Hiroshi Manya

Muscular Dystrophy and Neuronal Migration Disorder Caused by Mutations in a Glycosyltransferase, POMGnT1

Muscle-eye-brain disease (MEB) is an autosomal recessive disorder characterized by congenital muscular dystrophy, ocular abnormalities, and lissencephaly. Mammalian O-mannosyl glycosylation is a rare type of protein modification that is observed in a limited number of glycoproteins of brain, nerve, and skeletal muscle. Here we isolated a human cDNA for protein O-mannose beta-1,2-N-acetylglucosaminyltransferase (POMGnT1), which participates in O-mannosyl glycan synthesis. We also identified six independent mutations of the POMGnT1 gene in six patients with MEB. Expression of most frequent mutation revealed a great loss of the enzymatic activity. These findings suggest that interference in O-mannosyl glycosylation is a new pathomechanism for muscular dystrophy as well as neuronal migration disorder.

Loss-of-function of an N-acetylglucosaminyltransferase, POMGnT1, in muscle-eye-brain disease.

Muscle-eye-brain disease (MEB), an autosomal recessive disorder, is characterized by congenital muscular dystrophy, brain malformation, and ocular abnormalities. Previously, we found that MEB is caused by mutations in the gene encoding the protein O-linked mannose beta1,2-N-acetylglucosaminyltransferase 1 (POMGnT1), which is responsible for the formation of the GlcNAcbeta1-2Man linkage of O-mannosyl glycan. Although 13 mutations have been identified in patients with MEB, only the protein with the most frequently observed splicing site mutation has been studied. This protein was found to have no activity. Here, we expressed the remaining mutant POMGnT1s and found that none of them had any activity. These results clearly demonstrate that MEB is inherited as a loss-of-function of POMGnT1.

The twisted abdomen phenotype of Drosophila POMT1 and POMT2 mutants coincides with their heterophilic protein O-mannosyltransferase activity.

Walker-Warburg syndrome, caused by mutations in protein O-mannosyltransferase-1 (POMT1), is an autosomal recessive disorder characterized by severe brain malformation, muscular dystrophy, and structural eye abnormalities. As humans have a second POMT, POMT2, we cloned each Drosophila ortholog of the human POMT genes and carried out RNA interference (RNAi) knock-down to investigate the function of these proteins in vivo. Drosophila POMT2 (dPOMT2) RNAi mutant flies showed a “twisted abdomen phenotype,” in which the abdomen is twisted 30–60°, similar to the dPOMT1 mutant. Moreover, dPOMT2 interacted genetically with dPOMT1, suggesting that the dPOMTs function in collaboration with each other in vivo. We expressed dPOMTs in Sf21 cells and measured POMT activity. dPOMT2 transferred a mannose to the dystroglycan protein only when it was coexpressed with dPOMT1. Likewise, dPOMT1 showed POMT activity only when coexpressed with dPOMT2, and neither dPOMT showed any activity by itself. Each dPOMT RNAi fly totally reduced POMT activity, despite the specific reduction in the level of each dPOMT mRNA. The expression pattern of dPOMT2 mRNA was found to be similar to that of dPOMT1 mRNA using whole mount in situ hybridization. These results demonstrate that the two dPOMTs function as a protein O-mannosyltransferase in association with each other, in vitro and in vivo, to generate and maintain normal muscle development.

Physical and functional association of human protein o-mannosyltransferases 1 and 2

A defect of protein O-mannosylation causes congenital muscular dystrophy with brain malformation and structural eye abnormalities, so-called Walker-Warburg syndrome. Protein O-mannosylation is catalyzed by protein O-mannosyltransferase 1 (POMT1) and its homologue, POMT2. Coexpression of POMT1 and POMT2 is required to show O-mannosylation activity. Here we have shown that POMT1 forms a complex with POMT2 and the complex possesses protein O-mannosyltransferase activity. Results indicate that POMT1 and POMT2 associate physically and functionally in vivo. Recently, three mutations were reported in the POMT1 gene of patients who showed milder phenotypes than typical Walker-Warburg syndrome. We coexpressed these mutant POMT1s with POMT2 and found that none of them had any activity. However, all POMT1 mutants, including previously identified POMT1 mutants, coprecipitated with POMT2. These results indicate that the mutant POMT1s could form heterocomplexes with POMT2 but that such complexes are insufficient for enzymatic activity.

Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.

An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease

The β‐site amyloid precursor protein cleaving enzyme‐1 (BACE1), an essential protease for the generation of amyloid‐β (Aβ) peptide, is a major drug target for Alzheimers disease (AD). However, there is a concern that inhibiting BACE1 could also affect several physiological functions. Here, we show that BACE1 is modified with bisecting N‐acetylglucosamine (GlcNAc), a sugar modification highly expressed in brain, and demonstrate that AD patients have higher levels of bisecting GlcNAc on BACE1. Analysis of knockout mice lacking the biosynthetic enzyme for bisecting GlcNAc, GnT‐III (Mgat3), revealed that cleavage of Aβ‐precursor protein (APP) by BACE1 is reduced in these mice, resulting in a decrease in Aβ plaques and improved cognitive function. The lack of this modification directs BACE1 to late endosomes/lysosomes where it is less colocalized with APP, leading to accelerated lysosomal degradation. Notably, other BACE1 substrates, CHL1 and contactin‐2, are normally cleaved in GnT‐III‐deficient mice, suggesting that the effect of bisecting GlcNAc on BACE1 is selective to APP. Considering that GnT‐III‐deficient mice remain healthy, GnT‐III may be a novel and promising drug target for AD therapeutics.

Host-Soluble Galectin-1 Promotes HIV-1 Replication through a Direct Interaction with Glycans of Viral gp120 and Host CD4

ABSTRACT Sexual transmission of HIV-1 requires virus adsorption to a target cell, typically a CD4+ T lymphocyte residing in the lamina propria, beneath the epithelium. To escape the mucosal clearance system and reach its target cells, HIV-1 has evolved strategies to circumvent deleterious host factors. Galectin-1, a soluble lectin found in the underlayers of the epithelium, increases HIV-1 infectivity by accelerating its binding to susceptible cells. By comparison, galectin-3, a family member expressed by epithelial cells and part of the mucosal clearance system, does not perform similarly. We show here that galectin-1 directly binds to HIV-1 in a β-galactoside-dependent fashion through recognition of clusters of N-linked glycans on the viral envelope gp120. Unexpectedly, this preferential binding of galectin-1 does not rely on the primary sequence of any particular glycans. Instead, glycan clustering arising from the tertiary structure of gp120 hinders its binding by galectin-3. Increased polyvalency of a specific ligand epitope is a common strategy for glycans to increase their avidity for lectins. In this peculiar occurrence, glycan clustering is instead exploited to prevent binding of gp120 by galectin-3, which would lead to a biological dead-end for the virus. Our data also suggest that galectin-1 binds preferentially to CD4, the host receptor for gp120. Together, these results suggest that HIV-1 exploits galectin-1 to enhance gp120-CD4 interactions, thereby promoting virus attachment and infection events. Since viral adhesion is a rate-limiting step for HIV-1 entry, modulation of the gp120 interaction with galectin-1 could thus represent a novel approach for the prevention of HIV-1 transmission.

