Loss of Function of GALNT2 Lowers High-Density Lipoproteins in Humans, Nonhuman Primates, and Rodents

Abstract

Summary Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a novel regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, but the mechanisms relating GALNT2 to HDL-C remain unclear. We investigated the impact of homozygous GALNT2 deficiency on HDL-C in humans and mammalian models. We identified two humans homozygous for loss-of-function mutations in GALNT2 who demonstrated low HDL-C. We also found that GALNT2 loss-of-function in mice, rats, and nonhuman primates decreased HDL-C. O-glycoproteomics studies of a human GALNT2 deficient subject validated ANGPTL3 and ApoC-III as GalNAc-T2 targets. Additional glycoproteomics in rodents identified targets influencing HDL-C, including phospholipid transfer protein (PLTP). GALNT2 deficiency reduced plasma PLTP activity in humans and rodents, and in mice this was rescued by reconstitution of hepatic Galnt2. We also found that GALNT2 GWAS SNPs associated with reduced HDL-C also correlate with lower hepatic GALNT2 expression. These results posit GALNT2 as a direct modulator of HDL metabolism across mammals.

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

“…Genetic deficiency in GALNT11 has not been identified so far in man and it is predicted to be extremely rare if occurring (34), but GALNT11 has been identified as a GWAS candidate gene for chronic kidney decline (46). The GWAS signal for chronic kidney decline resides in intron one of the GALNT11 gene, suggesting that the genetic predisposition is a result of altered gene regulation, similar to what has been established for the role of GALNT2 in dyslipidemia (19,47). Our study did not directly address the functional role of Oglycans on other LDLR-related proteins including LRP2, however, GalNAc-T11 is highly expressed in proximal tubules of the kidney and LRP2 is the major endocytic receptor responsible for reabsorbtion of proteins from the glomerular filtrate (21, 48).…”

“…We previously did confirm some of the LDLR, VLDLR and LRP1 sites in WT CHO cells (18), as well as in rat liver and human plasma (19) (Fig. 1, labeled 2,3 and Suppl.…”

“…Many of the other 20 GALNT genes have been identified as candidate genes for dispositions to common diseases (25,34), and we were recently able to provide extensive validation for the role of GALNT2 in dyslipidemia in man, primates and rodents identified several sitespecifically regulated O-glycoprotein targets (19). The substrate specificities and functions of GalNAc-Ts have mainly been studied by in vitro enzyme assays using short peptides as acceptors in the past (35), and this approach has provided important insights into specific functions of many individual isoenzymes including GalNAc-T11.…”

“…Digests were desialylated with 1 U neuraminidase (N3001, Sigma) in 50 mM sodium citrate (pH 5.0) at 37C ON followed by SepPak purification and lyophilization. Samples were dissolved in lectin-binding buffer (175 mM Tris, pH7.4) and subjected to Jacalin-agarose (AL1153, Vector Laboratories) LWAC performed as described previously with PNA (19), with minor modifications. The glycopeptides were eluted with 3x1 column volume 0.7 M galactose in lectin buffer (175 mM Tris, pH7.4).…”

Site-specific O-Glycosylation by Polypeptide N-Acetylgalactosaminyltransferase 2 (GalNAc-transferase T2) Co-regulates β1-Adrenergic Receptor N-terminal Cleavage

“…Of the four ADAMS analyzed, we found that ADAM17 cleaved peptide II at 52 P2L 53 and that this was inhibited by GalNAc-T2 glycosylation ( Fig. 2A).…”