Apolipoprotein-CIII O-Glycosylation, a Link between GALNT2 and Plasma Lipids

Apolipoprotein-CIII (apo-CIII) is involved in triglyceride-rich lipoprotein metabolism and linked to beta-cell damage, insulin resistance, and cardiovascular disease. Apo-CIII exists in four main proteoforms: non-glycosylated (apo-CIII0a), and glycosylated apo-CIII with zero, one, or two sialic acids (apo-CIII0c, apo-CIII1 and apo-CIII2). Our objective is to determine how apo-CIII glycosylation affects lipid traits and type 2 diabetes prevalence, and to investigate the genetic basis of these relations with a genome-wide association study (GWAS) on apo-CIII glycosylation. We conducted GWAS on the four apo-CIII proteoforms in the DiaGene study in people with and without type 2 diabetes (n = 2318). We investigated the relations of the identified genetic loci and apo-CIII glycosylation with lipids and type 2 diabetes. The associations of the genetic variants with lipids were replicated in the Diabetes Care System (n = 5409). Rs4846913-A, in the GALNT2-gene, was associated with decreased apo-CIII0a. This variant was associated with increased high-density lipoprotein cholesterol and decreased triglycerides, while high apo-CIII0a was associated with raised high-density lipoprotein-cholesterol and triglycerides. Rs67086575-G, located in the IFT172-gene, was associated with decreased apo-CIII2 and with hypertriglyceridemia. In line, apo-CIII2 was associated with low triglycerides. On a genome-wide scale, we confirmed that the GALNT2-gene plays a major role i O-glycosylation of apolipoprotein-CIII, with subsequent associations with lipid parameters. We newly identified the IFT172/NRBP1 region, in the literature previously associated with hypertriglyceridemia, as involved in apolipoprotein-CIII sialylation and hypertriglyceridemia. These results link genomics, glycosylation, and lipid metabolism, and represent a key step towards unravelling the importance of O-glycosylation in health and disease.</p

Large-Scale Analysis of Apolipoprotein CIII Glycosylation by Ultrahigh Resolution Mass Spectrometry

Apolipoprotein-CIII (apo-CIII) is a glycoprotein involved in lipid metabolism and its levels are associated with cardiovascular disease risk. Apo-CIII sialylation is associated with improved plasma triglyceride levels and its glycosylation may have an effect on the clearance of triglyceride-rich lipoproteins by directing these particles to different metabolic pathways. Large-scale sample cohort studies are required to fully elucidate the role of apo-CIII glycosylation in lipid metabolism and associated cardiovascular disease. In this study, we revisited a high-throughput workflow for the analysis of intact apo-CIII by ultrahigh-resolution MALDI FT-ICR MS. The workflow includes a chemical oxidation step to reduce methionine oxidation heterogeneity and spectrum complexity. Sinapinic acid matrix was used to minimize the loss of sialic acids upon MALDI. MassyTools software was used to standardize and automate MS data processing and quality control. This method was applied on 771 plasma samples from individuals without diabetes allowing for an evaluation of the expression levels of apo-CIII glycoforms against a panel of lipid biomarkers demonstrating the validity of the method. Our study supports the hypothesis that triglyceride clearance may be regulated, or at least strongly influenced by apo-CIII sialylation. Interestingly, the association of apo-CIII glycoforms with triglyceride levels was found to be largely independent of body mass index. Due to its precision and throughput, the new workflow will allow studying the role of apo-CIII in the regulation of lipid metabolism in various disease settings