O-Fucosylation of ADAMTSL2 Thrombospondin Type 1 Repeats is Required for Secretion and is Impacted by Geleophysic Dysplasia-Causing Mutations

ABSTRACT: A disintegrin-like and metalloprotease domain with thrombospondin type 1 repeats-like 2 (ADAMTSL2) is a matricellular protein that interacts with latent TGF-b binding protein 1 and fibrillin-1. ADAMTSL2 mutations cause an autosomal recessive connective tissue disorder named geleophysic dysplasia 1 (GPHYSD1, OMIM #231050), which is characterized by short stature, small hands and feet, and cardiac defects. ADAMTSL2 contains seven thrombospondin type-1 repeats (TSRs), six of which contain the consensus sequence for serine or threonine glycosylation by protein O-fucosyltransferase 2 (POFUT2). O-fucose modified TSRs are subsequently elongated to a Glucose1-3Fucose (GlcFuc) disaccharide by b3-glucosyltransferase (B3GLCT). B3GLCT mutations cause Peters Plus Syndrome (PTRPLS, OMIM #261540), which is characterized by skeletal defects similar to those of GPHYSD1. Several ADAMTSL2 TSRs have consensus sequences for C-mannosylation, and one TSR contains a sequon for N-glycosylation that overlaps with an O-fucosylation site. 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 mass spectrometry to identify glycan modifications at the predicted consensus sequences. 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 knockout but not in B3GLCT knockout cells suggested that impaired ADAMTSL2 secretion is not responsible for skeletal defects in PTRPLS patients lacking B3GLCT. In contrast, secretion of mouse ADAMTSL2 carrying GPHYSD1 mutations (S641L in TSR3 and G817R in TSR6) was significantly reduced in HEK293T cells. Moreover, S641L eliminated O-fucosylation of TSR3. Combined these results provide strong evidence that developmental abnormalities in GPHYSD1 patients with this mutation are caused by loss of O-fucosylation on TSR3 and impaired ADAMTSL2 secretion.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: Ao Zhang  

LAB HEAD: Robert S. Haltiwanger

PROVIDER: PXD018902 | Pride | 2020-10-01

REPOSITORIES: Pride