Molecular shuttle between extracellular and cytoplasmic space allows for monitoring of GAG biosynthesis in human articular chondrocytes.

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.