Title : The Implications of the Identification of the Site-specific Glycosylation of
Lubricin and Its Role in Lubrication
Abstract :
- Sialylated and sulfated glycans will alter the charge of heavily glycosylated proteins
- Apomucins are usually neutral or acidic, secreted with a predicted pI of 2 to 4.7 (43, 44)
- 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
- 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)
- 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
- 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)
- An additional 10 sialic acids were required to render the whole lubricin negatively charged (Fig. 5A)
- 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
- This is likely the number of sialic acid residues required in order for lubricin to sustain its function on the cartilage surface
- 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
- 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
- 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
- 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)
- 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
- This shows that pathological alteration of the glycosylation of lubricin may contribute to an altered lubricating surface of articular joints
- 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
- 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
- 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):