Golgi N-glycan branching N-acetylglucosaminyltransferases I, V and VI promote nutrient uptake and metabolism.

Nutrient transporters are critical gate-keepers of extracellular metabolite entry into the cell. As integral membrane proteins, most transporters are N-glycosylated, and the N-glycans are remodeled in the Golgi apparatus. The Golgi branching enzymes N-acetylglucosaminyltransferases I, II, IV, V and avian VI (encoded by Mgat1, Mgat2, Mgat4a/b/c Mgat5 and Mgat6), each catalyze the addition of N-acetylglucosamine (GlcNAc) in N-glycans. Here, we asked whether N-glycan branching promotes nutrient transport and metabolism in immortal human HeLa carcinoma and non-malignant HEK293 embryonic kidney cells. Mgat6 is absent in mammals, but ectopic expression can be expected to add an additional ß1,4-linked branch to N-glycans, and may provide evidence for functional redundancy of the N-glycan branches. Tetracycline (tet)-induced overexpression of Mgat1, Mgat5 and Mgat6 resulted in increased enzyme activity and increased N-glycan branching concordant with the known specificities of these enzymes. Tet-induced Mgat1, Mgat5 and Mgat6 combined with stimulation of hexosamine biosynthesis pathway (HBP) to UDP-GlcNAc, increased cellular metabolite levels, lactate and oxidative metabolism in an additive manner. We then tested the hypothesis that N-glycan branching alone might promote nutrient uptake when glucose (Glc) and glutamine are limiting. In low glutamine and Glc medium, tet-induced Mgat5 alone increased amino acids uptake, intracellular levels of glycolytic and TCA intermediates, as well as HEK293 cell growth. More specifically, tet-induced Mgat5 and HBP elevated the import rate of glutamine, although transport of other metabolites may be regulated in parallel. Our results suggest that N-glycan branching cooperates with HBP to regulate metabolite import in a cell autonomous manner, and can enhance cell growth in low-nutrient environments.

N-glycan remodeling on glucagon receptor is an effector of nutrient sensing by the hexosamine biosynthesis pathway.

Glucose homeostasis in mammals is dependent on the opposing actions of insulin and glucagon. The Golgi N-acetylglucosaminyltransferases encoded by Mgat1, Mgat2, Mgat4a/b/c, and Mgat5 modify the N-glycans on receptors and solute transporter, possibly adapting activities in response to the metabolic environment. Herein we report that Mgat5(-/-) mice display diminished glycemic response to exogenous glucagon, together with increased insulin sensitivity. Glucagon receptor signaling and gluconeogenesis in Mgat5(-/-) cultured hepatocytes was impaired. In HEK293 cells, signaling by ectopically expressed glucagon receptor was increased by Mgat5 expression and GlcNAc supplementation to UDP-GlcNAc, the donor substrate shared by Mgat branching enzymes. The mobility of glucagon receptor in primary hepatocytes was reduced by galectin-9 binding, and the strength of the interaction was dependent on Mgat5 and UDP-GlcNAc levels. Finally, oral GlcNAc supplementation rescued the glucagon response in Mgat5(-/-) hepatocytes and mice, as well as glycolytic metabolites and UDP-GlcNAc levels in liver. Our results reveal that the hexosamine biosynthesis pathway and GlcNAc salvage contribute to glucose homeostasis through N-glycan branching on glucagon receptor.

"Add-on" domains of Drosophila ß1,4-N-acetylgalactosaminyltransferase B in the stem region and its pilot protein.

The glycolipid specific Drosophila melanogaster ß1,4-N-acetylgalactosaminyltransferase B (ß4GalNAcTB) depends on a zinc finger DHHC protein family member named GalNAcTB pilot (GABPI) for activity and translocation to the Golgi. The six-membrane spanning protein actually lacks the cysteine in the cytoplasmic DHHC motif, displaying DHHS instead. Here we show that the whole conserved region around the DHHS sequence, which is essential for palmitoylation in DHHC proteins, is not required for GABPI to interact with ß4GalNAcTB. In contrast, the two luminal loops between transmembrane domain 3-4 and 5-6 contain conserved amino acids, which are crucial for activity. Besides the dependence on GABPI, ß4GalNAcTB requires its exceptional short stem region for activity. A few hydrophobic amino acids positioned close to the transmembrane domain are essential for the interaction with GABPI. Along with its catalytic domain, ß4GalNAcTB, thus, requires an area in its own stem region and two small luminal loops of GABPI as "add-on" domains. Moreover, some inactive GABPI mutants could be rescued by fusion with ß4GalNAcTB, indicating their importance in direct GABPI-ß4GalNAcTB interaction.

Golgi targeting of Drosophila melanogaster beta4GalNAcTB requires a DHHC protein family-related protein as a pilot.

Drosophila melanogaster beta4GalNAcTB mutant flies revealed that this particular N-acetylgalactosaminyltransferase is predominant in the formation of lacdiNAc (GalNAcbeta1,4GlcNAc)-modified glycolipids, but enzymatic activity could not be confirmed for the cloned enzyme. Using a heterologous expression cloning approach, we isolated beta4GalNAcTB together with beta4GalNAcTB pilot (GABPI), a multimembrane-spanning protein related to Asp-His-His-Cys (DHHC) proteins but lacking the DHHC consensus sequence. In the absence of GABPI, inactive beta4GalNAcTB is trapped in the endoplasmic reticulum (ER). Coexpression of beta4GalNAcTB and GABPI generates the active enzyme that is localized together with GABPI in the Golgi. GABPI associates with beta4GalNAcTB and, when expressed with an ER retention signal, holds active beta4GalNAcTB in the ER. Importantly, treatment of isolated membrane vesicles with Triton X-100 disturbs beta4GalNAcTB activity. This phenomenon occurs with multimembrane-spanning glycosyltransferases but is normally not a property of glycosyltransferases with one membrane anchor. In summary, our data provide evidence that GABPI is required for ER export and activity of beta4GalNAcTB.