明胶酶A中不存在，对MMP-9的结构域组织有相当大的影响。
Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict control by many cell types including neutrophils, monocytes, macrophages, and tumor cells. MMP-9 is a key mediator in the physiological maintenance of the extracellular matrix both in tissue remodeling and development, while uncontrolled enzyme activity contributes to pathologies such as cancer and inflammation. Neutrophils release MMP-9 from granules in response to IL-8 stimulation. Human MMP-9 has three potential N-linked glycosylation sites and contains a Ser/Pro/Thr rich domain, known as the type V collagen-like domain, which is expected to be heavily O-glycosylated. Indeed, approximately 85% of the total sugars on human neutrophil MMP-9 are O-linked. This paper presents the detailed analysis of picomole amounts of these O-glycans using a novel HPLC-based strategy for O-glycan analysis that provides linkage and arm specific information in addition to monosaccharide sequence. The initial structural assignments were confirmed using HPLC with online MS/MS fragmentation analysis. Twelve sugars were identified that contained from two to nine monosaccharide residues. Most of these contained type 2 core structures with Gal beta1-4GlcNAc (N-acetyl lactosamine) extensions, with or without sialic acid or fucose. The O-glycans were modeled using the oligosaccharide structural database. On the basis of the structure of gelatinase A (MMP-2), a model of MMP-9 suggests that the type V collagen-like domain in gelatinase B is located on a loop remote from the active site. Fourteen potential O-glycosylation sites are multiply presented on this loop of 52 amino acids. Many of the O-glycans identified contain terminal galactose residues that may provide recognition epitopes. Importantly, heavy glycosylation of this loop region, absent in gelatinase A, has considerable implications for the domain organization of MMP-9.