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