O- Fucosylation of ADAMTSL2 is required for secretion and is impacted by geleophysic dysplasia-causing mutations

Abstract

ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder, geleophysic dysplasia 1 (GPHYSD1), which is characterized by short stature, small hands and feet, and cardiac defects. ADAMTSL2 is a matricellular protein previously shown to interact with latent transforming growth factor-β binding protein 1 and influence assembly of fibrillin 1 microfibrils. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for O-fucosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose-modified TSRs are subsequently elongated to a glucose β1-3-fucose (GlcFuc) disaccharide by β1,3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS), which is characterized by skeletal defects similar to GPHYSD1. Several ADAMTSL2 TSRs also have consensus sequences for C-mannosylation. Six reported GPHYSD1 mutations occur within the TSRs and two lie near O-fucosylation sites. To investigate the effects of TSR glycosylation on ADAMTSL2 function, we used MS to identify glycan modifications at predicted consensus sequences on mouse ADAMTSL2. We found that most TSRs were modified with the GlcFuc disaccharide at high stoichiometry at O-fucosylation sites and variable mannose stoichiometry at C-mannosylation sites. Loss of ADAMTSL2 secretion in POFUT2^-/- but not in B3GLCT^-/- cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients. In contrast, secretion was significantly reduced for ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6), and S641L eliminated O-fucosylation of TSR3. These results provide evidence that abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.

Keywords: ADAMTSL2; C-mannosylation; O-fucosylation; TSR; cell biology; extracellular matrix; geleophysic dysplasia; glycoprotein secretion; glycosylation; mass spectrometry.

Figure 1.

ADAMTSL2 domain structure and predicted glycosylation sites on TSRs. Top, sequences surrounding the POFUT2 consensus sequence (C-X-X-S/T-C) from mouse ADAMTSL2 (mADAMTSL2) TSRs are shown. Red C indicates conserved cysteines, blue S or T indicates predicted O-fucosylated serine or threonine, underlined W indicates predicted C-mannosylated tryptophan, purple N indicates predicted N-glycosylated asparagine, and green W, S, and G identify residues corresponding to those mutated in GPHYSD. TSR6 is unusual in that the POFUT2 site overlaps with the N-glycan site. Middle, domain organization of mADAMTSL2 including signal peptide (sp), cysteine-rich domain (cys), spacer domain (spa), N-glycan rich domain (N-gly), PLAC domain, and seven TSRs (ovals). The TSRs containing consensus sequences for modification with O-fucose (red oval), C-mannose W-X-X-(W/C) (hatched), or both modifications (red and hatched) are indicated.

Figure 2.

ADAMTSL2 TSRs were modified with O-fucose-glucose disaccharide and C-mannose in varying stoichiometries. A, relative abundance of GlcFuc disaccharide, O-fuc monosaccharide, and unmodified glycoforms on peptides from TSRs containing the C-X-X-(S/T)-C O-fucose consensus site analyzed by mass spectral analyses. See Fig. S2 for spectra and EICs of each peptide. Data can be found in Tables S3, S4, S7, and S8. B, plasmids encoding TSP1-TSR3-MycHis₆, ADAMTSL2-TSR6-MycHis₆, or an empty vector (EV) were transfected into HEK293T cells grown in the presence of 6AF as described under “Experimental procedures.” The proteins were purified from the medium, digested with or without PNGase F, click reaction was performed with azide-biotin, separated by SDS-PAGE, and probed with anti-myc antibody (red) or streptavidin (green). C, relative abundance of C-mannosylated forms of peptides from TSRs containing W-X-X-(W/C) consensus sequence. See Fig. S2 for spectra and EICs of each peptide. Data can be found in Tables S3, S4, S7, and S8. D, domain map of mADAMTSL2 summarizing the distribution of O-fucosylation and C-mannosylation on TSRs. Red triangle represents fucose. Blue circle represents glucose. Green circle represent mannose. Hollow icons represent less than 50% modification of fucose (red hollow triangle), glucose (blue hollow circle), and mannose (green hollow circle).

Figure 3.

ADAMTSL2 secretion required O-fucosylation but not N-glycan fucosylation. Plasmids encoding ADAMTSL2-MycHis₆ and hIgG were co-transfected into (A) Lec13-CHO cells grown in the presence (+) or absence (−) of 1 mm l-fucose or (B) into Lec1-CHO cells and Pro5-CHO cells. The medium and cell lysates were analyzed by Western blots probed with anti-myc (red) and anti-IgG (green) antibodies.

Figure 4.

POFUT2 is essential for mADAMTSL2 secretion, but B3GLCT is not. A, plasmids encoding ADAMTSL2-MycHis₆ (TSL2) and hIgG were co-transfected into WT, POFUT2-null (POFUT2^−/−), or B3GLCT-null (B3GLCT^−/−) HEK293T cells. Rescue experiments were performed by co-transfection with a plasmid encoding full-length POFUT2 or full-length B3GLCT (enzyme). Medium and cell lysate were analyzed by Western blots probed with anti-myc (red) to detect ADAMTSL2 (TSL2) and for transfection control anti-hIgG (green). B, quantification of Western blots from A. ADAMTSL2 signals were normalized to hIgG levels. Significance was analyzed with ordinary one-way ANOVA in Prism 7, n = 11 in biological replicates with independent experiments. ****, p < 0.0001; **, p < 0.01; n.s., not significant.

Figure 5.

GPHYSD1 equivalent mutations S641L and G817R reduced secretion of mADAMTSL2. A, plasmids encoding WT, S641L-mutant, and G817R-mutant ADAMTSL2-MycHis₆ were co-transfected with hIgG into WT HEK293T cells. The medium and cell lysate were analyzed by Western blots probed with anti-myc (red) and anti-IgG (green) antibodies. B, quantitation of Western blots from A as described in the legend to Fig. 4.

Figure 6.

GPHYSD1 S641L mutation reduced O-fucosylation resulting in reduced mADAMTSL2 secretion. A, relative abundance of glycoforms of the peptide containing the O-fucose consensus sequence from TSR3 generated from WT ADAMTSL2 or S641L-mutant ADAMTSL2 TSL2-TSR3 (S641L) chymotrypsin digestion. See Figs. S2D and S3 for spectra and EICs of each peptide, respectively. Data can be found in Tables S5, S6, S9, and S10. Statistics were performed with one-way ANOVA in Prism 7, n = 3 with biological replicates. B, plasmids encoding WT or T633A-mutant ADAMTSL2 were co-transfected with hIgG into WT HEK293T cells. The medium and cell lysate were analyzed by Western blots probed with anti-myc (red) and anti-IgG (green) antibodies. C, quantitation of Western blots from B using ordinary one-way ANOVA in Prism 7, n = 3 in biological replicates. **, p < 0.01; n.s., not significant.

Figure 7.

GPHYSD1 G817R mutation did not affect N-glycosylation in TSR6 and mutation of N-glycosylation site did not restore O-fucosylation. Plasmids encoding TSP1-TSR3-MycHis₆, WT, N813Q, T815V, G817R, double mutant N813Q/T815V forms of ADAMTSL2-TSR6-MycHis₆, or an empty vector (EV) were transfected into HEK293T cells grown in the presence of 6AF as described under “Experimental procedures.” The proteins were purified from the medium and cell lysate, click reaction performed with azide-biotin, separated by SDS-PAGE, and probed with anti-myc antibody (red) or streptavidin (green).