O-Glycosylation Modulates Proprotein Convertase Activation of Angiopoietin-like Protein 3

Abstract

The angiopoietin-like protein 3 (ANGPTL3) is an important inhibitor of the endothelial and lipoprotein lipases and a promising drug target. ANGPTL3 undergoes proprotein convertase processing (RAPR 224 2TT) for activation, and the processing site contains two potential GalNAc O-glycosylation sites immediately C-terminal (TT 226 ). We developed an in vivo model system in CHO ldlD cells that was used to show that O-glycosylation in the processing site blocked processing of ANGPTL3. Genome-wide SNP association studies have identified the polypeptide GalNAc-transferase gene, GALNT2, as a candidate gene for low HDL and high triglyceride blood levels. We hypothesized that the GalNAc-T2 transferase performed critical O-glycosylation of proteins involved in lipid metabolism. Screening of a panel of proteins known to affect lipid metabolism for potential sites glycosylated by GalNAc-T2 led to identification of Thr 226 adjacent to the proprotein convertase processing site in ANGPTL3. We demonstrated that GalNAc-T2 glycosylation of Thr 226 in a peptide with the RAPR 224 2TT processing site blocks in vitro furin cleavage. The study demonstrates that ANGPTL3 activation is modulated by O-glycosylation and that this step is probably controlled by GalNAc-T2.Site-specific O-glycosylation is emerging as a novel mechanism that regulates the action of proprotein convertase (PC) 2 processing of proteins (1). PC processing is crucial in regulating many fundamental biological pathways (2). O-Glycans in or immediately adjacent to the recognition sites of PCs can interfere with proteolysis required for activation or inactivation of proteins normally undergoing processing. We recently presented the first example of O-glycosylation-mediated regulation of the PC processing of the phosphaturic factor FGF23 (1), which results in functional inactivation of FGF23 and direct inhibition of the FGFR receptor by the released C-terminal fragment (3). Deficiency in intact FGF23 leads to the rare syndromes familial tumoral calcinosis and hyperostosis-hyperphosphatemia associated with hyperphosphatemia (4). Deficiency in FGF23 can be caused by either mutations in the UDP-GalNAc:polypeptide ␣-N-acetylgalactosaminyltransferase T3 (GalNAc-T3) that O-glycosylates the PC recognition site (RHTR 179 ) of FGF23 or by mutations in FGF23 resulting in loss of secretion (5). Importantly, mutations in the PC recognition site of FGF23 that completely block cleavage lead to the mirror disease autosomal dominant hypophosphatemic Ricketts (6). Thus, O-glycosylation in the PC site is essential for preventing inactivation, whereas some degree of inactivation at the PC site is equally important. More recently, it was also found that furin processing of the brain natriuretic peptide precursor, pro-brain natriuretic peptide, was blocked by O-glycosylation (7,8). These examples suggest that site-specific O-glycosylation directed by GalNAc-transferase isoforms play a direct regulatory role in PC processing.ANGPTL3 (angiopoietin-like protein 3) expressed by the liver is an imp...

A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

“…FGF23 PC-processing site, are not glycosylated by the endogenous GalNAc-Ts expressed in both CHO-K1 and ldlD cells (11,12). Although we could not directly confirm glycosylation of the P1Ј-P4Ј constructs by mobility shifts, the finding that constructs P1Ј-P3Ј exhibited complete or partial blocking of processing is in accord with efficient glycosylation of the target sequences.…”

“…We used a modification of a previously described chimeric reporter construct that facilitates rapid selection of stable clones as well as screening of processing and analysis of the influence of O-glycosylation ( Fig. 2A) (12). We initially analyzed glycosylation and processing of the reporter constructs by transient expression, but found that both glycosylation and processing of secreted products were quite incomplete so we used stable clones for all studies reported (Fig.…”

“…More recently, we demonstrated that deficient O-glycosylation of the threonine residue in the RTHR 179 PC-processing site of FGF23 is the cause of the rare human disease, familial tumoral calcinosis (11). Moreover, O-glycosylation immediately C-terminal to the processing site in ANGPTL3 (RAPR 224 -TT) was found to regulate processing and was possibly related to regulation of serum lipids (12). Other studies have also indicated that O-glycans in close proximity to PC-processing sites may affect cleavage (13,14).…”

“…Samples were dissolved in methanol/ water (1:1) containing 1% formic acid and introduced by direct infusion via a TriVersa NanoMate ESI-Chip interface (Advion BioSystems) at a flow rate of 100 nl/min and 1.4 kV spray voltage. Mass spectra were acquired in positive ion FT mode using parameters similar to previous studies (12), except at a nominal resolving power of either 30,000 or 60,000. Electron transfer dissociation-MS 2 spectra were analyzed by comparison with theoretical c and z ⅐ fragment m/z values calculated for all positional combinations of one HexNAc residue distributed on the all potential S and T glycosylation sites in the sequence.…”

Proteolytic Processing of Angiopoietin-like Protein 4 by Proprotein Convertases Modulates Its Inhibitory Effects on Lipoprotein Lipase Activity

“…This effect also seems LPL-specific (26). ANGPTL3 cleavage is reported to be affected by the O-glycosylation state of threonine at amino acid position 226 by polypeptide N-acetylgalactosaminyltransferase 2, adjacent to the proprotein convertase-processing site (27). However, this O-glycosylation site is not conserved among ANGPTL4 proteins.…”