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. 2015 Feb 2;25(3):286-295.
doi: 10.1016/j.cub.2014.11.049. Epub 2014 Dec 24.

Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism

Deepika Vasudevan  1 , Hideyuki Takeuchi  1 , Sumreet Singh Johar  1 , Elaine Majerus  2 , Robert S Haltiwanger  3
Affiliations

Peters plus syndrome mutations disrupt a noncanonical ER quality-control mechanism

Deepika Vasudevan et al. Curr Biol. .

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 cotranslationally, as TSRs fold. Mass spectral analysis reveals that TSRs from mature, secreted protein are stoichiometrically modified with the disaccharide, whereas 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 noncanonical 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.

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Figures

Figure 1
Figure 1. B3GLCT is required for secretion of some but not all POFUT2 targets
A) Domain map of POFUT2 targets tested. B) siRNA specific for POFUT2, B3GLCT or non-specific control were co-transfected into HEK293T cells with plasmids encoding tagged POFUT2 targets (ADAMTS13, ADAMTSL1, ADAMTSL2 and TSP1(TSR1-3) or human IgG heavy chain. Media and cell lystates were evaluated by Western blot (red= anti-V5 or -myc; green= anti-IgG). C) Quantitation of data from panel A. Data are mean +/− standard deviation (n=3).
Figure 2
Figure 2. O-Fucosylation occurs in the ER and is at least partly co-translational
A) 6AF-labeled ADAMTS13-V5-His6 or ADAMTSL1-myc-His6 was immunoprecipitated from cell lysates or Ni-NTA enriched from media (sufficient ADAMTS13 could not be obtained from media fractions). Purified proteins were subjected to EndoH or PNGaseF digest and analyzed by western blots with indicated antibodies (red= anti-V5 or -myc; green=Streptavidin). B) Ribosomes were isolated from HEK293T cells transfected with either empty vector or plasmid encoding 3XFlag-ADAMTS13 and labeled with 6AF. Nascent polypeptides were detected with anti-Flag (red) and 6AF was detected with Streptavidin (green). * indicates background bands that appear in the empty vector controls.
Figure 3
Figure 3. Properly folded TSRs are marked with O-fucose disaccharide in cells
A) ADAMTS13 was purified from Media or Cell Lysate fractions of HEK293T cells transfected with plasmid encoding ADAMTS13. Purified ADAMTS13 was subjected to reduction/alkylation, trypsin digestion, and analysis by nano-LC-MS/MS as described in Methods. Extracted ion chromatograms (EICs) of the ions corresponding to unmodified (black), O-fucose mono- (red) and disaccharide (blue) glycoforms of the peptides containing the O-fucosylation site from TSR5, 7 and 8 derived from ADAMTS13 are shown. Details of the ions representing each glycoform used to prepare the EICs are listed in Table S1 and Figure S2A. B) HEK293T cells transfected with 3XFlag-ADAMTS13 were lysed in the presence of 100 mM iodoacetamide. Aliquots from the cell lysates were analyzed by Western blot under non-reducing (top panel) and reducing (bottom panel) conditions. Treating the samples with reducing agents (bottom panel) led to complete reduction of ADAMTS13, making the two populations indistinguishable. Mature form of ADAMTS13 isolated from medium is provided as a control for fully folded protein (migration position marked with *). C) EICs (prepared as in A) of peptides from unfolded aggregates (top of gel from B) and mostly folded (migrating with mature protein at * from B) cell-associated ADAMTS13 showing predominantly unmodified peptides from TSR5, 7 and 8 in the aggregate, but majority glycosylated peptide from TSR5 and 7 in the mostly folded fraction. D) Lec13 CHO cells were transfected with plasmids encoding the tagged POFUT2 targets shown or IgG as a negative control. Each target was immunoprecipitated from cell lysates and western blotted with anti-tag antibodies or with anti-BiP. The cells were rescued by addition of 1mM fucose exogenously. Quantitation represents relative amounts of BiP/Protein with -Fuc normalized to 1. Note that BiP was not detected in the IgG immunoprecipitate. Data are mean +/− standard deviation from 3 independent experiments.
Figure 4
Figure 4. Fucose and glucose stabilize folded TSRs in an additive manner
A) Equivalent amounts of unmodified, Fuc-TSR and GlcFuc-TSR (all TSP1-TSR3) were incubated with denaturants in an in vitro melting reaction. Aliquots were removed at the indicated times and analyzed by reverse phase HPLC. TSR levels were monitored by absorbance at 214 nm (U= unfolded isoform; F= folded isoform). Note that Fuc-TSR and GlcFuc-TSR were not completely unfolded within the 80 min time course, so were completely denatured by incubating for 80 min in 30 mM DTT. B) U and F were quantified by determining area-under-curve (AUC) for each population of TSR in the melting reaction in panel A. Values were normalized to U at t=80min for TSR and completely denatured Fuc-TSR and GlcFuc-TSR (30 mM DTT) set to 100%. Schematic on the right shows the unfolding reactions as equilibria, with weighted arrows representing the favored reaction (orange line=disulfide bond; red triangle= fucose; blue circle= glucose). Data are mean +/− standard deviation from 3 or more independent experiments.
Figure 5
Figure 5. O-Fucosylation by POFUT2 accelerates the net folding rate of TSRs
A) Bacterially expressed (unmodified) TSP1-TSR3 was denatured and refolded in vitro in the presence of redox agents (black), redox and POFUT2 (blue) or redox, POFUT2 and GDP-fucose (red). Aliquots were removed at the indicated times and analyzed using reverse-phase HPLC (U= unfolded TSR; F=folded TSR; I= folding intermediates). B) In-vitro refolding assay (as in A) in the presence of GDP-fucose using POFUT1 (black), a closely related O-fucosyltransferase that modifies properly folded Epidermal Growth Factor-like repeats [37], as a negative control, wt POFUT2 (red) and the catalytically inactive POFUT2-E54A (blue). C) Quantitation of unfolded and folded products by AUC in B shows the rate of disappearance of unfolded protein (top) and rate of formation of folded protein (bottom).
Figure 6
Figure 6. A model for POFUT2 in the folding of TSRs
See discussion.
See this image and copyright information in PMC

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