Molecular basis of congenital disorder of glycosylation type 1N

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N-glycosylation is essential for life. Glycoproteins such as cadherins, integrins and laminins are key to development, organogenesis, and tissue organization. Defects in N-glycosylation underlie numerous human genetic disorders including a heterogeneous group of autosomal-recessive, metabolic diseases termed Congenital Disorders of Glycosylation (CDGs). CDGs present with a range of devastating symptoms, including failure to thrive, developmental and speech delays, vision loss, hypotonia, microcephaly, seizures, and stroke- like episodes. Children with CDGs suffer cognitive and physical disabilities. While some of these symptoms can be treated, there is no cure for CDGs. Our goal in this R21 application is to explore the molecular basis of CDG type 1N, a poorly understood disease caused by missense mutations in the endoplasmic reticulum membrane protein RFT1. Patients, identified thus far in North America, U.K./Europe, the Middle East, and India, often present with a unique sensorineural deafness in addition to typical severe CDG symptoms. There is no therapy for CDG1N. Unlike most CDGs for which the underlying molecular defect is well understood, e.g., deficiency of a glycosyltransferase or sugar-processing enzyme, CDG1N is an enigma because the function of RFT1 is not known. The pathway of N-glycosylation is blocked at a critical stage in CDG1N cells, resulting in the build-up of a lipid intermediate (termed M5-DLO) that cannot be utilized by the glycosylation machinery. We hypothesize that the critical role of RFT1 in cells is to catalyze utilization of M5-DLO either by overcoming an impediment posed by endoplasmic reticulum structure/morphology to facilitate its handoff to downstream machinery, or by preventing its mis-localization. We will test this hypothesis in two specific aims. The first aim will focus on the role of RFT1 in M5-DLO utilization, making use of RFT1-deficient cells (including CDG1N patient fibroblasts), cell-free systems, and a novel approach to sub-fractionating the endoplasmic reticulum. We will also test whether RFT1 is an M5-DLO binding protein. Recognizing that structural information is critical to understand the function of RFT1 at a molecular level, the second aim will develop a structure-function model of RFT1. Here we will define its membrane topology, identify key functional residues, and initiate a program to elucidate its 3-dimensional structure. In the long term, our results will illuminate why RFT1 deficiency results in M5-DLO accumulation and consequently CDG1N, thereby paving the way for the development of treatment strategies and therapeutics that could subserve RFT1's function to restore glycosylation and improve clinical symptoms.