RESUMEN
Human genetics studies have implicated GALNT2, encoding GalNAc-T2, as a 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.
Asunto(s)
Lipoproteínas HDL/metabolismo , N-Acetilgalactosaminiltransferasas/deficiencia , Secuencia de Aminoácidos , Proteína 3 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/metabolismo , Animales , Secuencia de Bases , HDL-Colesterol/sangre , Técnicas de Silenciamiento del Gen , Glicoproteínas/metabolismo , Homocigoto , Humanos , Hígado/enzimología , Ratones , Ratones Noqueados , Modelos Animales , Mutación/genética , N-Acetilgalactosaminiltransferasas/química , N-Acetilgalactosaminiltransferasas/genética , N-Acetilgalactosaminiltransferasas/metabolismo , Fenotipo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Polimorfismo de Nucleótido Simple/genética , Primates , Proteómica , Ratas , Triglicéridos/metabolismo , Polipéptido N-AcetilgalactosaminiltransferasaRESUMEN
Alternative splicing of the human beta-aspartyl (asparaginyl) hydroxylase (BAH) gene results in the expression of humbug, a truncated form of BAH that lacks the catalytic domain of the enzyme. Overexpression of BAH and humbug has been associated with a variety of human cancers, and although humbug lacks enzymatic activity, it is expressed at levels comparable with that of BAH in various cancer cell lines. Phosphorothioate antisense oligonucleotides (ONs) were designed to dissect out the function of these hydroxylase protein isoforms. In A549 cells, these ONs differentially down-regulated BAH and humbug at the mRNA and protein level. Phosphorothioate ON uptake and antisense studies were conducted in parallel in nude mice bearing A549 tumor xenografts. Microscopic examination of the tumor after administration of a fluorescein-labeled ON showed strong labeling of the outer layers of the tumor connective tissue but cells within the interior of the tumor were sparsely labeled. A modest but significant effect on tumor growth was observed in animals treated with an antisense ON directed against both BAH and humbug transcripts. However, Northern analysis of tumor RNA did not indicate a down-regulation of the targeted mRNA species. These results demonstrate the successful development of antisense ONs that selectively differentiate between the closely related beta-hydroxylase protein isoforms. However, determination of the biological function of these proteins in vivo was limited by the poor uptake properties of phosphorothioate ONs in A549 tumors.