Regulation of Mammalian Protein O-Mannosylation PREFERENTIAL AMINO ACID SEQUENCE FOR O-MANNOSE MODIFICATION

O-Mannosyl glycans are important in muscle and brain development. Protein O-mannosyltransferase (POMT) catalyzes the initial step of O-mannosyl glycan biosynthesis. To understand which serine (Ser) and threonine (Thr) residues POMT recognizes for mannosylation, we prepared a series of synthetic peptides based on a mucin-like domain in α-dystroglycan (α-DG), one of the best known O-mannosylated proteins in mammals. In α-DG, the mucin-like domain spans amino acid residues 316 to 489. Two similar peptide sequences, corresponding to residues 401–420 and 336–355, respectively, were strongly mannosylated by POMT, whereas other peptides from α-DG and peptides of various mucin tandem repeat regions were poorly mannosylated. Peptides 401–420 and 336–355 contained four and six Ser and Thr residues, respectively. Substitution of Ala residues for the Ser or Thr residues showed that Thr-414 of peptide 401–420 and Thr-351 of peptide 336–355 were prominently modified by O-mannosylation. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Edman degradation analysis of the mannosylated peptide 401–420 indicated that Thr-414 was the Thr residue that was most prominently modified by O-mannosylation and that O-mannosylation occurred sequentially rather than at random. Based on these results, we propose a preferred amino acid sequence for mammalian O-mannose modification.

Congenital muscular dystrophy type 1D (MDC1D) due to a large intragenic insertion/deletion, involving intron 10 of the LARGE gene.

Mutation of the LARGE gene is the rarest of the six known genetic causes of α-dystroglycanopathy. We report further a family with MDC1D due to a complex genomic rearrangement that was not apparent on standard sequencing of LARGE. Two sisters in a consanguineous family had moderate mental retardation and cerebellar malformations, together with dystrophic changes and markedly reduced α-dystroglycan glycosylation staining on muscle biopsy. There was homozygous linkage to the LARGE locus but sequencing of LARGE coding regions was normal. Analysis of LARGE cDNA showed an abnormal sequence inserted between exons 10 and 11, in most of the transcripts, predicted to introduce a premature stop codon. The abnormal sequence mapped to a spliced EST (DA935254) of unknown function, normally located at 100 kb centromeric of LARGE on chromosome 22q12.3. Quantitative PCR analysis of the EST and adjacent regions showed twice the normal copy number in patients’ genomic DNA samples, consistent with a large intra-chromosomal duplication inserted into intron 10 of LARGE in a homozygous state. This insertion was associated with deletion of a central region of intron 10, but the exact break points of the deletion/duplication were not found, suggesting that an even more complex rearrangement may have occurred. The exact function of LARGE, a golgi protein, remains uncertain. POMT and POMGnT enzyme activities were normal in patients’ lymphoblast cells, suggesting that defects in LARGE do not affect the initiation of O-mannosyl glycans.

Protective effect of N-glycan bisecting GlcNAc residues on β-amyloid production in Alzheimer's disease

Alteration of glycoprotein glycans often changes various properties of the target glycoprotein and contributes to a wide variety of diseases. Here, we focused on the N-glycans of amyloid precursor protein whose cleaved fragment, beta-amyloid, is thought to cause much of the pathology of Alzheimers disease (AD). We previously determined the N-glycan structures of normal and mutant amyloid precursor proteins (the Swedish type and the London type). In comparison with normal amyloid precursor protein, mutant amyloid precursor proteins had higher contents of bisecting GlcNAc residues. Because N-acetylglucosaminyltransferase III (GnT-III) is the glycosyltransferase responsible for synthesizing a bisecting GlcNAc residue, the current report measured GnT-III mRNA expression levels in the brains of AD patients. Interestingly, GnT-III mRNA expression was increased in AD brains. Furthermore, beta-amyloid treatment increased GnT-III mRNA expression in Neuro2a mouse neuroblastoma cells. We then examined the influence of bisecting GlcNAc on the production of beta-amyloid. Both beta-amyloid 40 and beta-amyloid 42 were significantly decreased in GnT-III-transfected cells. When secretase activities were analyzed in GnT-III transfectant cells, alpha-secretase activity was increased. Taken together, these results suggest that upregulation of GnT-III in AD brains may represent an adaptive response to protect them from additional beta-amyloid production.