PMCID: PMC4256492

 

    Legend: Gene, Sites, Suger

Section : Accessibility and Characterization of the Glycosylated Region of Lubricin

Content :

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

Section : Identification of Lubricin Mucin Glycopeptides Using CID and ETD

Content :

1. In order to analyze the STP-rich region and identify the number of glycosylation sites on synovial lubricin , we adopted a combined approach using both CID- and ETD- MS2 to identify the types of glycans attached, as well as their position
2. Tryptic glycopeptides of both RA and OA samples were generated both before and after partial de-glycosylation (Fig. 1B) for subsequent mass spectrometric analysis
3. The LC-CID/ETD- MS2 approach successfully identified 185 O-glycosylated peptides
4. They are presented, together with the identified O-linked glycans, glycan attachment sites , and annotation method (software or manual) of each individual glycopeptide , in Table I (and in supplemental Table S1)
5. Predominantly core 1 and monosialylated core 1 (NeuAcα2–3Galβ1–3GalNAcα1-) O-linked glycopeptides were identified
6. A small proportion of disialylated core 1 (NeuAcα2–3Galβ1–3(NeuAcα2–6)GalNAcα1-) peptides were also detected
7. The peptides (KPAPTTPK) identified as non-glycosylated in the previously defined mucin-like domain and in the STP-rich region (VLAKPTPK and KPAPTTPK) were also shown to be glycosylated
8. The glycosylation of the threonine in the KPAPTTPK repeat was also shown, as indicated by the data presented in this report (supplemental Table S1)
9. In addition to core 1, a small proportion of core 2 (Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1-) and monosialylated core 2 (e.g. NeuAcα2–3(Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1-)) glycopeptides were also identified
10. These findings are consistent with the O-linked glycans identified in our and others' previous studies (7, 26, 38)
11. The CID- MS2 approach effectively identified the nature of the glycans attached to lubricin
12. CID- MS2 spectra of four different O-linked isoforms of the same amino acid sequence (EPAPTTPK) located in the STP-rich region are presented in Figs. 2A–2D
13. The spectrum of the [M+2H]2+ ions at m/z 603.3 (Galβ1–3GalNAcα1-O-[EPAPTTPK]) resulted in fragmentation of the glycan component into y-type ions (Domon and Costello nomenclature) and b-type ions corresponding to [HexNAc+H]+ and [HexHexNAc+H]+ oxonium ions at m/z 204 and 366, respectively (Fig. 2A)
14. This corresponded to a glycopeptide with a core 1 glycan at one of the Thr residues
15. The spectrum showed the neutral loss of Hex residue (m/z 1043.6), which was followed by a loss of HexNAc residue (m/z 840.3), establishing the glycan sequence as Hex-HexNAc
16. However, the CID- MS2 spectrum did not show which of the Thr residues was glycosylated
17. The sialylated version of this glycopeptide was also identified (Fig. 2B)
18. The presence of an oxonium ion at m/z 292 (sialic acid, NeuAc) in the CID- MS2 spectrum of the [M+2H]2+ ions at m/z 748.8 (NeuAc-Hex-HexNAc-O-[EPAPTTPK]) showed that this glycopeptide can also be sialylated (Fig. 2B)
19. The loss of a NeuAc residue (m/z 1205.3) followed by the loss of Hex (m/z 1043.3) and finally the loss of HexNAc (m/z 840.5) indicated a NeuAc-Hex-HexNAc- structure attached to a Thr residue in the peptide sequence (Fig. 2B)
20. This, together with previous O-glycan analysis, suggested that the attached structure was NeuAcα2–3Galβ1–3GalNAcα1-O-Thr Thr
21. The CID- MS2 spectra allowed the identification of isomeric glycopeptides , showing differences in the number of glycosylation sites and glycan sequences
22. The presence of diagnostic ions at m/z 407 [HexNAc2\+H]\+ nd 569 [Hex(HexNAc)HexNAc\+H]\+ Fig. 2D) were used to differentiate a single substituted core 2 O-glycan from a doubly substituted core 1 O-glycan on both Thr residues in EPAPTTPK repeats
