PMID: PMC4256492-1-8

 

    Legend: Gene, Sites

Title : The Implications of the Identification of the Site-specific Glycosylation of Lubricin and Its Role in Lubrication

Abstract :
  1. Sialylated and sulfated glycans will alter the charge of heavily glycosylated proteins
  2. Apomucins are usually neutral or acidic, secreted with a predicted pI of 2 to 4.7 (43, 44)
  3. The predicted pI of apo lubricin is exceptional in this respect in that it can be as high as 9.8, but the protein can end up acidic after glycosylation
  4. With a detailed glycosylation map, the dependence of glycosylation and the amount of sialylation for the charge and pI of the lubricin can be modeled (Fig. 4B)
  5. Given that the majority of glycans of lubricin are mono- rather than disialylated (Table I and supplemental Table S1), an upper limit of 168 possible sialic acid residues was suggested
  6. The positive charge buffering capacity of lubricin required ∼60 sialic acids to give the STP-rich region of lubricin a negative charge at the physiological pH (7.2–7.4) of SF (43)
  7. An additional 10 sialic acids were required to render the whole lubricin negatively charged (Fig. 5A)
  8. Beyond 80 sialic acids, lubricin and its STP-rich region both were negatively charged and capable of maintaining the negative charge during pH shifts of SF and/or limited chemical/enzymatical agents that partially lowered the sialic acid content of lubricin
  9. This is likely the number of sialic acid residues required in order for lubricin to sustain its function on the cartilage surface
  10. We carried out isoelectric focusing before and after de-sialylation in order to better understand the contribution of sialic acid to the physical properties of lubricin
  11. The pI of lubricin before de-sialylation ranged from 4 to 7.5 in a chaotropic environment (Fig. 5B), whereas after de-sialylation the pI of lubricin was ∼7.5
  12. This suggests that the removal of sialic acids changed the molecule from highly acidic to basic and that in addition to the N and C termini , the mucin domain also became positively charged because of the presence of abundant Lys residues and the loss of sialic acid
  13. This analysis showed that lubricin is an amphoteric, mucin-like molecule with a negatively charged central domain that can become highly hydrated due to its glycosylation and is flanked by positively charged unglycosylated regions ( pI 9 .49–9.98) (Fig. 5C)
  14. The substantial change in the pI and the drastic alteration of the charge of lubricin with around 60 to 70 sialic acids indicates that there is a critical point where the number of glycosylation sites (controlled by the ppGalNAc Ts) and the amount of sialic acid (controlled by sialyltransferases) will significantly alter the properties of lubricin
  15. This shows that pathological alteration of the glycosylation of lubricin may contribute to an altered lubricating surface of articular joints
  16. Overall, in this study we used a combined CID/ETD- MS2 fragmentation approach to successfully characterize the heavily glycosylated STP-rich region of lubricin and identify an unprecedented 168 glycosylation sites on a single protein
  17. This approach allowed the identification of not only the site of glycosylation, but also its nature, providing a new understanding of the nature of this unique zwitterionic protein
  18. The use of prediction software uncovered the potential importance of novel transferases, which was confirmed by GALNT expression showing that the less understood GALNT5 and -15 were highly expressed in FLSs
Output (sent_index, trigger, protein, sugar, site):
  • 0. Glycosylation, , Lubricin, -, -
  • 1. glycosylated, , proteins, -, -
  • 13. unglycosylated, , pI 9, -, regions
  • 14. glycosylation, , -, sialic acid, sites
  • 15. glycosylation, , lubricin, -, -
  • 16. glycosylated, , -, -, region
  • 16. glycosylation, , -, -, sites
  • 17. glycosylation, , -, -, site
Output(Part-Of) (sent_index, protein, site):
  • 13. pI 9, regions
  • 16. STP, region
  • 16. lubricin, region
  • 16. protein, sites
  • 6. STP, region
  • 6. lubricin a, region
  • 8. STP, region
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX