Hoshi Hiroko, Shimawaki Ken, Takegawa Yasuhiro, Ohyanagi Tatsuya, Amano Maho, Hinou Hiroshi, Nishimura Shin-Ichiro
Field of Drug Discovery Research, Faculty of Advanced Life Science and Graduate School of Life Science, Hokkaido University, N21, W11, Kita-ku, Sapporo 001-0021, Japan.
Biochim Biophys Acta. 2012 Sep;1820(9):1391-8. doi: 10.1016/j.bbagen.2012.01.004. Epub 2012 Jan 14.
Cell 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.
Fluorescence (FITC)-labeled tetrapeptide (H-Ser(1)-Gly(2)-Ser(3)-Gly(4)-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.
PGI 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.
Synthetic serglycin-type PGIs allow for live cell imaging during proteoglycan biosynthesis and structural characterization of GAG-primed PGIs by an antibody-based enrichment protocol.
Novel 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.
细胞表面蛋白聚糖在多种生物学过程中发挥着至关重要的功能作用，如细胞增殖、分化、黏附、炎症、免疫反应、软骨组织维持及组织强度等。我们在此表明，类丝甘霉素合成糖肽可有效地作为分子穿梭体，跨越质膜劫持细胞内的糖胺聚糖（GAG）生物合成途径。
携带Galβ(1➝4)Xylβ1➝的荧光（FITC）标记四肽（H-Ser(1)-Gly(2)-Ser(3)-Gly(4)-OH）被定义为蛋白聚糖起始物（PGI）单体，其串联重复的PGI聚合物用于通过共聚焦激光扫描显微镜对细胞摄取和细胞内运输进行直接成像。建立了一种通过联合使用抗FITC抗体和液相色谱/质谱对GAG引发的PGI进行富集分析的新方法。
PGI单体迅速被纳入人关节软骨细胞，并分布于包括内质网/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。结果表明，PGI在细胞内成为GAG生物合成的良好底物，由PGI引发的高分子量GAG主要成分是硫酸软骨素，其中N-乙酰-d-半乳糖胺残基被4-O-硫酸盐或6-O-硫酸盐基团取代。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可作为一类新的细胞因子样分子来刺激细胞生长。
合成的丝甘霉素型PGI可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。
用于研究蛋白聚糖生物合成的新型糖组学，即使用合成糖肽作为PGI的实时GAG组学，应极大地促进活细胞中GAG的动态分析。本文是名为“糖蛋白质组学”的特刊的一部分。
