Principal Investigator: Anant K. Menon
Grant Title: Molecular mechanism of glucosylation of the dolichol-linked N-glycan
precursor
Abstract
Protein glycosylation is essential for all eukaryotes, from disease-causing
protists such as malaria and trypanosomes, to yeast and mammals. Secretory
proteins are almost invariably N-glycosylated, O- and C-mannosylated,
and/or GPI-anchored as they enter the lumen of the endoplasmic reticulum(ER). Yeast cells that cannot synthesize N-glycoproteins or GPI-anchoredproteins are inviable, and mice with the same defects die as embryos. Specificdefects in glycosylation cause human disease. For example, defective Omannosylation is a major underlying cause of muscular dystrophies, and a
GPI anchoring defect in multipotent hematopoietic human stem cells causes paroxysmal
nocturnal hemoglobinuria, an acquired hemolytic disease. Congenital Disorders of
Glycosylation (CDGs) are a family of severe inherited diseases characterized by predominantly
neurological symptoms.
Protein N-glycosylation occurs in the lumen of the ER. The oligosaccharide moiety fromdolichol-diphosphate-oligosaccharide (the 'dolichol-linked N-glycan precursor' or 'dolichollinked oligosaccharide (DLO)') is transferred to specific asparagine residues of secretory
proteins as they emerge from the protein translocation channel into the ER lumen. Synthesis of
the dolichol-linked N-glycan precursor is initiated on the cytoplasmic face of the ER and after
seven glycosyltransfer reactions, the resulting glycolipid is flipped across the ER membrane to
be further elaborated before participating in the protein N-glycosylation reaction. The optimal
N-glycan precursor oligosaccharide in yeast and mammals contains a terminal cap of threeglucose residues that is sourced from the glycolipid glucosyl-phosphoryl dolichol (GPD) in theER lumen. Paradoxically, this lipid is synthesized on the cytoplasmic face of the ER and must
be flipped across the ER membrane to provide a source of lumenal glucose. As the spontaneous
rate of GPD flipping is extremely low, a specific transporter is needed to move GPD across theER membrane at a physiological rate. The molecular identity of this important transport
protein - herein termed GPD scramblase - is not known and its discovery remains a major
challenge for molecular cell biology. The central role of glucosylation in optimal ER protein
N-glycosylation was recognized more than 3 decades ago, and its relevance for cellularphysiology and human disease is abundantly clear as glucosylation deficiency results inCongenital Disorders of Glycosylation. Our aim is to identify GPD scramblase via an
unprecedented approach that combines bioinformatics and biochemistry. Our approach is wellsuited to the 1-year period of the Mizutani Foundation grant.
Although GPD is important in yeast and humans, not all N-glycosylation-competentorganisms have glucose in their N-glycan precursor. Taking advantage of this fact, we
implemented an innovative bioinformatics approach for assignment of protein function. Using
this method, termed phylogenetic profiling, we identified a number of polytopic ER membraneproteins as GPD scramblase candidates in yeast. We will test these candidates by in vivo
approaches, for example by comparing the phenotypes of the corresponding yeast mutants toGPD synthase deficiency, as well as by using reconstitution-based assays of GPD scrambling
using previously described radiolabeled GPD analogs. Identifying GPD scramblase is a highly
significant overall objective that is expected to open new directions for study of the molecularmechanism of lipid scrambling, reveal new genetic loci associated with CDGs, and illuminate
potential targets for treatment of N-glycan deficiency.