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

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O-glycan sites. Moreover, we found that O-glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O-glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O-glycosylation of LDLR-related proteins and identified conserved O-glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease.

The glucagon-like peptide 2 receptor is expressed in enteric neurons and not in the epithelium of the intestine.

Glucagon-like peptide 2 (GLP-2) is a potent intestinotrophic growth factor with therapeutic potential in the treatment of intestinal deficiencies. It has recently been approved for the treatment of short bowel syndrome. The effects of GLP-2 are mediated by specific binding of the hormone to the GLP-2 receptor (GLP-2R) which was cloned in 1999. However, consensus about the exact receptor localization in the intestine has never been established. By physical, chemical and enzymatic tissue fragmentation, we were able to divide rat jejunum into different compartments consisting of: (1) epithelium alone, (2) mucosa with lamina propria and epithelium, (3) the external muscle coat including myenteric plexus, (4) a compartment enriched for the myenteric plexus and (5) intestine without epithelium. Expression of Glp2r; chromogranin A; tubulin, beta 3; actin, gamma 2, smooth muscle, enteric and glial fibrillary acidic protein in these isolated tissue fractions was quantified with qRT-PCR. Expression of the Glp2r was confined to compartments containing enteric neurons and receptor expression was absent in the epithelium. Our findings provide evidence for the expression of the GLP-2R in intestinal compartments rich in enteric neurons and, importantly they exclude significant expression in the epithelium of rat jejunal mucosa.

Precision mapping of the human O-GalNAc glycoproteome through SimpleCell technology.

Glycosylation is the most abundant and diverse posttranslational modification of proteins. While several types of glycosylation can be predicted by the protein sequence context, and substantial knowledge of these glycoproteomes is available, our knowledge of the GalNAc-type O-glycosylation is highly limited. This type of glycosylation is unique in being regulated by 20 polypeptide GalNAc-transferases attaching the initiating GalNAc monosaccharides to Ser and Thr (and likely some Tyr) residues. We have developed a genetic engineering approach using human cell lines to simplify O-glycosylation (SimpleCells) that enables proteome-wide discovery of O-glycan sites using 'bottom-up' ETD-based mass spectrometric analysis. We implemented this on 12 human cell lines from different organs, and present a first map of the human O-glycoproteome with almost 3000 glycosites in over 600 O-glycoproteins as well as an improved NetOGlyc4.0 model for prediction of O-glycosylation. The finding of unique subsets of O-glycoproteins in each cell line provides evidence that the O-glycoproteome is differentially regulated and dynamic. The greatly expanded view of the O-glycoproteome should facilitate the exploration of how site-specific O-glycosylation regulates protein function.

Probing isoform-specific functions of polypeptide GalNAc-transferases using zinc finger nuclease glycoengineered SimpleCells.

Our knowledge of the O-glycoproteome [N-acetylgalactosamine (GalNAc) type] is highly limited. The O-glycoproteome is differentially regulated in cells by dynamic expression of a subset of 20 polypeptide GalNAc-transferases (GalNAc-Ts), and methods to identify important functions of individual GalNAc-Ts are largely unavailable. We recently introduced SimpleCells, i.e., human cell lines made deficient in O-glycan extension by zinc finger nuclease targeting of a key gene in O-glycan elongation (Cosmc), which allows for proteome-wide discovery of O-glycoproteins. Here we have extended the SimpleCell concept to include proteome-wide discovery of unique functions of individual GalNAc-Ts. We used the GalNAc-T2 isoform implicated in dyslipidemia and the human HepG2 liver cell line to demonstrate unique functions of this isoform. We confirm that GalNAc-T2-directed site-specific O-glycosylation inhibits proprotein activation of the lipase inhibitor ANGPTL3 in HepG2 cells and further identify eight O-glycoproteins exclusively glycosylated by T2 of which one, ApoC-III, is implicated in dyslipidemia. Our study supports an essential role for GalNAc-T2 in lipid metabolism, provides serum biomarkers for GalNAc-T2 enzyme function, and validates the use of GALNT gene targeting with SimpleCells for broad discovery of disease-causing deficiencies in O-glycosylation. The presented glycoengineering strategy opens the way for proteome-wide discovery of functions of GalNAc-T isoforms and their role in congenital diseases and disorders.

