Role of O ‐linked glucose‐fucose disaccharide modification of thrombospondin type I repeats in protein folding and embryo development

Abstract

We propose that Protein O-fucosyltransferase 2 (POFUT2) and β3-glucosyltransferase (B3GLCT) function to ensure efficient folding of forty-nine proteins containing tandemly repeated Thrombospondin Type 1 Repeats (TSRs). In the endoplasmic reticulum (ER), POFUT2 and B3GLCT recognize properly folded TSRs and act sequentially to add the unusual O-linked Glucoseβ1-3Fucose disaccharide to either a serine or threonine residue located within the POFUT2 consensus sequence (C-X-X-S/T-C). Once all TSRs are modified the target protein exits the ER. In crystal structures the disaccharide forms H-bonds and van der Waals interactions with underlying amino acids in the TSR, raising the possibility that the disaccharide functions as a surrogate amino acid to stabilize the correctly folded TSR. Results from in vitro folding/refolding assays provide evidence that the disaccharide stabilizes the TSR fold and accelerates the overall rate of protein folding. Consistent with this prediction, loss of POFUT2 or B3GLCT in cell culture impairs trafficking of POFUT2/B3GLCT targets. To begin to evaluate the function of the disaccharide in vivo, we generated mouse loss of function mutations in Pofut2 and B3glct. Loss of Pofut2 causes early embryo lethality due to a block in gastrulation, resulting from functional reduction of ADAMTS9 in extraembryonic tissue. In contrast, B3glct animals survive, but have craniofacial and skeletal abnormalities, defects in pigmentation, soft tissue syndactyly, and develop hydrocephalus. The mouse B3glct phenotype shares similarities to Peters Plus syndrome in humans, caused by recessive mutations in B3GLCT. We propose that hydrocephalus in B3glct mutants results from the combined effects of the mutation on ADAMTS9 and ADAMTS20 in ependymal cells and Sco-Spondin (SSPO) in the subcommissural organ. The distinct differences in the effects of these mutations during mouse development support a model whereby the O-linked fucose is essential for folding and secretion of all POFUT2/B3GLCT target proteins and that B3GLCT-mediated extension to the glucose-fucose disaccharide is only required by a subset of sensitive targets.