PMID: PMC4256492-1-3

 

    Legend: Gene, Sites

Title : Accessibility and Characterization of the Glycosylated Region of Lubricin

Abstract :
  1. Human synovial lubricin was purified from SF of RA and OA patient samples (n = 5)
  2. 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)
  3. The antibody also detected an additional, faint high-mass band that was due to lubricin complexes (37)
  4. All bands were previously confirmed to contain lubricin when subjected to in-gel trypsin digestion and subsequent LC- MS2 analysis (38)
  5. 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)
  6. 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)
  7. 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)
  8. 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
  9. 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
  10. 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)
  11. This was further verified by partial de-glycosylation using sialidase and O-glycanase to remove sialylated and unsialylated core 1 structures
  12. 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)
  13. The dominating lubricin band seen in SDS-PAGE was subjected to in-gel trypsin digestion, and unmodified lubricin peptides were identified via LC- MS2
  14. The identified peptides were predominately from the N- and C-terminal regions
  15. 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)
  16. 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
  17. 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%)
  18. 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
  19. 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
  20. 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
  21. 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
  22. 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):

 

 

Protein NCBI ID SENTENCE INDEX