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.

Genotype–phenotype investigation of 35 patients from 11 unrelated families with camptodactyly–arthropathy–coxa vara–pericarditis (CACP) syndrome

“…In vitro experiments have shown that this mutated protein does not undergo the normal process of SPC‐mediated cleavage within the PEX domain (Rhee et al., ), meaning that the PRG4 protein is nonfunctional. Indeed, full‐length protein presents an optimal lubricating function when the intact negatively charges STP‐rich region and positively charged at the N‐ and C‐terminal regions are intact (Ali et al., ; Lee, Muller, Rezwan, & Spencer, ; Swann, Hendren, Radin, Sotman, & Duda, ). In our cohort, we report three siblings with homozygous p.Y1367X (c.4101C>G) mutation predicted to escape the NMD.…”

Reduced Mucin-7 (Muc7) Sialylation and Altered Saliva Rheology in Sjögren's Syndrome Associated Oral Dryness

“…The electrostatic repulsion properties of the central mucin domain have been suggested to confer essential boundary lubrication properties, not just for salivary mucins but other similar lubricating glycoproteins such as lubricin found in the synovial fluid of the joint (16). Lubricin has positively charged terminal regions flanking the central negatively charged domain, and these hydrophobic regions are suggested to be important for the adherence of lubricin to the cartilage surface (59). A similar mechanism has been proposed for the boundary lubricating layer of the oral mucosa (60).…”

Sialidase Unmasks Mucin Domain Epitopes of Lubricin

“…2,7,13,14 Lubricin's central, long mucin-like domain is extensively O-glycosylated with Gal(β1-3)GalNAc-O-Ser/Thr, and about two thirds of lubricin's O-glycosylated sites are capped with sialic acid as (α2,3)NeuAc-(β1-3)Gal-GalNAc-O-Ser/ Thr. 2,6,7,[11][12][13][14] Given lubricin's ~200 O-glycosylated sites, 14 there are approximately 133 sialic acid residues per lubricin molecule. Ali et al…”

“…9 The central, long mucin domain of lubricin is extensively O-glycosylated with Gal(β1-3)GalNAc-OSer/Thr, and about two thirds of the O-glycosylated sites are capped with sialic acid as (α2,3)NeuAc-(β1-3)GalGalNAc-O-Ser/Thr. 2,6,7,[11][12][13][14] Our aim was to determine whether removal of sialic acid by sialidase could enhance the detection of lubricin with the S6.79 antibody in human tissues by IHC and Western blot analysis. Our data show that the S6.79 lubricin epitope is highly masked by sialic acid in human pericardium, splenic capsule and trabeculae, plasma, serum, eye sleep extract, and liver sinusoids compared with the epitope in normal synovial tissue and fluid.…”