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

Ao Zhang,Steven J Berardinelli,Christina Leonhard-Melief,Deepika Vasudevan,Ta-Wei Liu,Andrew Taibi,Sharee Giannone,Suneel S Apte,Bernadette C Holdener,Robert S Haltiwanger

doi:10.1074/jbc.ra120.014557

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.

Highlights

ADAMTSL2 is a member of the A Disintegrin-like And Metalloprotease with Thrombospondin type I repeats (ADAMTS)
ADAMTSL2 mutations were identified in patients with autosomal recessive geleophysic dysplasia (GPHYSD1, OMIM number 231050), a syndrome characterized by short stature, short tubular bones, thick skin, and early death caused by cardiopulmonary abnormalities [6]
We examined the effects of two geleophysic dysplasia 1 (GPHYSD1) mutations in ADAMTSL2 occurring within protein O-fucosyltransferase 2 (POFUT2) consensus sites, one in TSR3 (S641L) and the other in TSR6 (G817R)

Summary

Edited by Peter Cresswell

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 contains 10 N-glycosylation sites, one of which falls within TSR6 and overlaps with a predicted protein O-fucosyltransferase 2 (POFUT2) consensus sequence (Fig. 1). Several GPHYSD1 mutations reduce secretion of ADAMTSL2, including a GPHYSD1 mutation adjacent to the O-fucosylation site in TSR6 (G811R) [6] Another GPHYSD1 mutation, S635L, falls within the POFUT2 consensus sequence in TSR3 from ADAMTSL2 [8]. These results raised the possibility that altered O-fucosylation is responsible for reduced secretion of ADAMTSL2 in GHPYSD1 S635L and G811R patients. We showed that the reduction in secretion of the S641L mutation results from reduced O-fucosylation of TSR3, providing a molecular explanation for GPHYSD1 in patients with this mutation

Results

Discussion

Protein expression and purification

Statistical methodsd

Western blotting

Mass spectral analysis