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The O-glycomap of Lubricin, a Novel Mucin Responsible for Joint Lubrication, Identified by Site-specific Glycopeptide Analysis

Journal article
Authors Liaqat Ali
Sarah A. Flowers
Chunsheng Jin
E. P. Bennet
Anna-Karin H Ekwall
Niclas G. Karlsson
Published in Molecular & Cellular Proteomics
Volume 13
Issue 12
Pages 3396-3409
ISSN 1535-9476
Publication year 2014
Published at Institute of Medicine, Department of Rheumatology and Inflammation Research
Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 3396-3409
Language en
Links dx.doi.org/10.1074/mcp.M114.040865
Keywords ARTICULAR-CARTILAGE DEGRADATION, ZONE PROTEIN SZP, RHEUMATOID-ARTHRITIS, LINKED OLIGOSACCHARIDES, GALNAC-TRANSFERASE, SYNOVIAL-FLUID, BOUNDARY, LUBRICATION, GLYCAN BIOSYNTHESIS, MASS-SPECTROMETRY, GLYCOSYLATION
Subject categories Rheumatology and Autoimmunity, Biochemistry

Abstract

The lubricative, heavily glycosylated mucin-like synovial glycoprotein lubricin has previously been observed to contain glycosylation changes related to rheumatoid and osteoarthritis. Thus, a site-specific investigation of the glycosylation of lubricin was undertaken, in order to further understand the pathological mechanisms involved in these diseases. Lubricin contains an serine/threonine/proline (STP)-rich domain composed of imperfect tandem repeats (EPAPTTPK), the target for O-glycosylation. In this study, using a liquid chromatography-tandem mass spectrometry approach, employing both collision-induced and electron-transfer dissociation fragmentation methods, we identified 185 O-glycopeptides within the STP-rich domain of human synovial lubricin. This showed that adjacent threonine residues within the central STP-rich region could be simultaneously and/or individually glycosylated. In addition to core 1 structures responsible for biolubrication, core 2 O-glycopeptides were also identified, indicating that lubricin glycosylation may have other roles. Investigation of the expression of polypeptide N-acetylgalactosaminyltransferase genes was carried out using cultured primary fibroblast-like synoviocytes, a cell type that expresses lubricin in vivo. This analysis showed high mRNA expression levels of the less understood polypeptide N-acetylgalactosaminyltransferase 15 and 5 in addition to the ubiquitously expressed polypeptide N-acetylgalactosaminyltransferase 1 and 2 genes. This suggests that there is a unique combination of transferase genes important for the O-glycosylation of lubricin. The site-specific glycopeptide analysis covered 82% of the protein sequence and showed that lubricin glycosylation displays both micro-and macroheterogeneity. The density of glycosylation was shown to be high: 168 sites of O-glycosylation, predominately sialylated, were identified. These glycosylation sites were focused in the central STP-rich region, giving the domain a negative charge. The more positively charged lysine and arginine residues in the N and C termini suggest that synovial lubricin exists as an amphoteric molecule. The identification of these unique properties of lubricin may provide insight into the important low-friction lubricating functions of lubricin during natural joint movement.

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