Molecular shuttle between extracellular and cytoplasmic space allows for monitoring of GAG biosynthesis in human articular chondrocytes ☆ , ☆☆ Hiroko Hoshi a , Ken Shimawaki a , Yasuhiro Takegawa a , Tatsuya Ohyanagi a , Maho Amano a , Hiroshi Hinou a , Shin-Ichiro Nishimura a, b, ⁎ a 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 b Medicinal Chemistry Pharmaceuticals, LLC, 1-715, N21, W12, Kita-ku, Sapporo 001-0021, Japan abstract article info Article history: Received 4 November 2011 Received in revised form 20 December 2011 Accepted 7 January 2012 Available online 14 January 2012 Keywords: Molecular shuttle Artificial proteoglycan initiator Serglycin Glycosaminoglycan biosynthesis Nuclear localization Background: Cell surface proteoglycans play vital functional roles in various biological processes such as cell pro- liferation, differentiation, adhesion, inflammation, immune response, sustentation of cartilage tissue and inten- sity 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. Methods: Fluorescence (FITC)-labeled tetrapeptide (H-Ser 1 -Gly 2 -Ser 3 -Gly 4 -OH) carrying Galβ(1➝4)Xylβ1➝ de- fined 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 en- richment analysis of GAG-primed PGIs by combined use of anti-FITC antibody and LC/mass spectrometry was established. Results: 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 expres- sion level of type II collagen, suggesting that PGIs can function as new class cytokine-like molecules to stimulate cell growth. Conclusion: Synthetic serglycin-type PGIs allow for live cell imaging during proteoglycan biosynthesis and struc- tural characterization of GAG-primed PGIs by an antibody-based enrichment protocol. General significance: 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. © 2012 Elsevier B.V. All rights reserved. 1. Introduction Proteoglycans (PGs) are complex glycoconjugates consisted of a core polypeptide with a number of covalently linked glycosaminoglycan (GAG) chains. Cell surface and extracellular matrix GAG of PGs are known to play vital functional roles in various biological processes such as cell proliferation, differentiation, adhesion, inflammation, im- mune response, wound repair, sustentation of cartilage tissue and inten- sity of tissues [1–3]. The matured GAG chains synthesis is supposed to be initiated from the core tetrasaccharide moiety, namely [GlcAβ(1→3) Galβ(1→3)Galβ(1→4)Xylβ1→], which is attached to the serine or thre- onine residue of core polypeptides. To investigate the functional role of this core area in the specific and controlled biosynthetic pathway to pro- duce two major classes of GAGs, chondroitin sulfate/dermatan sulfate and heparin/heparan sulfate, extensive efforts have been paid toward chemical synthesis of GAGs related to the core structure [4–6]. In 1973, Suzuki et al. reported in a pioneering work [7] that a simple p-nitrophenyl β-D-xyloside can be incorporated into embryonic chick cartilages and functioned as artificial substrates of GAG biosynthesis within the cells. The importance of this finding to predict the existence of specific mechanism in the endocytic incorporation of synthetic glyco- sides has been demonstrated widely by the results that various β-D- xylosides bearing hydrophobic/aromatic aglycons are also internalized into cells and modified as proteoglycan initiators (PGIs) by this biosyn- thetic pathway while the aglycons appeared to influence GAG struc- tures as well as efficacy in the glycosylation of PGIs [8]. It deserves to be noted that the mechanism in GAG synthesis from the PGIs involves Biochimica et Biophysica Acta 1820 (2012) 1391–1398 ☆ This article is part of a Special Issue entitled Glycoproteomics. ☆☆ The authors declare no conflict of interest. ⁎ Corresponding author at: 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. E-mail address: shin@sci.hokudai.ac.jp (S.-I. Nishimura). 0304-4165/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2012.01.004 Contents lists available at SciVerse ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen