PMID: PMC4084840-2-2

 

    Legend: Gene, Sites

Title : Site-SpecificMicroheterogeneity of ITIH4 N-Glycoforms

Abstract :
  1. Contrary to siteoccupancy, the microheterogeneity of N-glycoforms differs substantiallybetween recombinant and serum-derived ITIH4
  2. On the basis of CID MS/MSof untreated and exoglycosidase-treated glycopeptides and MALDI-TOFanalysis of detached, permethylated N-glycans, we conclude that complex,core fucosylated, asialo-glycoforms are dominant in recombinant ITIH4
  3. Fully sialylated forms represent a minor component of all N-glycoformsat all sites except N81
  4. This is also the only canonical glycosylationsite with detectable high mannose glycans
  5. Core-fucosylated glycansat N81 represent a minor component of the total microheterogeneity
  6. This is in contrast to the glycoforms at the three other canonicalglycosylation sites ( N207, N517, and N577 ) of recombinant ITIH4 , wherecore-fucosylated forms dominate
  7. We also observed more highly branchedN-linked glycans, including tetra-antennary glycans, at N517 and N577
  8. Of the four canonical sites , we observed the highest microheterogeneityof glycoforms at N517
  9. This may be due in part to the efficiency ofour analytical workflow for this glycosylation site ; for example,the LPTQNITFQTE peptide is the only proteolytic productwe observe at the N517 N-glycosylation site , but we observe semispecificproteolytic cleavage (peptides AFINTF, KAFITNF) near the N81 site
  10. The observation of multiple proteolytic products at site N81 may limitour ability to detect minor glycoforms
  11. However, in trypsin- GluC digestswe observe only one proteolytic product containing site N207 , andin trypsin-chymotrypsin digests we only observe one proteolytic productcontaining site N577
  12. Even at these sites we observe fewer glycoformsthan at N517 , which suggests that the site-specific differences inmicroheterogeneity are likely of biological or structural origin
  13. However, we cannot rule out the possibility that differences in peptideionization may also play a role in these observations
  14. Serum-derived ITIH4 N-linked glycopeptides demonstrate higher levels of sialylation,lower levels of fucosylation, differences in fucose linkage, and lessmicroheterogeneity compared to recombinant ITIH4
  15. Complex, sialylatedN-glycans are present at all four canonical N-glycosylation sitesof serum-derived ITIH4
  16. MALDI-analysis confirmed the predominanceof sialylated N-linked glycans and also corroborated the presenceof minor fucosylated glycoforms
  17. On the basis of our site-specificcharacterization of glycopeptides from serum ITIH4 , it appears thatmost fucosylated glycoforms are restricted to sites N517 and N577 ,although we also detected a triantennary fucosylated N-glycan at N81after treatment with a broad-specificity neuraminidase
  18. We also detectedmore branching (tri- and tetra-antennary glycoforms) at N517 and N577than at N81 and N207 , a pattern that is consistent with recombinant ITIH4
  19. This is consistent with the fact that site specificity of N-glycanstructures, including branching, depends on the structure of the matureprotein
  20. Processing of N-linked glycans by exoglycosidasesand glycosyltransferases, including extension and capping, take placeprimarily in the Golgi apparatus after the protein is already folded;access of glycosyltransferases to N-linked glycans is thought to beinfluenced by the protein structure
  21. Therefore,processes governed by protein structure should have similar outcomesin recombinant and serum-derived ITIH4 if the enzymes are presentin active form
  22. This is consistent with our findings of site-specificN-glycan branching and suggests that activity of neuraminidases islower and activity of FUT8 is higher in the HEK293 compared to humanliver, the major source of the serum derived ITIH4
  23. This conclusionhas to be viewed, however, with caution given many unknown factorsincluding distribution and stability of the glycoforms invivo
Output (sent_index, trigger, protein, sugar, site):
  • 14. glycopeptides, , -, -, glycopeptides
  • 17. glycopeptides, , ITIH4, -, glycopeptides
  • 17. sites, , -, -, sites N517 and N577
  • 2. glycopeptides, , -, -, glycopeptides
  • 5. Core-fucosylated, , -, -, N81
  • 6. canonicalglycosylation, , -, -, N207, N517, and N577
  • 6. canonicalglycosylation, , ITIH4, -, N207, N517, and N577
  • 6. canonicalglycosylation, , ITIH4, -, sites
  • 6. glycoforms, , -, -, N207, N517, and N577
  • 6. glycoforms, , -, -, sites
  • 6. sites, , -, -, N207, N517, and N577
  • 6. sites, , -, -, sites
  • 6. wherecore-fucosylated, , ITIH4, -, -
  • 9. N-glycosylation, , -, -, site
  • 9. glycosylation, , -, -, site
Output(Part-Of) (sent_index, protein, site):
  • 14. ITIH4, glycopeptides
  • 17. ITIH4, glycopeptides
  • 17. ITIH4, site-specificcharacterization
  • 6. ITIH4, N207, N517, and N577
  • 6. ITIH4, sites
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 17. ITIH4, -, sites N517 and N577
  • 5. ITIH4, -, N81
  • 6. ITIH4, -, N207, N517, and N577

 

 

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