Title : Accessibility and Characterization of the Glycosylated Region of
Lubricin
Abstract :
- Human synovial lubricin was purified from SF of RA and OA patient samples (n = 5)
- After purification, lubricin was detected as a major band in both RA and OA samples through the use of lubricin-specific antibody with an apparent molecular mass of >200 kDa after SDS-PAGE (Fig. 1A, lane 1)
- The antibody also detected an additional, faint high-mass band that was due to lubricin complexes (37)
- All bands were previously confirmed to contain lubricin when subjected to in-gel trypsin digestion and subsequent LC- MS2 analysis (38)
- In order to indicate the localization of the glycosylated mucin-like domain within lubricin , the samples (reduced and alkylated) were subjected to in-solution trypsin digestion and separated on SDS-PAGE gels prior to subsequent Western blotting using lubricin-specific antibody and biotinylated lectins (PNA, Galβ1–3GalNAcα1-O-Ser/Thr and WGA, sialic acid , and terminal GlcNAc)
- The effectiveness of the trypsin digestion was shown by the fact that the generated peptides were too small to be detected on the gel (Fig. 1)
- This showed that both the less glycosylated N- and C-terminals and the mucin-like domain were accessible for digestion (Fig. 1A, PNA and WGA)
- The lubricin mucin domain is different from traditional indigestible mucin domains , allowing Lys residues ( trypsin cleavage site ) in the imperfect repeat EPAPTTPK to be protease accessible
- This also suggested that the glycans of lubricin were smaller and/or less frequent than other mucins, allowing the trypsin site to be accessible despite the surrounding glycosylation
- The positive lectin (PNA and WGA) binding of the reduced and alkylated but not trypsin digested samples suggested that SF lubricin predominantly contained short core 1 and sialylated core 1 structures (Fig. 1A)
- This was further verified by partial de-glycosylation using sialidase and O-glycanase to remove sialylated and unsialylated core 1 structures
- This treatment resulted in a substantial decrease in size (Fig. 1B), with an apparent mass of >155 kDa, close to the predicted size of apo lubricin (151 kDa)
- The dominating lubricin band seen in SDS-PAGE was subjected to in-gel trypsin digestion, and unmodified lubricin peptides were identified via LC- MS2
- The identified peptides were predominately from the N- and C-terminal regions
- Even though the mucin-like domain was indicated to be less extensively glycosylated, only a few non-modified peptides from the mucin domain could be identified (Fig. 1C, black)
- In a stretch of 507 amino acids in the central region (aa 348–855) there were only two (one unique) peptides (KPAPTTPK) (3% coverage) identified
- After partial de-glycosylation, a total of 99 (13 unique) unmodified peptides from this lubricin mucin-like domain were identified via LC- MS2 (Fig. 1C, gray), providing a coverage of 84% of the mucin-like domain (aa 348–855) rich in Thr (29.5%), Pro (30.5%), and, to a lesser extent, Ser (2.4%)
- These data suggested that this entire region was highly glycosylated with small glycans, as even though tryptic peptides could be created, unmodified peptides could not be identified
- The current domain model of lubricin consists of less glycosylated N and C terminals separated by a glycosylated mucin-like domain (aa 348–855) region of a tandemly repeated amino acid sequence
- However, the data shown here indicate that lubricin consists of an extended glycosylated STP-rich region (aa 232–1056) (Fig. 4A) larger than the mucin-like domain previously defined by UniProt
- The molecular mass of glycosylated lubricin is estimated to be ∼350 kDa, and that of apomucin to be >151 kDa, indicating glycosylation constitutes 57% of the total protein mass
- Given that the estimated average mass of an oligosaccharide on lubricin is 600 to 1000 Da (38), it is expected that lubricin holds 200 to 300 oligosaccharide chains
Output (sent_index, trigger,
protein,
sugar,
site):
- 15. glycosylated, , -, -, domain
- 18. glycosylated, , -, -, region
- 19. glycosylated, , -, -, region
- 20. glycosylated, , -, -, region
- 21. glycosylated, , lubricin, -, -
- 5. glycosylated, , -, -, domain
Output(Part-Of) (sent_index,
protein,
site):
- 0. Lubricin, -
- 13. lubricin, peptides
- 15. -, peptides
- 17. -, peptides
- 17. lubricin, domain
- 20. STP, region
- 8. -, domain
- 8. lubricin, domain
- 8. trypsin, Lys residues
- 8. trypsin, site
- 9. trypsin, site
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):