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.

Fig. 1.

Accessibility and characterization of the glycosylated region of lubricin. A, the enriched synovial lubricin samples, before and after trypsin digestion, were separated on a 3–8% Tris acetate gel, blotted onto PVDF membrane, and then probed with lubricin-specific antibody (mouse anti-lubricin) and carbohydrate-specific biotinylated lectins PNA, specific for core 1 (Galβ1–3GalNAc) O-glycan, and WGA, specific for sialic acid and terminal GlcNAc. B, SDS-PAGE (3–8% Tris acetate gel) of the acidic glycoprotein fractions of the SF before (−) and after (+) partial de-glycosylation stained with Coomassie Brilliant Blue. C, SF lubricin was in-solution digested, and non-modified peptides were identified via mass spectrometry for protein coverage determination (black). The low protein coverage (in particular the mucin domain) suggests that the mucin domain is extensively glycosylated. Some of the core 1 structures were removed by partial de-glycosylation, and the previously glycosylated peptides were identified for protein coverage (gray). The results suggest that lubricin contains an extended STP-rich region relative to the mucin domain previously defined by UniProt.

Fig. 2.

Identification of lubricin mucin glycopeptides using CID. The figure shows the different glycoforms of the same tryptic peptide within the STP-rich region. A, CID-MS² spectrum of the tandem repeat (EPAPTTPK) containing core 1 glycan (Galβ1–3GalNAcα1-) of the [M+2H]²⁺ ions at m/z 603.3. B, CID-MS² spectrum of the same tandem repeat containing sialyl T antigen (NeuAcα2–3Galβ1–3GalNAcα1-) of the [M+2H]²⁺ ions at m/z 748.8. C, CID-MS² spectrum of the same tandem repeat (EPAPTTPK) containing two separate core 1 glycans (Galβ1–3GalNAcα1-) on two adjacent threonines of the [M+2H]²⁺ ions at m/z 785.8. D, CID-MS² spectrum of the same tandem repeat containing core 2 glycan Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1- of the [M+2H]²⁺ ions at m/z 785.8. This shows that the same peptide (A, B, and D) can be differently glycosylated, indicating the complexity associated with lubricin O-glycosylation, and that CID was able to provide information about the nature of glycans and peptides. In the figure, yellow squares and circles represent N-acetylgalactosamine and galactose, respectively, and blue squares and pink diamonds represent N-acetylglucosamine and N-acetylneuramininc acid (sialic acid), respectively.

Fig. 3.

Identification of glycosylation sites using electron transfer dissociation (ETD). A, ETD-MS² spectrum of the [M+3H]³⁺ ions at m/z 673.3 indicates a tryptic peptide (ITTLKTTTLAPK) with two separate core 1 glycans (Galβ1–3GalNAcα1-) on two adjacent threonine residues. B, ETD-MS² spectrum of the [M+3H]³⁺ ions at m/z 673.3 reveals the same tryptic peptide (ITTLKTTTLAPK) with two separate core 1 glycans (Galβ1–3GalNAcα1-) on two separate threonine residues, indicating that lubricin glycosylation displays macroheterogeneity within the same peptide sequence. M denotes the glycopeptide parent mass.

Fig. 4.

The O-glycosylation map of lubricin. A, the glycosylated peptides identified in the STP-rich region and non-glycosylated peptides (light gray) in the end domains of the lubricin sequence before (black) and after (dark gray) partial de-glycosylation using CID and ETD fragmentation. B, graphic representation of all of the identified glycans and their positions in the protein sequence. The figure includes the identified glycopeptide containing GalNAcα1- (dark orange, 130), core 1 Galβ1–3GalNAcα1- (chocolate, 161), core 2 Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1- (blue, 85), and sialylated core 1 and 2 (pink, 73) glycans with their positions in the protein sequence. The graph also includes the total number of identified glycosylation sites (168 sites) in the protein sequence (black). The total number of sites is based on the identified peptides and the fact that two threonines in the imperfect tandem repeat variants (e.g. EPAPTTPK, SAPTTPK, EPAPTTTK) can be glycosylated. It indicates a high number of glycosylations in the STP-rich region, whereas the N- and C-terminal regions are scarcely glycosylated. C, the combined CID and ETD identified glycopeptides in the STP-rich region (black and dark gray) and non-glycosylated peptides (light gray) in the end domains of lubricin before (black) and after partial de-glycosylation (dark gray). The software used (NetOGlyc4.0 and ISOGlyP) predicted sites of lubricin O-glycosylated Ser/Thr in the protein sequence (yellow) and the total number of potential Ser/Thr sites (370). The results indicate that the gene-based ISOGlyP software predicted a similar number of glycosylation sites (191 sites) as identified by the data presented in this report (168 sites). D, the GALNT profiling expression analysis of the 20 known human GALNT genes (top) in primary FLSs and the ISOGlyP-predicted sites by the available GALNT genes identified by the data presented in this report (bottom). The genes were arranged in descending order of their expression. RQ, relative quantification. Asterisk denotes genes not included in the ISOGlyP software.

Fig. 5.

The implication of the type and distribution of O-glycosylation on the charge and role of lubricin as a lubricating agent. A, the predicted isoelectric point (pI) of full-length (black) and STP-rich regions (dark gray) for varying numbers of sialic acids identified, indicating that the pI decreases as the number of sialic acids increases. B, enriched acidic SF fractions, before and after sialidase treatment, were separated on pH 3–10 IPG gels and blotted to PVDF membrane. Western blot analysis using lubricin-specific antibody (mAb13) indicated a pH range of synovial lubricin before sialidase treatment (pH 4–7.5) and a drastic increase in pH (pH 7.5) after sialidase treatment. C, the pI of the N- (9.45–9.6) and C-terminal regions (9.69–9.98) (light gray), where very few glycosylation sites were identified, and the extended STP-rich region, which contained the majority of the protein glycosylation (4–7.5).