PMID: PMC4084840-2-4

 

    Legend: Gene, Sites

Title : Newly Described O-Glycopeptides of ITIH4

Abstract :
  1. Both recombinant and serum ITIH4 are glycosylated on peptides IPKPEASFSPRand ASFSPR containing serine residues S640 and S642
  2. Peptides LAILPASAPPATSNPDPAVSR and LAILPASATPATSNPDPAVSR from two ITIH4 variants are glycosylatedin serum and have similar associated glycan com positions
  3. The presenceof an additional threonine in the variant form does not impact theglycan com positions associated with the peptide
  4. In recombinant ITIH4 ,LAILPASATPATSNPDPAVSR contains smaller andless sialylated glycan com positions compared to serum ITIH4
  5. PeptideGPDVLTATVSGK is glycosylated exclusively in recombinant ITIH4
  6. Initiation of O-GalNAc glycosylation through the transfer ofGalNAc to serine or threonine residues is controlled by a family ofat least 20 UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts).
  7. Differences in glycosylation site- and tissue-specificity are expectedto be more pronounced for O-GalNAc glycosylation compared to N-glycosylation
  8. Our observation of site-specific differences in O-glycosylation betweenserum-derived and recombinant ITIH4 is consistent with this observation
  9. We observe the same glycan com positions (HexNAc1Hex1NeuAc1 and HexNAc1Hex1NeuAc2) on peptides IPKPEASFSPR andASFSPR in serum and recombinant ITIH4
  10. In serum ITIH4 , we detectedglycosylation at S640 but not at S642
  11. Glycosylation of this peptidein different contexts suggests that glycosylation at this site mayplay an important role in ITIH4 stability or function
  12. Serum ITIH4 peptide LAILPASAPPATSNPDPAVSR ( residues691–710 ) is O-glycosylated at three sites , on residues S696,T701, and S709, and we have characterized multiple glycoforms of thispeptide
  13. Our glycopeptide data indicate that this peptide containsglycan com positions as large as HexNAc3Hex3NeuAc4
  14. When we analyzed detached O-glycans in serumand recombinant ITIH4 , we observed HexNAc1Hex1NeuAc1 and HexNAc1Hex1NeuAc2, and additional low-abundance glycoforms
  15. Together, this suggests that S696, T701, and S709 are occupied withsimple core-1 O-glycans
  16. While we do not observe this peptide in recombinant ITIH4 , we detect the PAVSRVMNMK (residues 706–715)+ HexNAc1Hex1NeuAc2 glycopeptide (containing S709) in trypsin-chymo trypsin digests of recombinant ITIH4
  17. This suggests that S709 is glycosylated in both serum and recombinant ITIH4 , while additional glycosylation occurs in serum ITIH4
  18. We alsoobserve multiple glycoforms of peptide GPDVLTATVSGK (withpotentially glycosylated residues T506, T508, and S510) in recombinant ITIH4 , but not in serum-derived ITIH4 , and we detected two fucosylatedO-glycopeptides on GPDVLTATVSGK from recombinant ITIH4but did not detect any fucosylated O-glycopeptides in serum-derived ITIH4
  19. In addition to the glycopeptides discussed above, evidenceof ITIH4 glycosylation on residues 719–725 has been reportedin a study of glycopeptides from urinary protein digests
  20. However, we do not observe any glycopeptidesat this site ; this may be due to the different sources of ITIH4 orpossibly reflect proteolytic resistance of the ITIH4 sequence
  21. Itis also possible that high levels of O-glycosylation in this regionof ITIH4 may protect ITIH4 from proteolysis in its native environmentbut could also prevent us from observing glycopeptides if high-densityglycosylation blocks access of trypsin , endoproteinase Glu-C, andchymo trypsin to proteolytic sites in this region
  22. The ITIH4 sequenceis atypical in that it contains regions without frequent sites forcleavage of trypsin , chymo trypsin , and other common proteases
  23. Thismakes bottom-up analyses challenging and necessitates the use of multipleproteases for observation of different glycopeptides
  24. We detectedseveral glycoforms of glycopeptide IPKPEASFSPR in serumand recombinant ITIH4 , and we have confirmed that S640 is glycosylatedin serum ITIH4
