B3GLCT (b3-glucosyltransferase) adds a b3-linked glucose to O-fucose on Thrombospondin type 1 Repeats (TSRs), forming the disaccharide Glucoseb1-3Fucose. The O-fucose is added by Protein O-fucosyltransferase 2 (POFUT2) to a Serine/Threonine located in a proposed consensus sequence within the TSR: C1XX(S/T)C2. Database searches with this consensus reveal 49 potential POFUT2 targets (and thus predicted B3GLCT targets) in humans. Nearly half of these targets are members of the ADAMTS or ADAMTS-like super-families, many of which are known to play essential biological roles in remodeling extracellular matrix. Elimination of Pofut2 in mice results in early embryonic lethality, consistent with an essential function for O-fucosylation for some or all of these proteins. In contrast, mutations in B3GLCT result in Peters plus syndrome (PPS, OMIM #261540), a rare autosomal recessive disorder characterized by structural malformations including Peters anomaly of the eye, short stature, brachydactyly, developmental delay, and characteristic craniofacial abnormalities. Several other abnormalities are commonly seen in patients including defects in heart, cleft lip/palate, genitourinary system, ear, and CNS. Elimination of B3glct in mice results in several similar phenotypes, including craniofacial and long bone growth defects, suggesting the mutants will be an excellent in vivo model to study B3GLCT function. Our recent publication suggests that both POFUT2 and B3GLCT are important for the quality control of TSR folding. Using RNAi-mediated knockdown, we demonstrated that loss of POFUT2 causes a secretion defect for all targets analyzed, while knockdown of B3GLCT is only necessary for secretion of some targets. These results provide a potential explanation for the difference in phenotype between Pofut2 null mice and PPS patients. Together these observations have led to our central hypothesis, that B3GLCT-mediated addition of glucose is required for efficient folding of a subset of TSR-proteins, and that the anomalies seen in PPS results from impaired secretion of a small number of sensitive targets. Here we will test this hypothesis in three Aims. Aim 1 examines how identified mutations in PPS patients affect B3GLCT activity and stability using cell-based and biochemical assays. Aim 2 examines which predicted POFUT2 targets require B3GLCT for secretion and why. We will test whether B3GLCT is required for secretion of POFUT2 targets relevant to PPS, and examine whether some TSRs require B3GLCT for folding and others do not. Finally, Aim 3 investigates whether loss of B3glct impairs secretion of POFUT2 targets in vivo and whether the B3glct knockout has different affects on targets that vary in number of TSRs. In addition, this aim tests whether effects on protein secretion are cell-type specific and whether the bone growth abnormalities observed in B3glct mutants result from reduction of functional protein or alternatively to unresolved unfolded protein response d...