23. These results indicated that there were heavily glycosylated regions of the STP-rich region , such as the doubly glycosylated EPAPTTPK repeat shown in Fig. 2C
24. The STP region also displayed more complex O-glycosylation such as the core 2 structure shown in Fig. 2D
25. ETD- MS2 analysis was used for the identification of glycosylation sites within the STP-rich region , particularly for the identification of non-consensus repeats
26. Generally, the ETD- MS2 highly charged glycopeptide precursor ions fragmented efficiently, allowing the site of glycosylation to be further narrowed down, in most cases to single amino acid residues (Table I and supplemental Table S1)
27. All ETD- MS2 spectra were manually annotated for verification of the location of uniquely modified Ser/Thr residues in order to remove all possible ambiguity
28. The ETD- MS2 spectrum of the [M+3H]3+ ions at m/z 673.3 glycopeptide (K972ITTLKTTTLAPK985V) allowed the identification of the glycan-modified Thr residues within the peptide sequence
29. The ETD- MS2 spectrum, [M+3H]3+ ions at m/z 673.3 (Fig. 3A), displayed the z10+1-, z9+1-, and c7-ions at m/z 1788.6, 1687.8, and 1506.6, respectively, indicating that Thr974, Thr975, and Thr980 were unmodified
30. However, the c6-ion at m/z 1040.4 was observed with the addition of a Hex-HexNAc residue (365 Da), indicating that the Thr979 was modified with an unsialylated core 1 structure
31. The c5-ion at m/z 574.4 showed that the adjacent Thr978 was modified with a second Hex-HexNAc residue (Fig. 3A)
32. The z7-ion at m/z 1444.7 denoted that the two core 1 glycans (Hex-HexNAc) were still intact and attached to the peptide
33. However, the z5+1-ion at m/z 513.2 confirmed that both Thr978 and Thr979 were core 1 modified, as it was the loss of two threonines and two Hex-HexNAc- units
34. Lubricin displayed macroheterogeneity as shown by the identification of an isomeric glycopeptide (Fig. 3B)
35. The ETD- MS2 spectrum of the [M+3H]3+ ions at m/z 673.3 indicated the same peptide sequence (K972ITTLKTTTLAPK985V) with two core 1 glycans on separate threonine residues (Fig. 3B)
36. The c7-ion at m/z 1141.6 indicated the loss of the Hex-HexNAc Thr980
37. The c6-ion at m/z 1040.6 was the loss of unmodified Thr979
38. The Thr978 was modified with the second Hex-HexNAc residue identified by the c5-ion at m/z 574.5, the loss of Thr978 with a HexHexNAc
39. The c5-ion at m/z 574.5, a peptide with two core 1 glycans, indicated that Thr974 and Thr975 were unmodified
40. The ETD- MS2 spectra of the isomeric [M+3H]3+ ion at m/z 673.3 (Figs. 3A and 3B) revealed the site occupancy within the glycopeptide K972ITTLKTTTLAPK985V of SF lubricin
41. Overall, the dual fragmentation approach identified 185 lubricin glycopeptides , primarily from the STP-rich region
42. This allowed us to characterize 168 glycosylation sites , predominantly in the STP-rich region (aa 232–1056), covering 71% of the Ser/Thr in this STP-rich region
43. This, together with the identified non-glycosylated Ser/Thr (mainly in the N and C termini ), covered 72% of the Ser/Thr (266 out of 370 Ser/Thr were identified) in the entire protein sequence
44. The Ser/Thr coverage provided one of the most extensive O-glycosylation maps of a mucin-type protein (supplemental Fig
45. S2 and supplemental Table S1)
46. The identified glycosylated and non-glycosylated Ser/Thr (both in the N and C termini and in the STP-rich region ) are shown in supplemental Fig
47. S2
48. In addition to the Ser/Thr coverage, the mass spectrometric approach covered 82% of the entire protein sequence , and the coverages for the N terminus (aa 1–231), STP-rich region (aa 232–1056), and C terminus (aa 1057–1404) were 79%, 80%, and 85%, respectively

