PMID: PMC4256492-2

 

    Legend: Gene, Sites

Title : Decreased expression and increased degradation of lubricin have been suggested in the joints of RA and OA patients, making the changing characteristics of lubricin a potential indicator of arthritic disease progression

Abstract :

1. In addition to boundary lubrication, suggested to be established by core 1 O-glycosylation, the multiple protein domains of lubricin may serve other biological functions, such as the protection of chondrocytes and signaling (45)
2. The method adopted in this study allowed us to investigate the specific location of glycans on lubricin
3. The combination of CID and ETD methods not only enabled evaluation of the protein component and the location of the glycosylation, but also provided details of the attached glycan
4. Although this was a very effective approach, manual interpretation of CID/ETD- MS2 data was essential because of the lack of universal software in the field of glycoproteomics
5. The detailed analysis allowed further understanding of the zwitterionic nature of the protein
6. The inclusion of molecular biology to evaluate the expression of important glycosyltransferases of this highly specialized tissue showed that it has a very different profile from other tissues of the body
7. Western blot analysis revealed that unlike that of traditional mucous-forming mucin (46), the mucin-like domain of lubricin could be completely digested by trypsin (Figs. 1A and 1C)
8. Extensive degradation of lubricin by papain and Pronase and partial degradation by pepsin have also been reported previously (47)
9. In addition to these, neutrophil elastase (a serine protease ) and cathepsin B (a cysteine protease ) have also been shown to degrade lubricin in vitro (13, 48)
10. Given that lubricin was found to have an abundance of closely located occupied glycosylation sites , this suggests that it was the smaller size, rather than a smaller number of glycans, that made lubricin more enzyme accessible than other heavily glycosylated proteins such as mucins
11. The glycans identified included the previously reported released O-linked glycans of lubricin (7, 26, 38)
12. However, the confirmation of core 2 O-linked glycans, identified as core 2 glycopeptides from the previously defined mucin-like domain (aa 348–855), and the site-specific glycopeptide characterization of lubricin (in particular the STP-rich region ) are shown for the first time in this report
13. Core 2 structures are the oligosaccharide precursors of inflammatory epitopes such as sialyl Lewis x and sulfated sialylated type 2 structures (49)
14. These types of structures on lubricin have previously been indicated to influence joint inflammation (38)
15. Core 2 structures can also have other functions—for example, cell surface glycans reduce cell–cell interaction (50) and can even be used as cell surface markers to distinguish effector and memory CD8+ T cells (51)
16. CID- MS2 fragmentation of the O-linked glycopeptides produced sequence information for different glycoforms of the same tryptic peptide (EPAPTTPK) (Figs. 2A–2D)
17. This showed that lubricin glycosylation displayed both macro- (two separate core 1–like glycans) and site-specific micro-heterogeneity (different glycans at a single amino acid position ) (Figs. 2C and 2D)
18. However, because CID- MS2 resulted in extensive glycosidic fragment ions, it was not always possible to identify the site-specific location of the glycan in peptides with more than one Thr or Ser (Figs. 2A, 2B, and 2D)
19. To overcome this, ETD was used as an additional technique for the site-specific identification of glycans because it induces peptide backbone cleavage, leaving the glycan unaffected
20. An additional complication of ETD fragmentation in this study was the abundance of the small repeat (EPAPTTPK), as its low mass reduces the higher charge state advantages of ETD
21. Therefore, it was the novel combined use of CID and ETD that allowed the site-specific glycan localization and glycan determination of this difficult protein
22. The site-specific glycopeptide analysis (Fig. 4B) redefined the mucin-like domain to an extended STP-rich region (aa 232–1056)
23. This was due to the identification of extensive O-linked glycopeptides (e.g. peptide K972ITTLKTTTLAPK985V found outside the repeat domain showing four out of five glycosylated sites ) in the vicinity of the tandem repeat region of the previously defined mucin repeat domain suggested by UniProt (Figs. 4A and 4B)
24. In contrast to N-linked glycosylation, the identification of O-glycan attachment sites is made more difficult by the lack of a consensus sequence and the heterogeneity associated with extensive O-glycosylation
25. The recent increase in O-glycan data has allowed the development of prediction tools including NetOGlyc4.0 and ISOGlyP
26. It was obvious for lubricin glycosylation that without knowledge about the types of transferases present, the specificity of software such as NetOGlyc4.0 (39), based on neural network predictions of mucin type O-glycosylation sites from all 20 GalNAc Ts, will have some limitations (Fig. 4C)
27. In contrast, software such as ISOGlyP (40), based on individual glycosyl transferase prediction specificity, is likely to be more successful (Figs. 4C and 4D)
28. ISOGlyP predicted 191 O-glycosylation sites , more similar to the data presented in this report (168 Ser/Thr O-linked glycosylation sites )
29. Interestingly, the MS data identified a GalNAc and core 2 modified Thr (1159NGTLVAFR1166) in the hemopexin 1 domain of the C-terminal region (Figs. 4A and 4B; Table I; supplemental Fig
30. S1), which was not predicted to be glycosylated by either of the software programs (supplemental Table S2)
31. This might indicate a potential regulatory role associated with a particular ppGalNAc T, as the C-terminal recombinant construct of lubricin has been shown to be involved in binding to the cartilage surface (43)
32. The GALNT profiling expression analysis using primary FLSs showed high expression of the ubiquitous GALNT1 and -2 genes
33. In addition, high expression levels of GALNT5 and, particularly, GALNT15 were also shown
34. GALNT5 has been shown to exhibit a restricted expression pattern (21), including expression in chondrocytes (neXtProt)
35. GALNT15 has been suggested to have a broader expression pattern; its dominant expression in the FLSs indicated a particular role in the synovial tissue
36. The identification of GALNT15 as the 17th most abundant enzyme in chondrocytes (42) indicated that this less studied enzyme could be particularly important for the glycosylation of synovial lubricin
37. The site-specific glycopeptide analysis showed that majority of lubricin O-glycans were composed of core 1 structures with terminal galactose (Table I and supplemental Table S1)
38. Terminal galactose is a ligand for galectins, known to increase expression during RA (52) and suggested, along with fibrinogen, to play a pro-inflammatory role by regulating neutrophil activation and degranulation (53)
39. The high proportion of sialylated core 1 glycopeptides identified has biosynthetic importance, as the sialic-acid-terminated glycan end cannot be extended any further by glycosyltransferases in the Golgi/endoplasmic reticulum (54)
40. This might also explain the low proportion of core 2 structures identified as core 2 glycopeptides , which could be a consequence of low core 2 GlcNAc transferase activity or high sialyl transferase activity, or both
41. The high proportion of sialylated core 1 glycans on lubricin reduces the possibility of the formation of larger, potentially immunologically reactive glycans, restricting lubricin to short, negatively charged glycans
42. The terminal domains of lubricin have a large number of positively charged arginine and lysine residues , whereas the STP-rich region is negatively charged because of the attached sialic acid , making lubricin an amphoteric polyelectrolyte
43. Lubricin is suggested to be a good lubricant for negatively charged surfaces such as the surface of the outermost layers (lamina splendens) of the articular cartilage
44. This is mainly due to an increase in repellent charge forces between the negatively charged STP-rich region and the negatively charged components of the outermost layers of the cartilage such as hyaluronic acid, lipids, and proteoglycans (55)
45. The pI of synovial lubricin ranged from 4 to 7.5 as measured by isoelectric focusing (Figs. 5B and 5C)
46. De-sialylation increased the pI substantially to close to 7.5 (Fig. 5B)
47. The lower pI relative to the theoretical calculation for apolubricin ( pI 9 .8) is likely due to the influence of the pKa of individual amino acid residues by the chaotropic reagants and the remaining sulfated residues on the lubricin oligosaccharides (38)
48. Although lubrication might not be totally dependent on sialic acid , it might be enhanced through an increase in repellent charge forces due to an increase in negative charges around the STP-rich region domain (55)
49. The terminal somatomedin B-like and hemopexin-like domains have been shown to promote integrin-mediated attachment of cells to the extracellular matrix (6, 8)
50. It has also been reported that lubricin lacking these end domains binds only weakly to the cartilage surface (56)
51. This weak binding is suggested to result in inefficient lubrication (43)
52. Therefore, it can be speculated that for efficient lubricin function, both positively charged end domains and the negatively charged STP-rich region are essential

