Title : Site-SpecificMicroheterogeneity of
ITIH4 N-Glycoforms
Abstract :
- Contrary to siteoccupancy, the microheterogeneity of N-glycoforms differs substantiallybetween recombinant and serum-derived ITIH4
- 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
- Fully sialylated forms represent a minor component of all N-glycoformsat all sites except N81
- This is also the only canonical glycosylationsite with detectable high mannose glycans
- Core-fucosylated glycansat N81 represent a minor component of the total microheterogeneity
- This is in contrast to the glycoforms at the three other canonicalglycosylation sites ( N207, N517, and N577 ) of recombinant ITIH4 , wherecore-fucosylated forms dominate
- We also observed more highly branchedN-linked glycans, including tetra-antennary glycans, at N517 and N577
- Of the four canonical sites , we observed the highest microheterogeneityof glycoforms at N517
- 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
- The observation of multiple proteolytic products at site N81 may limitour ability to detect minor glycoforms
- 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
- 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
- However, we cannot rule out the possibility that differences in peptideionization may also play a role in these observations
- 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
- Complex, sialylatedN-glycans are present at all four canonical N-glycosylation sitesof serum-derived ITIH4
- MALDI-analysis confirmed the predominanceof sialylated N-linked glycans and also corroborated the presenceof minor fucosylated glycoforms
- 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
- 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
- This is consistent with the fact that site specificity of N-glycanstructures, including branching, depends on the structure of the matureprotein
- 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
- Therefore,processes governed by protein structure should have similar outcomesin recombinant and serum-derived ITIH4 if the enzymes are presentin active form
- 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
- 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