PMID: 24884609

 

    Legend: Gene, Sites

Title : Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4

Abstract :
  1. Inter-alpha-trypsin inhibitor heavy chain H4 ( ITIH4 ) is a 120 kDa acute-phase glycoprotein produced primarily in the liver, secreted into the blood, and identified in serum
  2. ITIH4 is involved in liver development and stabilization of the extracellular matrix ( ECM ), and its expression is altered in liver disease
  3. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry
  4. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4
  5. First, we confirmed that the four ITIH4 N-X-S/T sequons ( N81, N207, N517, and N577 ) were glycosylated by treating ITIH4 tryptic/ GluC glycopeptides with PNGaseF in the presence of (18)O water
  6. Next, we performed glycosidase-assisted LC-MS/MS analysis of ITIH4 trypsin- GluC glycopeptides enriched via hydrophilic interaction liquid chromatography to characterize ITIH4 N-glycoforms
  7. While microheterogeneity of N-glycoforms differed between ITIH4 protein expressed in HEK293 cells and protein isolated from serum, occupancy of N-glycosylation sites did not differ
  8. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif
  9. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4
  10. We also identified isoform-specific ITIH4 O-glycoforms and documented that utilization of O-glycosylation sites on ITIH4 differed between the cell line and serum
Output (sent_index, trigger, protein, sugar, site):
  • 1. glycoprotein, , Inter-alpha-trypsin inhibitor heavy chain H4, -, -
  • 1. glycoprotein, , glycoprotein, -, -
  • 10. O-glycoforms, , ITIH4, -, -
  • 10. O-glycosylation, , -, -, sites
  • 3. glycosylation, , ITIH4, -, -
  • 4. glycopeptide, , -, -, glycopeptide
  • 5. glycopeptides, , -, -, glycopeptides
  • 5. glycosylated, , -, -, N81, N207, N517, and N577
  • 6. N-glycoforms, , ITIH4, -, -
  • 6. glycopeptides, , -, -, glycopeptides
  • 7. N-glycosylation, , -, -, sites
  • 7. occupancy, , -, -, sites
  • 8. N-glycosylation, , -, -, site
  • 9. contained, , ITIH4, high-mannose N-linked glycans, N274
Output(Part-Of) (sent_index, protein, site):
  • 5. GluC, glycopeptides
  • 5. ITIH4, N81, N207, N517, and N577
  • 5. ITIH4, glycopeptides
  • 6. GluC, glycopeptides
  • 6. ITIH4, glycopeptides
  • 9. ITIH4, N274
*Output_Site_Fusion* (sent_index, protein, sugar, site):
  • 5. ITIH4, -, N81, N207, N517, and N577
  • 9. ITIH4, high-mannose N-linked glycans, N274

 

 

Protein NCBI ID SENTENCE INDEX
ITIH4 protein 3700 7
Recombinant ITIH4 3700 4
ITIH4 3700 1,2,3,4,5,6,9,10
GluC 57733 5,6
Inter-alpha-trypsin inhibitor heavy chain H4 3700 1