Porcine ITIH4 (Inter Alpha-Globulin Inhibitor H4) ELISA Kit

Factors predisposing to the phenotypic features of higher total cholesterol (T-Cho) have not been clearly defined. Here we report an association between a C/T single nucleotide polymorphism at IVS17+8 in the inter-alpha-trypsin inhibitor heavy chain 4 gene (ITIH4) and plasma total cholesterol levels in 351 adult individuals from an east-central area of Japan. Age and gender-adjusted levels of plasma T-Cho, LDL-cholesterol, triglyceride, and HDL-cholesterol were analyzed. When we separate the subjects into two genotypic groups regarding this single nucleotide polymorphism (SNP), those who lack the T-allele had significantly higher plasma T-Cho levels than the others who bear T-allele (mean 252.3 mg/dl versus 241.7 mg/dl; p=0.009). Of the 309 individuals without the T-allele, approximately 90% presented with hypercholesterolemia, whereas only 10% were hypercholesterolemic among 42 individuals with the T-allele (p <0.0001). These data suggest that genetic variation at ITIH4 locus is one of the likely candidate determinants for plasma cholesterol metabolisms.

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 O-18 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.