Output (sent_index, trigger, protein, sugar, site):

• 10. glycosylated, , proteins, -, -
• 10. glycosylation, , -, -, sites
• 12. glycopeptide, , -, -, glycopeptide
• 12. glycopeptides, , -, -, domain
• 12. glycopeptides, , -, -, glycopeptides
• 12. particular, , -, -, region
• 16. glycoforms, , -, -, peptide
• 16. glycopeptides, , -, -, glycopeptides
• 22. glycopeptide, , -, -, glycopeptide
• 23. glycopeptides, , -, -, glycopeptides
• 23. glycosylated, , -, -, sites
• 24. heterogeneity, , -, -, sequence
• 26. O-glycosylation, , -, -, sites
• 28. O-glycosylation, , -, -, sites
• 28. glycosylation, , -, -, sites
• 28. presented, , -, -, sites
• 36. glycosylation, , lubricin, -, -
• 37. glycopeptide, , -, -, glycopeptide
• 39. glycopeptides, , -, -, glycopeptides
• 39. sialylated, , -, -, glycopeptides
• 40. glycopeptides, , -, -, glycopeptides

Output(Part-Of) (sent_index, protein, site):

• 1. lubricin, domains
• 1. protein, domains
• 12. -, glycopeptides
• 12. STP, region
• 18. -, Ser
• 18. -, Thr
• 22. STP, region
• 29. hemopexin 1, domain
• 29. hemopexin 1, region
• 42. STP, region
• 42. lubricin, domains
• 44. STP, region
• 48. STP, domain
• 49. hemopexin, domains
• 52. STP, region
• 7. lubricin, domain

*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

Protein	NCBI ID	SENTENCE INDEX