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

• 38. Lubricin, a HexHexNAc, Thr978

Section : The O-glycosylation Map of Lubricin

Content :

1. The identified O-glycopeptides , glycan com position, fragmentation technique, annotation technique (software/manual), and glycosylation sites are listed in Table I (and in supplemental Table S1)
2. Regions of glycosylation sites identified via CID and ETD both before and after partial de-glycosylation are shown in Fig. 4A
3. The identified O-glycopeptides characterized 168 glycosylation sites
4. This indicated that 63% of the identified Ser/Thr residues (266 Ser/Thr , both glycosylated and non-glycosylated, were identified) in lubricin were O-glycan modified (Fig. 4B) with a bias toward Thr glycosylation due to the high Thr content (supplemental Fig
5. S2)
6. An extended STP-rich region was also apparent spanning amino acids 232–1056, larger than the previously defined mucin-like domain suggested in UniProt
7. The entire lubricin molecule has in total 370 potential O-glycosylation sites
8. Of these, 35% of Ser/Thr (130 glycans) were HexNAc (GalNAc) modified with a distribution throughout the extensively glycosylated STP-rich region (Fig. 4B)
9. Core 1 modified (43%, 161 glycans) and larger core 2 modified (23%, 85 glycans) glycopeptides were also identified (Fig. 4B)
10. The high-abundant core 1 modified Ser/Thr were uniformly distributed throughout the STP-rich region , whereas the low-abundant core 2 modified residues were limited to the previously defined mucin-like domain (aa 348–856) (Fig. 4B)
11. This was likely because the accumulative nature of glycopeptides from the repeat region made them easier to detect, and it might not necessarily be a reflection of core 2 enrichment in the repeat area
12. Outside the STP region , only a single GalNAc-modified Thr (1159NGTLVAFR1166) was identified
13. This residue , in the hemopexin 1 domain (1148–1191), was also shown to be glycosylated with core 2 structures (Figs. 4A and 4B; supplemental Fig
14. S1; supplemental Table S2)
15. The identification of core 2 glycopeptides confirmed the presence of core 2 structures on lubricin
16. The identification of core 2 together with single HexNAc (GalNAc)-modified glycopeptides suggested that lubricin glycosylation might also have other roles in addition to lubrication
17. The majority of the GalNAc extended into either core 1 or core 2 sialylated structures (73 glycans; Fig. 4B)
18. Both mono- and disialylated core 1 and core 2 modified Ser/Thr were identified (Table I and supplemental Table S1), but monosialylation was more prevalent, which is consistent with previously identified synovial lubricin O-glycans (38)

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

Section : Comparison of the Lubricin O-glycomap with Predicted O-glycosylation Sites

Content :

1. The glycosylations identified here were compared with currently available O-glycosylation prediction tools
2. The glycosylation prediction tool NetOGlyc4.0 (39) is based on in vivo identified O-glycosylation sites and predicted almost double the number of O-glycosylation sites identified here (almost 90% of all Ser/Thr residues ) (Fig. 4C)
3. In addition to the STP-rich region , most of the Ser/Thr residues in the N-terminal region were predicted to be O-glycosylated (Fig. 4C)
4. An individual ppGalNAc T in vitro enzymatic specificity–based prediction tool, ISOGlyP (40), predicted that 51% (191) of the sites were glycosylated (Figs. 4C and 4D) utilizing all ppGalNAc T specificities available in the tool
5. This prediction is closer to the 168 detected in this study (Fig. 4B)
6. ISOGlyP predicted that a high proportion of the Thr in the STP-rich region would be glycosylated, as was also shown by the MS analysis (Table I, supplemental Fig
7. S2, supplemental Table S1)
8. However, no single ppGalNAc T of those included in the tool was able to glycosylate all the sites identified via MS. The ubiquitously expressed GALNT1 and -2 were suggested to glycosylate only 76% of the total sites found via MS (Fig. 4D)
9. In total, 166 out of 191 (87%) sites predicted by the nine available genes in the software were identified through MS analysis (Fig. 4D)
10. This suggested that a GALNT not included in ISOGlyP might be responsible for at least some of the glycosylation on synovial lubricin (Fig. 4D and supplemental Table S2)

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

Section : Investigating the Expression of Glycosyltransferase Genes and Glycosylation

Content :

