Yang Xiaojing, Lehotay Michael, Anastassiades Tassos, Harrison Mark, Brockhausen Inka
Department of Medicine, The Arthritis Center and Human Mobility Research Center, Queen's University, Kingston, ON K7L 3N6, Canada.
Biochem Cell Biol. 2004 Oct;82(5):559-68. doi: 10.1139/o04-058.
Synoviocytes are fibroblastic cells that line joint cavities. These cells synthesize numerous cell-surface and extracellular-matrix glycoproteins that are required for maintenance of the joint. Joint inflammation, such as occurs in arthritis, has been shown to have major effects on synoviocyte proliferation and on the biosynthesis of glycoproteins. The structures of the carbohydrate moieties of glycoproteins, however, and the enzymes involved in their synthesis have not yet been described for synoviocytes. Therefore, to characterize the cell-surface glycoconjugates, synoviocytes were isolated from bovine ankles, and the cells were grown in primary cultures. Lectin-binding assays were used to identify exposed N- and O-glycan carbohydrate determinants on synoviocytes, and specific enzyme assays were used to identify some of the glycosyltransferases involved in the synthesis of the glycan chains. A number of the enzymes that synthesize N- and O-linked oligosaccharides were found to be active in cell-free extracts of synoviocytes, including those that synthesize core-1-based O-glycans and the more complex bi-antennary N-glycans. To understand the molecular events underlying the inflammatory response in the synovium of arthritis patients, we examined the effect of the inflammatory cytokine tumour necrosis factor alpha (TNF-alpha) on synoviocytes and on glycosylation profiles. TNF-alpha treatment, which induces apoptosis in synoviocytes, was accompanied by changes in lectin-binding patterns, indicating alterations in the expression of cell-surface oligosaccharides. Concurrently, changes in specific enzyme activities were observed in treated cells. Two enzymes potentially important to the inflammatory process, core 2 beta6-GlcNAc-transferase and beta4-Gal-transferase, increased after TNF-alpha treatment. This is the first study of glycoprotein biosynthesis in synoviocytes, and it shows that synoviocytes have a characteristic glycosylation phenotype that is altered in the presence of inflammatory cytokines.
滑膜细胞是衬于关节腔的成纤维细胞。这些细胞合成维持关节所需的多种细胞表面和细胞外基质糖蛋白。已证明，诸如关节炎中出现的关节炎症对滑膜细胞增殖和糖蛋白生物合成有重大影响。然而，滑膜细胞糖蛋白碳水化合物部分的结构及其合成过程中涉及的酶尚未见报道。因此，为了表征细胞表面糖缀合物，从牛踝关节分离出滑膜细胞，并在原代培养中培养这些细胞。凝集素结合试验用于鉴定滑膜细胞上暴露的N-和O-聚糖碳水化合物决定簇，特异性酶试验用于鉴定参与聚糖链合成的一些糖基转移酶。发现许多合成N-和O-连接寡糖的酶在滑膜细胞的无细胞提取物中具有活性，包括那些合成基于核心-1的O-聚糖和更复杂的双天线N-聚糖的酶。为了了解关节炎患者滑膜炎症反应背后的分子事件，我们研究了炎性细胞因子肿瘤坏死因子α（TNF-α）对滑膜细胞和糖基化谱的影响。TNF-α处理可诱导滑膜细胞凋亡，同时伴有凝集素结合模式的改变，表明细胞表面寡糖表达发生了变化。同时，在处理过的细胞中观察到特定酶活性的变化。两种对炎症过程可能重要的酶，核心2 β6-N-乙酰葡糖胺转移酶和β4-半乳糖转移酶，在TNF-α处理后增加。这是首次对滑膜细胞中糖蛋白生物合成进行的研究，结果表明滑膜细胞具有特征性的糖基化表型，在炎性细胞因子存在时会发生改变。
