Analyzing the Effects of Peters Plus Syndrome Mutations on Human β1,3‐glucosyltransferase

Abstract

Peters plus syndrome (PPS) is a severe autosomal recessive congenital disorder of glycosylation characterized by multiple structural anomalies. PPS patients have classic phenotypes, including Peters anomaly (an ocular anterior segment malformation), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable other systemic abnormalities, including cleft lip/palate, and anomalies in heart, skeletal and genitourinary system. PPS patients have mutations in the β1,3‐glucosyltransferase (B3GLCT) gene, which encodes an enzyme that catalyzes the addition of a glucose to an O‐linked fucose on thrombospondin type‐1 repeats (TSRs), forming an unusual Glucoseβ1‐3Fucose (GlcFuc) disaccharide on TSRs. Protein O‐fucosyltransferase 2 (POFUT2) is the enzyme that adds the O‐fucose to a serine or threonine in the consensus sequence C1XX(S/T)C2 found in TSRs, where C1 and C2 are the first two of six conserved cysteines in TSRs. Currently 65 human proteins are predicted to contain TSRs, and 49 out of these proteins contain the consensus sequence for this GlcFuc disaccharide modification. Half of these proteins belong to ADAMTS/ADAMTS‐like family of extracellular matrix proteins and have critical roles in mediating angiogenesis, re‐structuring extracellular matrix, and developmental processes. Previous results demonstrate that secretion of several ADAMTS/L proteins, in particular ADAMTS20 TSR2‐8, is dependent on the presence of POFUT2 and B3GLCT. Since both POFUT2 and B3GLCT are localized in endoplasmic reticulum and only modify folded TSRs, this leads to the proposal that both POFUT2 and B3GLCT assist the folding of TSRs, modulating the secretion of TSR‐containing proteins. Many identified PPS mutations in B3GLCT are splicing mutations upstream of the catalytic domain and are predicted to result in non‐functional proteins. To analyze the missense and truncation PPS mutations downstream of the catalytic domain, we introduced these mutations separately into a plasmid encoding human B3GLCT and analyzed for their ability to rescue secretion of ADAMTS20 TSR2‐8 from B3GLCT‐null cells. Our results showed that these PPS missense and truncation mutants not only abolished their ability to rescue ADAMTS20 TSR2‐8 secretion, but also greatly reduced mutant protein levels compared to wild type B3GLCT within the cells. Mutant enzymes harboring mutations associated with Peters Plus Spectrum, which is a milder form of PPS with patients who have a subset of PPS phenotypes, all rescued ADAMTS20 TSR2‐8 secretion and had the same protein levels within the cells as wild type B3GLCT. On‐going experiments are comparing the enzymatic activity and protein stability of mutants versus wild type enzyme. Overall, by analyzing the impact of PPS and Peters Plus Spectrum mutations on B3GLCT, we are aiming to discover novel therapeutic targets and advancing the development of novel treatments to alleviate the symptoms of PPS as well as other congenital disorders of glycosylation. This project is supported by NIH grant HD096030.