1. An alternate method for understanding glycosylation is to investigate the Golgi apparatus glycosyltransferases responsible for glycosylating lubricin
2. Because the ISOGlyP results suggested less common transferases were necessary for lubricin glycosylation, the expression of ppGalNAc Ts from human primary FLSs isolated from RA and OA patients was investigated
3. These types of cell lines are known to produce lubricin ( 1 )
4. The relative quantifications of all transcripts were normalized against β-actin expression
5. The average (n = 4, except for GALNT8 , where n = 3) expression of the GALNT genes is arranged in descending order of expression in Fig. 4D
6. High mRNA expression was observed for GALNT1 , -2, -5, and 15, and lower expression was noted for the GALNT8 , -10, -12, and 16 genes
7. The high expression of GALNT1 and -2 was in agreement with the suggestion that these two genes are ubiquitously expressed
8. In contrast, GALNT5 and GALNT15 have been shown to display restricted expression profiles, suggesting these isoforms serve unique functions in the tissue where they are expressed (2 1 )
9. The high expression of GALNT5 in FLSs indicated a potential role of this gene, and its relevance is increasing, as the expression of this gene has also been shown in chondrocytes (neXtProt)
10. The GALNT5 gene was also able to correctly predict 54% of the sites identified via MS (Fig. 4D), which also indicates potential involvement of this gene in lubricin glycosylation
11. The data showed that the highest expression was of the GALNT15 gene in the FLS cultures
12. The specificity of this enzyme toward mucin-type domains is not currently understood (4 1 ), making its further investigation essential, especially as the gene has been shown to be one of the most expressed genes in chondrocytes and bone (2 1 , 42)

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

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

Content :

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

Section : Accessibility and characterization of the glycosylated region of lubricin

Content :

1. A, the enriched synovial lubricin samples, before and after trypsin digestion, were separated on a 3–8% Tris acetate gel, blotted onto PVDF membrane, and then probed with lubricin-specific antibody (mouse anti- lubricin ) and carbohydrate-specific biotinylated lectins PNA, specific for core 1 (Galβ1–3GalNAc) O-glycan, and WGA, specific for sialic acid and terminal GlcNAc
2. B, SDS- PAGE (3–8 % Tris acetate gel) of the acidic glycoprotein fractions of the SF before (−) and after (+) partial de-glycosylation stained with Coomassie Brilliant Blue
3. C, SF lubricin was in-solution digested, and non-modified peptides were identified via mass spectrometry for protein coverage determination (black)
4. The low protein coverage (in particular the mucin domain ) suggests that the mucin domain is extensively glycosylated
5. Some of the core 1 structures were removed by partial de-glycosylation, and the previously glycosylated peptides were identified for protein coverage (gray)
6. The results suggest that lubricin contains an extended STP-rich region relative to the mucin domain previously defined by UniProt

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

Section : Identification of lubricin mucin glycopeptides using CID

Content :

1. The figure shows the different glycoforms of the same tryptic peptide within the STP-rich region
2. A, CID-MS2 spectrum of the tandem repeat (EPAPTTPK) containing core 1 glycan (Galβ1–3GalNAcα1-) of the [M+2H]2+ ions at m/z 603.3
3. B, CID-MS2 spectrum of the same tandem repeat containing sialyl T antigen (NeuAcα2–3Galβ1–3GalNAcα1-) of the [M+2H]2+ ions at m/z 748.8
4. C, CID-MS2 spectrum of the same tandem repeat (EPAPTTPK) containing two separate core 1 glycans (Galβ1–3GalNAcα1-) on two adjacent threonines of the [M+2H]2+ ions at m/z 785.8
5. D, CID-MS2 spectrum of the same tandem repeat containing core 2 glycan Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1- of the [M+2H]2+ ions at m/z 785.8
6. This shows that the same peptide (A, B, and D) can be differently glycosylated, indicating the complexity associated with lubricin O-glycosylation , and that CID was able to provide information about the nature of glycans and peptides
7. In the figure, yellow squares and circles represent N-acetylgalactosamine and galactose, respectively, and blue squares and pink diamonds represent N-acetylglucosamine and N-acetylneuramininc acid (sialic acid) , respectively

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

Section : Identification of glycosylation sites using electron transfer dissociation (ETD)

Content :

