Peters Plus Syndrome Mutations Disrupt a Noncanonical ER Quality-Control Mechanism

Abstract

Background O-Fucose is added to cysteine-rich domains called Thrombospondin type 1 repeats (TSRs) by Protein O-fucosyltransferase 2 (POFUT2) and is elongated with glucose by β3-glucosyltransferase (B3GLCT). Mutations in B3GLCT result in Peters Plus Syndrome (PPS), an autosomal recessive disorder characterized by eye and other developmental defects. Although 49 putative targets are known, the function of the disaccharide and its role in PPS remain unexplored. Results Here we show that while POFUT2 is required for secretion of all targets tested, B3GLCT only affects the secretion of a subset, consistent with the observation that B3GLCT mutant phenotypes in PPS patients are less severe than embryonic lethal phenotypes of Pofut2-null mice. O-Glycosylation occurs co-translationally, as TSRs fold. Mass spectral analysis reveals that TSRs from mature, secreted protein are stoichiometrically modified with the disaccharide, while TSRs from protein still folding in the ER are partially modified, suggesting that O-glycosylation marks folded TSRs and promotes ER exit. In vitro unfolding assays demonstrate that fucose and glucose stabilize folded TSRs in an additive manner. In vitro refolding assays under redox conditions showed that POFUT2 recognizes, glycosylates, and stabilizes the folded form of TSRs, resulting in a net acceleration of folding. Conclusions While known ER quality control machinery rely on identifying and tagging unfolded proteins, we find that POFUT2 and B3GLCT mediate a non-canonical ER quality control mechanism that recognizes folded TSRs and stabilizes them by glycosylation. Our findings provide a molecular basis for the defects observed in PPS and potential targets that contribute to the pathology.

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking

“…HPLC-based unfolding assays were performed essentially as described (3). EGF proteins (5-10 M) were denatured in 100 mM Tris-HCl, pH 7.4, containing 0.6 M GdnHCl, 2 mM dithiothreitol (DTT), and 10 mM EDTA at room temperature for various times.…”

“…Folded proteins move on, and unfolded proteins are recognized and re-glucosylated by the UDP-glucose glycoprotein glucosyltransferase (UGGT) to allow another folding cycle. In addition to this well-known pathway, we recently identified a non-canonical quality control pathway for folding of thrombospondin type 1 repeats (TSRs) (3). TSRs are small protein motifs (ϳ60 amino acids) typically found as tandem repeats in proteins (4).…”

“…Most TSRs contain a consensus sequence for modification by protein O-fucosyltransferase 2 (POFUT2), a soluble enzyme in the ER lumen (5,6). POFUT2 only modifies folded TSRs (6), and knockdown of POFUT2 in cells results in loss of secretion of multiple target proteins (3). The addition of O-fucose appears to stabilize the folded form of a TSR, accelerating its folding and secretion (3).…”

“…POFUT2 only modifies folded TSRs (6), and knockdown of POFUT2 in cells results in loss of secretion of multiple target proteins (3). The addition of O-fucose appears to stabilize the folded form of a TSR, accelerating its folding and secretion (3). Thus, in contrast to the classical N-glycan quality control pathway where UGGT recognizes an unfolded substrate, POFUT2 recognizes and modifies a folded structure.…”

Distinct C- mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3

“…Both modifications have been shown to be required for secretion of at least some proteins containing TSRs (12)(13)(14)(15)(16)(17)(18). Initially, C-mannosylation was described as an endoplasmic reticulum (ER) localized, enzyme-catalyzed process that modifies the first tryptophan in the consensus motif WxxW (19)(20)(21).…”