Vasopressin induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter in the distal convoluted tubule.

The thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) is important for renal electrolyte balance and its phosphorylation causes an increase in its transport activity and cellular localization. Here, we generated phospho-specific antibodies against two conserved N-terminal phosphorylation sites (Thr53, Thr58 and Thr53/Thr58) to assess the role of arginine vasopressin (AVP) in regulating NCC in rodent kidney in vivo. Immunohistochemistry showed distinct staining of phosphorylated NCC (pNCC) at the apical plasma membrane domain of distal convoluted tubule (DCT) cells. Unlike total NCC, pNCC was localized only to the apical plasma membrane as determined by immunogold electron microscopy. In AVP-deficient Brattleboro rats, acute deamino-Cys-1, d-Arg-8 vasopressin (dDAVP) exposure significantly increased pNCC abundance at the apical plasma membrane by about threefold, whereas total NCC and its cellular distribution were not affected. dDAVP significantly increased the abundance of phosphorylated STE20/SPS1-related proline-alanine-rich kinase and oxidative stress-response kinase (SPAK and OSR1), kinases implicated in NCC phosphorylation. Intracellular calcium levels in early and late DCTs were increased in response to 1 min superfusion of dDAVP, confirming that these segments are AVP responsive. In rats fed a high-salt diet with angiotensin (ANG) type 1-receptor blockade, similar increases in pNCC and active SPAK and OSR1 were detected following chronic or acute dDAVP, thus indicating the effects of AVP are independent of ANGII. Our results show that AVP is a potent regulator of NCC activity.

Porcine glucagon-like peptide-2: structure, signaling, metabolism and effects.

Mass spectrometry of HPLC-purified porcine glucagon-like peptide-2 (pGLP-2)(1) revealed a 35 amino acid sequence with C-terminal Ser and Leu, in contrast to the 33 amino acids of human, cow, rat and mouse GLP-2. Synthetic pGLP-2 stimulated cAMP-production in COS-7 cells expressing human GLP-2 (hGLP-2) receptor with the same potency and efficacy as hGLP-2. In anesthetized pigs (n=9) given intravenous pGLP-2 infusions, the half life (t1/2) of intact pGLP-2 (8.4+/-0.9 min) was shorter (p<0.01) than that of the primary metabolite pGLP-2 (3-35) (34.0+/-5.2 min), generated by dipeptidyl peptidase-4 (DPP-4) cleavage. Adding the DPP-4 inhibitor valine-pyrrolidide prolonged t1/2 of intact pGLP-2 (p<0.05). The metabolic clearance rate (MCR) of intact pGLP-2 (23.9+/-3.82 mL/(kg x min)) was greater (p<0.0001) than that of pGLP-2 (3-35) (6.36+/-1.45 mL/(kg x min)) and larger than the previously reported MCR of hGLP-2 in pig. The MCR of intact pGLP-2 was reduced by valine-pyrrolidide (p<0.05), but was still greater than that of intact hGLP-2 previously reported. In the isolated perfused porcine pancreas, pGLP-2 stimulated glucagon release (p<0.05), but had no effect on insulin or somatostatin release. Exocrine secretion was unaffected and there was no apparent vasoactive effect. In mice (n=8), both subcutaneous hGLP-2 and pGLP-2 given twice daily for 10 days, significantly and equally increased small intestinal weight, length and cross-sectional area of proximal ileum. In conclusion, pGLP-2 and hGLP-2 have similar effects in vivo and in vitro in spite of the structural differences. However, pGLP-2 is cleared more rapidly in pigs than hGLP-2.