title: Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4.
abstract: 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. ITIH4 is involved in liver development and stabilization of the extracellular matrix (ECM), and its expression is altered in liver disease. In this study, we aimed to characterize glycosylation of recombinant and serum-derived ITIH4 using analytical mass spectrometry. Recombinant ITIH4 was analyzed to optimize glycopeptide analyses, followed by serum-derived ITIH4. 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. 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. 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. A fifth N-glycosylation site was discovered at N274 with the rare nonconsensus NVV motif. Site N274 contained high-mannose N-linked glycans in both serum and recombinant ITIH4. 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.
authors.affiliation: Department of Biochemistry and Molecular & Cellular Biology, Georgetown University , Washington, D.C. 20057, United States.
authors.forename: Kevin Brown
authors.initials: KB
authors.lastname: Chandler
authors.forename: Zuzana
authors.initials: Z
authors.lastname: Brnakova
authors.forename: Miloslav
authors.initials: M
authors.lastname: Sanda
authors.forename: Shuo
authors.initials: S
authors.lastname: Wang
authors.forename: Stephanie H
authors.initials: SH
authors.lastname: Stalnaker
authors.forename: Robert
authors.initials: R
authors.lastname: Bridger
authors.forename: Peng
authors.initials: P
authors.lastname: Zhao
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.forename: Nathan J
authors.initials: NJ
authors.lastname: Edwards
authors.forename: Radoslav
authors.initials: R
authors.lastname: Goldman
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D053491:Proteinase Inhibitory Proteins, Secretory / Q000097:blood* / Q000737:chemistry
chemical_list: D001798:Blood Proteins; D006023:Glycoproteins; C091236:ITIH4 protein, human; D020033:Protein Isoforms; D053491:Proteinase Inhibitory Proteins, Secretory