  25. This sequence is specific to ITIH4 isoform 1 1 and thereforecontributes to isoform-specific glycosylation that may influence ITIH4stability or interaction with the ECM
  26. On the basis of expressionstudies, ITIH4 is primarily synthesized in the liver, and four isoforms of ITIH4 have been predicted based onmRNA and/or cDNA sequencing experiments
  27. Three ITIH4 isoforms have been describedin adult liver tissue, while the fourth was found in fetal human liver
  28. Isoform 1 was the first to be described and has been selected as the“canonical” sequence in UniProt
  29. In this discussion,all references to amino acid residue locations are made in referenceto ITIH4 isoform 1 1
  30. Isoforms 2–4 are missing small regions(all in the C-terminal half of the protein) compared to isoform 1 1
  31. Isoform 2 also has a unique sequence “ACPSCSRSRAPAVPA”starting at residue 727
  32. Specifically , isoforms 2 –4 do notcontain residues 621–650 of ITIH4 isoform 1 1 , and intriguingly,our proteomic analyses of serum-derived ITIH4 show that the peptideIPKPEASFSPR (and ASFSPR withone missed GluC cleavage at E) from this region of ITIH4 is O-glycosylated
  33. Indeed , glycoprotein interactions with the ECM are frequently modulatedby the presence of O-glycans and isoform-specific O-glycosylationcould have important implications for ECM stability
  34. We have alsoselected ITIH4 isoform 1 1 for overexpression in HEK293 cells and confirmthat this region is glycosylated in the cell line as well, and identicalglycan com positions are present in serum and recombinant ITIH4
  35. In addition to detecting isoform-specific glycosylation, we alsodetected several O-glycopeptides that flank the proline-rich regionof ITIH4 , which may also have an impact on ITIH4 stability or interactionswith other proteins , or the ECM
  36. ITIH4 can undergo cleavage by plasmakallikrein between R688 and R689, andmay undergo additional proteolytic degradation after this initialcleavage based on detection of native peptides in serum and plasma
  37. ASFSPR (residues 639–644) and LAILPASA(P/T)PATSNPDPAVSR(residues 690–710), both O-glycopeptides described in our currentstudy, flank the plasma kallikrein cleavage site and the potentiallybioactive peptide
  38. Due to the potential of glycosylation to impactproteolytic processing, these findings deserve further investigation
Output (sent_index, trigger, protein, sugar, site):
  • 1. glycosylated, , ITIH4, -, -
  • 1. residues, , -, -, serine residues
  • 10. detectedglycosylation, , ITIH4, -, -
  • 11. glycosylation, , -, -, site
  • 12. O-glycosylated, , -, -, residues691–710
  • 12. O-glycosylated, , -, -, sites
  • 12. glycoforms, , -, -, thispeptide
  • 12. residues, , -, -, residues S696,T701
  • 13. glycopeptide, , -, -, glycopeptide
  • 16. glycopeptide, , -, -, glycopeptide
  • 17. glycosylated, , ITIH4, -, -
  • 18. GPDVLTATVSGK, , -, -, residues
  • 18. O-glycopeptides, , -, -, O-glycopeptides
  • 18. fucosylated, , -, -, O-glycopeptides
  • 18. fucosylatedO-glycopeptides, , -, -, fucosylatedO-glycopeptides
  • 18. glycosylated, , -, -, residues
  • 18. residues, , -, -, residues
  • 19. glycopeptides, , -, -, glycopeptides
  • 19. glycosylation, , -, -, residues
  • 21. glycopeptides, , -, -, glycopeptides
  • 23. glycopeptides, , -, -, glycopeptides
  • 24. glycopeptide, , -, -, glycopeptide
  • 32. O-glycosylated, , -, -, peptideIPKPEASFSPR
  • 33. glycoprotein, , glycoprotein, -, -
  • 34. glycosylated, , -, -, region
  • 35. O-glycopeptides, , -, -, O-glycopeptides
  • 37. O-glycopeptides, , -, -, O-glycopeptides
  • 5. glycosylated, , ITIH4, -, -
  • 6. serine, , -, -, threonine residues
  • 9. compositions, , -, -, positions
Output(Part-Of) (sent_index, protein, site):
  • 0. ITIH4, O-Glycopeptides
  • 12. ITIH4, residues691–710
  • 2. ITIH4 variants, -
  • 20. ITIH4, sequence
  • 32. -, peptideIPKPEASFSPR
  • 32. ITIH4 isoform 1, residues
  • 32. ITIH4, peptideIPKPEASFSPR
  • 34. ITIH4, positions
  • 37. plasma kallikrein, site
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 12. ITIH4, -, residues S696,T701

 

 

Protein NCBI ID SENTENCE INDEX