Molecular shuttle between extracellular and cytoplasmic space: Glycosaminoglycan biosynthesis promoted by serglycin precursor glycopeptides in human articular chondrocytes.
Molecular shuttle between extracellular and cytoplasmic space: Glycosaminoglycan biosynthesis promoted by serglycin precursor glycopeptides in human articular chondrocytes.
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细胞外和细胞质空间之间的分子穿梭：人关节软骨细胞中丝甘氨酸前体糖肽促进糖胺聚糖生物合成。
DOI：
10.1016/j.bbagen.2012.01.004
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Nishimura S.
中科院分区：
文献类型：
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作者：
Hoshi H;Shimawaki K;Takegawa Y;Ohyanagi T;Amano M;Hinou H;Nishimura S.
背景细胞表面蛋白聚糖在细胞增殖、分化、粘附、炎症、免疫反应、软骨组织的维持和组织强度等多种生物过程中发挥着重要的功能作用。我们在此表明，类甘氨酸合成糖肽可有效地作为分子穿梭机，跨质膜劫持细胞内的糖胺聚糖（GAG）生物合成途径。方法荧光（FITC）标记的四肽（H-Ser1-Gly2-Ser3-Gly4-OH）携带Galβ（1➝4）Xylβ1➝定义为蛋白聚糖引发剂(PGI) 单体及其串联重复 PGI 聚合物用于通过共焦激光扫描显微镜对细胞摄取和细胞内交通进行直接成像。建立了结合使用抗FITC抗体和LC/质谱法对GAG引发的PGI进行富集分析的新方法。结果SPGI单体迅速掺入人关节软骨细胞中并分布在包括ER/高尔基体在内的整个细胞质中，而PGI聚合物则特异性定位于细胞核中。研究表明，PGI 成为细胞内 GAG 生物合成的良好底物，由 PGI 引发的高分子量 GAG 是硫酸软骨素，其中 N-乙酰基-d-半乳糖胺残基被 4-O-硫酸酯或 6-O-硫酸酯基团取代作为主要成分。 PGI 激活软骨细胞增殖并诱导 II 型胶原表达水平上调，表明 PGI 可以作为新型细胞因子样分子刺激细胞生长。结论合成的丝甘氨酸型 PGI 允许在蛋白聚糖生物合成过程中进行活细胞成像，并通过基于抗体的富集方案对 GAG 引发的 PGI 进行结构表征。一般意义指定用于研究的新型糖组学蛋白多糖生物合成，即使用合成糖肽作为 PGI 的实时 GAG 组学，应有利于活细胞中 GAG 的动态分析。本文是题为糖蛋白组学的特刊的一部分。
BACKGROUNDCell surface proteoglycans play vital functional roles in various biological processes such as cell proliferation, differentiation, adhesion, inflammation, immune response, sustentation of cartilage tissue and intensity of tissues. We show here that serglycin-like synthetic glycopeptides function efficiently as a molecular shuttle to hijack glycosaminoglycan (GAG) biosynthetic pathway within cells across the plasma membrane.METHODSFluorescence (FITC)-labeled tetrapeptide (H-Ser1-Gly2-Ser3-Gly4-OH) carrying Galβ(1➝4)Xylβ1➝ defined as proteoglycan initiator (PGI) monomer and its tandem repeating PGI polymer was employed for direct imaging of cellular uptake and intracellular traffic by confocal laser-scanning microscopy. Novel method for enrichment analysis of GAG-primed PGIs by combined use of anti-FITC antibody and LC/mass spectrometry was established.RESULTSPGI monomer was incorporated promptly into human articular chondrocytes and distributed in whole cytoplasm including ER/Golgi while PGI polymer localized specifically in nucleus. It was demonstrated that PGIs become good substrates for GAG biosynthesis within the cells and high molecular weight GAGs primed by PGIs is chondroitin sulfate involving N-acetyl-d-galactosamine residues substituted by 4-O-sulfate or 6-O-sulfate group as major components. PGIs activated chondrocytes proliferation and induced up-regulation of the expression level of type II collagen, suggesting that PGIs can function as new class cytokine-like molecules to stimulate cell growth.CONCLUSIONSynthetic serglycin-type PGIs allow for live cell imaging during proteoglycan biosynthesis and structural characterization of GAG-primed PGIs by an antibody-based enrichment protocol.GENERAL SIGNIFICANCENovel glycomics designated for investigating proteoglycan biosynthesis, namely real-time GAGomics using synthetic glycopeptides as PGIs, should facilitate greatly dynamic profiling of GAGs in the living cells. This article is part of a Special Issue entitled Glycoproteomics.