title: The O-glycomap of lubricin, a novel mucin responsible for joint lubrication, identified by site-specific glycopeptide analysis.
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.
authors.affiliation: From the ‡Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Box 440, Medicinaregatan 9A, 405 30, Gothenburg, Sweden;
authors.forename: Liaqat
authors.initials: L
authors.lastname: Ali
authors.affiliation: From the ‡Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Box 440, Medicinaregatan 9A, 405 30, Gothenburg, Sweden;
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Flowers
authors.affiliation: From the ‡Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Box 440, Medicinaregatan 9A, 405 30, Gothenburg, Sweden;
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Jin
authors.affiliation: §Department of Odontology, Copenhagen Center for Glycomics, University of Copenhagen, Norre Alle 20, DK-2200 Copenhagen N, Denmark;
authors.forename: Eric Paul
authors.initials: EP
authors.lastname: Bennet
authors.affiliation: ¶Department of Rheumatology and Inflammation Research, Institute of Medicine, University of Gothenburg, Guldhedsgatan 10A, SE-41346, Gothenburg, Sweden.
authors.forename: Anna-Karin H
authors.initials: AK
authors.lastname: Ekwall
authors.affiliation: From the ‡Department of Medical Biochemistry, Institute of Biomedicine, University of Gothenburg, Box 440, Medicinaregatan 9A, 405 30, Gothenburg, Sweden; niclas.karlsson@medkem.gu.se.
authors.forename: Niclas G
authors.initials: NG
authors.lastname: Karlsson
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D002240:Carbohydrate Sequence; D005347:Fibroblasts / Q000737:chemistry* / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006020:Glycopeptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008153:Lubrication; D008239:Lysine / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010449:Peptide Mapping; D061251:Primary Cell Culture; D017434:Protein Structure, Tertiary; D055672:Static Electricity; D013582:Synovial Fluid / Q000737:chemistry / Q000166:cytology; D013912:Threonine / Q000737:chemistry* / Q000378:metabolism; D000097763:Polypeptide N-acetylgalactosaminyltransferase; D000099158:Proteoglycan 4
chemical_list: D001120:Arginine; D006020:Glycopeptides; D006023:Glycoproteins; D007527:Isoenzymes; D008239:Lysine; D017350:N-Acetylgalactosaminyltransferases; D013912:Threonine; D000097763:Polypeptide N-acetylgalactosaminyltransferase; D000099158:Proteoglycan 4