1. A, ETD- MS2 spectrum of the [M+3H]3+ ions at m/z 673.3 indicates a tryptic peptide (ITTLKTTTLAPK) with two separate core 1 glycans (Galβ1–3GalNAcα1-) on two adjacent threonine residues
2. B, ETD- MS2 spectrum of the [M+3H]3+ ions at m/z 673.3 reveals the same tryptic peptide (ITTLKTTTLAPK) with two separate core 1 glycans (Galβ1–3GalNAcα1-) on two separate threonine residues , indicating that lubricin glycosylation displays macroheterogeneity within the same peptide sequence
3. M denotes the glycopeptide parent mass

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

Section : The A, the glycosylated peptides identified in the STP-rich region and non-glycosylated peptides (light gray) in the end domains of the lubricin sequence before (black) and after (dark gray) partial de-glycosylation using CID and ETD fragmentation

Content :

1. B, graphic representation of all of the identified glycans and their positions in the protein sequence
2. The figure includes the identified glycopeptide containing GalNAcα1- (dark orange, 130), core 1 Galβ1–3GalNAcα1- (chocolate, 161), core 2 Galβ1–3(Galβ1–4GlcNAcβ1–6)GalNAcα1- (blue, 85), and sialylated core 1 and 2 (pink, 73) glycans with their positions in the protein sequence
3. The graph also includes the total number of identified glycosylation sites (168 sites ) in the protein sequence (black)
4. The total number of sites is based on the identified peptides and the fact that two threonines in the imperfect tandem repeat variants (e.g. EPAPTTPK, SAPTTPK, EPAPTTTK) can be glycosylated
5. It indicates a high number of glycosylations in the STP-rich region , whereas the N- and C-terminal regions are scarcely glycosylated
6. C, the combined CID and ETD identified glycopeptides in the STP-rich region (black and dark gray) and non-glycosylated peptides (light gray) in the end domains of lubricin before (black) and after partial de-glycosylation (dark gray)
7. The software used (NetOGlyc4.0 and ISOGlyP) predicted sites of lubricin O-glycosylated Ser/Thr in the protein sequence (yellow) and the total number of potential Ser/Thr sites (370)
8. The results indicate that the gene-based ISOGlyP software predicted a similar number of glycosylation sites (191 sites ) as identified by the data presented in this report (168 sites )
9. D, the GALNT profiling expression analysis of the 20 known human GALNT genes (top) in primary FLSs and the ISOGlyP-predicted sites by the available GALNT genes identified by the data presented in this report (bottom)
10. The genes were arranged in descending order of their expression
11. RQ, relative quantification
12. Asterisk denotes genes not included in the ISOGlyP software

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

Section : The implication of the type and distribution of A, the predicted isoelectric point (pI) of full-length (black) and STP-rich regions (dark gray) for varying numbers of sialic acids identified, indicating that the pI decreases as the number of sialic acids increases

Content :

1. B, enriched acidic SF fractions, before and after sialidase treatment, were separated on pH 3 –10 IPG gels and blotted to PVDF membrane
2. Western blot analysis using lubricin-specific antibody (mAb13) indicated a pH range of synovial lubricin before sialidase treatment ( pH 4 –7.5) and a drastic increase in pH (pH 7.5) after sialidase treatment
3. C, the pI of the N- (9.45–9.6) and C-terminal regions (9.69–9.98) (light gray), where very few glycosylation sites were identified, and the extended STP-rich region , which contained the majority of the protein glycosylation (4–7.5)

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

Section : 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

Content :

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

Section : Concluding Remarks

Content :

1. The mass spectrometric site-specific glycopeptide characterization performed in this study mapped the glycosylation profile of lubricin within the STP-rich region and indicates that lubricin glycosylation displays both micro- and macroheterogeneity
2. The presence of two adjacent simultaneously glycosylated Thr residues in the consensus repeat unit EPAPTTPK indicated that there are regions within the lubricin domain that are highly glycosylated
3. The data presented here redefine an extended STP-rich region relative to the mucin domain previously defined by UniProt
4. Screening of ppGalNAc Ts from primary FLSs showed high expression of the less understood GALNT15 and GALNT5 genes, indicating that lubricin glycosylation is unique
5. Overall, this study showed that heavy glycosylation, particularly sialylation, is essential for creating the amphoteric nature of lubricin , a property that may facilitate its efficient biolubrication function

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

 

Protein	NCBI ID	SENTENCE INDEX