POMGNT 2 , a gatekeeper enzyme 1 Protein O-linked Mannose β1 , 4N-acetylglucosaminyltransferase 2 ( POMGNT 2 ) is a Gatekeeper Enzyme for Functional Glycosylation of α-Dystroglycan

Disruption of the O-mannosylation pathway involved in functional glycosylation of αdystroglycan gives rise to congenital muscular dystrophies. Protein O-linked mannose β-1,4-Nacetylglucosaminyltransferase 2 (POMGNT2) catalyzes the first step towards the functional matriglycan structure on α-dystroglycan that is responsible for binding extracellular matrix proteins and certain arenaviruses. Alternatively, protein O-linked mannose β-1,2-Nacetylglucosaminyltransferase 1 (POMGNT1) catalyzes the first step towards other various glycan structures present on α-dystroglycan of unknown function. Here, we demonstrate that POMGNT1 is promiscuous for O-mannosylated peptides while POMGNT2 displays significant primary amino acid selectivity near the site of Omannosylation. We define a POMGNT2 acceptor motif, conserved among fifty-nine vertebrate species, in α-dystroglycan that when engineered into a POMGNT1-only site is sufficient to convert the O-mannosylated peptide to a substrate for POMGNT2. Additionally, an acceptor glycopeptide is a less efficient substrate for POMGNT2 when two of the conserved amino acids are replaced. These findings begin to define the selectivity of POMGNT2 and suggest this enzyme functions as a gatekeeper enzyme to prevent the vast majority of O-mannosylated sites on proteins from becoming modified with glycan structures functional for binding LG-domain containing proteins. INTRODUCTION Congenital muscular dystrophy (CMD) describes a family of genetic, degenerative diseases characterized by contractures, myopathy, and in some cases, central nervous system abnormalities. Many CMDs are caused by defects in the formation of a functional dystrophin glycoprotein complex (DGC) that links the actin cytoskeleton to the extracellular matrix (ECM). αDystroglycan (α-DG), encoded by the DAG1 gene, provides the physical link to LG-domain containing proteins in the ECM (1), however, there are only a few known mutations in the DAG1 coding sequence that lead to CMD (2). A subset of CMDs, termed secondary dystroglycanopathies, is caused by mutations in genes encoding enzymes responsible for glycosylating α-DG in its mucin-like domain (residues 313-489). These secondary dystroglycanopathies range in severity from mild Limb-Girdle muscular dystrophy to the more severe Walker-Warburg syndrome (3-5). The causal genes for secondary dystroglycanopathies have been identified as encoding enzymes in the pathway associated with the biosynthesis of the Omannosyl (O-Man) glycans (6,7). The O-mannosylation pathway begins in the endoplasmic reticulum (ER) where a complex of POMT1 and POMT2 catalyze the transfer of mannose from dolicholphosphomannose to serine and threonine residues in an α-linkage to α-DG (8), and presumably a handful of other proteins (9). Bifurcation of the pathway then occurs by the http://www.jbc. org/cgi/doi/10.1074/jbc.M116.764712 The latest version is at JBC Papers in Press. Published on December 8, 2016 as Manuscript M116.764712 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from by gest on D ecem er 3, 2016 hp://w w w . jb.org/ D ow nladed from POMGNT2, a gatekeeper enzyme