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.

Pancreatic beta cell-surface expression of glucose transporter 2 (Glut-2) is essential for glucose-stimulated insulin secretion, thereby controlling blood glucose homeostasis in response to dietary intake. We show that the murine GlcNAcT-IVa glycosyltransferase is required for Glut-2 residency on the beta cell surface by constructing a cell-type- and glycoprotein-specific N-glycan ligand for pancreatic lectin receptors. Loss of GlcNAcT-IVa, or the addition of glycan-ligand mimetics, attenuates Glut-2 cell-surface half-life, provoking endocytosis with redistribution into endosomes and lysosomes. The ensuing impairment of glucose-stimulated insulin secretion leads to metabolic dysfunction diagnostic of type 2 diabetes. Remarkably, the induction of diabetes by chronic ingestion of a high-fat diet is associated with reduced GlcNAcT-IV expression and attenuated Glut-2 glycosylation coincident with Glut-2 endocytosis. We infer that beta cell glucose-transporter glycosylation mediates a link between diet and insulin production that typically suppresses the pathogenesis of type 2 diabetes.

Defects in O-mannosylation of ␣-dystroglycan are thought to cause certain types of congenital muscular dystrophies with neuronal migration disorders. Among these muscular dystrophies, WalkerWarburg syndrome is caused by mutations in the gene encoding putative protein O-mannosyltransferase 1 (POMT1), which is homologous to yeast protein O-mannosyltransferases. However, there is no evidence that POMT1 has enzymatic activity. In this study, we first developed a method to detect protein O-mannosyltransferase activity in mammalian cells. Then, using this method, we showed that coexpression of both POMT1 and POMT2 (another gene homologous to yeast protein O-mannosyltransferases) was necessary for the enzyme activity, but expression of either POMT1 or POMT2 alone was insufficient. The requirement of an active enzyme complex of POMT1 and POMT2 suggests that the regulation of protein O-mannosylation is complex. Further, protein Omannosylation appears to be required for normal structure and function of ␣-dystroglycan in muscle and brain. In view of the potential importance of this form of glycosylation for a number of developmental and neurobiological processes, the ability to assay mammalian protein O-mannosyltransferase activity should greatly facilitate progress in the identification and localization of Omannosylated proteins and the elucidation of their functional roles.

Inflammation, protease/anti-protease imbalance and oxidative stress play important roles in the pathogenesis of emphysema. Nrf2 counteracts oxidative tissue damage and inflammation through transcriptional activation via the anti-oxidant responsive element (ARE). To clarify the protective role of Nrf2 in the development of emphysema, the susceptibility of Nrf2-knockout mice to cigarette smoke (CS)-induced emphysema was examined. In Nrf2-knockout mice, emphysema was first observed at 8 weeks and exacerbated by 16 weeks following CS-exposure, whereas no pathological abnormalities were observed in wild-type mice. Neutrophilic lung inflammation and permeability lung damage were significantly enhanced in Nrf2-knockout mice 8 weeks after CSexposure. Importantly, neutrophil elastase activity in bronchoalveolar lavage fluids was markedly higher in Nrf2-knockout mice preceding the pronounced neutrophil accumulation. The expression of secretory leukoprotease inhibitor, a potent inhibitor of neutrophil elastase, was inducible in wild-type, but not in Nrf2-knockout mice. This protease/anti-protease imbalance, together with the lack of inducible expression of ARE-regulated anti-oxidant/anti-inflammatory genes, may explain the predisposition of Nrf2-knockout mice to neutrophilic inflammation. Indeed, specific activators of Nrf2 induced the expression of the SLPI gene in macrophages. These results indicate that Nrf2 protects against the development of emphysema by regulating not only the oxidant/ anti-oxidant balance, but also inflammation and the protease/anti-protease balance.

ABSTRACT

No abstract

No abstract

A yeast mutant capable of producing Man 5 GlcNAc 2 human compatible sugar chains on glycoproteins was constructed. An expression vector for ␣-1,2-mannosidase with the "HDEL" endoplasmic reticulum retention/ retrieval tag was designed and expressed in Saccharomyces cerevisiae. An in vitro ␣-1,2-mannosidase assay and Western blot analysis showed that it was successfully localized in the endoplasmic reticulum. A triple mutant yeast lacking three glycosyltransferase activities was then transformed with an ␣-1,2-mannosidase expression vector. The oligosaccharide structures of carboxypeptidase Y as well as cell surface glycoproteins were analyzed, and the recombinant yeast was shown to produce a series of high mannose-type sugar chains including Man 5 GlcNAc 2 . This is the first report of a recombinant S. cerevisiae able to produce Man 5 GlcNAc 2 -oligosaccharides, the intermediate for hybrid-type and complex-type sugar chains.Saccharomyces cerevisiae is useful for the production of recombinant proteins of biological interest because of the established expression system, and it can be easily grown in large quantities. Moreover, yeast share the early steps of the mammalian Asn-linked glycosylation pathway. However, the mature Asn-linked oligosaccharides of yeast are mannan glycans and are highly antigenic against mammals. Thus, it would be necessary to eliminate the antigenicity of the sugar chains when recombinant therapeutic glycoproteins are produced in yeast.Several genes concerned with the biosynthesis of yeast sugar chains have been cloned, and the glycosylation pathway of yeast has been clarified. The OCH1 gene encodes an ␣-1,6-mannosyltransferase that initiates ␣-1,6-polymannose outer chain formation on the Asn-linked inner oligosaccharide Man 8 GlcNAc 2 in S. cerevisiae (1). MNN1 has been proposed as the structural gene for the ␣-1,3-mannosyltransferase that elongates the outer chain and the inner core oligosaccharide (2, 3). The ⌬och1 mnn1 double mutant accumulated a single oligosaccharide moiety, Man 8 GlcNAc 2 , a high mannose-type structure (1). This mutant may be useful to produce recombinant therapeutic glycoproteins without any antigenicity toward humans.On the other hand, some glycoproteins of therapeutic value require complex-type sugar chains for their efficacy. Erythropoietin (EPO), 1 a hematopoietic glycoprotein factor produced in the kidney, has three complex-type Asn-linked sugar chains and one mucin-type sugar chain. It is reported that the composition and structure of each sugar chain affected the biological activity, the efficiency of secretion, and had profound effects on the half-life of EPO in the blood circulation (4). It seems that the most active form of the EPO molecule requires tetraantennary Asn-linked sugar chains (5) with full sialylation, to prevent serum clearance by the action of the hepatic asialoglycoprotein binding protein (6, 7). When EPO was expressed in the ⌬och1 mnn1 mutant yeast, the recombinant EPO should have high mannose-type oligosaccharides, which are trapped by the...