The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Abstract Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3∆ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation

Glycoprotein biosynthesis in Saccharomyces cerevisiae: ngd29, an N-glycosylation mutant allelic to och1 having a defect in the initiation of outer chain formation

AbstractOuter chain glycosylation in Saccharomyces cerevisiae leads to heterogeneous and immunogenic asparagine-linked saccharide chains containing more than 50 mannose residues on secreted glycoproteins. Using a [3H]mannose suicide selection procedure a collection of N-glycosylation defective mutants (designated ngd) was isolated. One mutant, ngd29, was found to have a defect in the initiation of the outer chain and displayed a temperature growth sensitivity at 37°C allowing the isolation of the corresponding gene by complementation. Cloning, sequencing and disruption of NGD29 showed that it is a non lethal gene and identical to OCH1. It complemented both the glycosylation and growth defect. Membranes isolated from an ngd29 disruptant or an ngd29mnn1 double mutant were no longer able, in contrast to membranes from wild type cells, to transfer mannose from GDPmannose to Man8GlcNAc2, the in vivo acceptor for building up the outer chain. Heterologous expression of glucose oxidase from Aspergillus niger in an ngd29mnn1 double mutant produced a secreted uniform glycoprotein with exclusively Man8GlcNAc2 structure that in wild type yeast is heavily hyperglycosylated. The data indicate that this mutant strain is a suitable host for the expression of recombinant glycoproteins from different origin in S. cerevisiae to obtain mammalian oligomannosidic type N-linked carbohydrate chains

Autosomal Recessive Dilated Cardiomyopathy due to DOLK Mutations Results from Abnormal Dystroglycan O-Mannosylation

Genetic causes for autosomal recessive forms of dilated cardiomyopathy (DCM) are only rarely identified, although they are thought to contribute considerably to sudden cardiac death and heart failure, especially in young children. Here, we describe 11 young patients (5–13 years) with a predominant presentation of dilated cardiomyopathy (DCM). Metabolic investigations showed deficient protein N-glycosylation, leading to a diagnosis of Congenital Disorders of Glycosylation (CDG). Homozygosity mapping in the consanguineous families showed a locus with two known genes in the N-glycosylation pathway. In all individuals, pathogenic mutations were identified in DOLK, encoding the dolichol kinase responsible for formation of dolichol-phosphate. Enzyme analysis in patients' fibroblasts confirmed a dolichol kinase deficiency in all families. In comparison with the generally multisystem presentation in CDG, the nonsyndromic DCM in several individuals was remarkable. Investigation of other dolichol-phosphate dependent glycosylation pathways in biopsied heart tissue indicated reduced O-mannosylation of alpha-dystroglycan with concomitant functional loss of its laminin-binding capacity, which has been linked to DCM. We thus identified a combined deficiency of protein N-glycosylation and alpha-dystroglycan O-mannosylation in patients with nonsyndromic DCM due to autosomal recessive DOLK mutations

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics, or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase complex, the key enzyme of N-glycosylation. Deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity towards compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas deletion of OST6, a functional homolog of OST3, has no effect. Oligosaccharyltransferase activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared to wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase CPY, Gas1 or DPAP B, displayed an underglycosylation pattern. By affinity chromatography a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the Omannosylation process, as glycosylation of distinct glycoproteins of this type were affected as well. Altogether the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation. Keywords: ROT1/ N‐glycosylation/ O‐glycosylation/ oligosaccharyltransferase/ dolichol/ Saccharomyces cerevisiae Downloaded from http://glycob.oxfordjournals.org/ at Instytut Biochemii i Biofizyki PAN on April 17, 201

Deficiency of dolichyl-P-Man:Man7GlcNAc2-PP-dolichyl mannosyltransferase causes congenital disorder of glycosylation type Ig.

Deficiency of the endoplasmic reticulum enzyme dolichyl-phosphate mannose (Dol-P-Man):Man(7)GlcNAc(2)-PP-dolichyl mannosyltransferase leads to a new type of congenital disorder of glycosylation, designated type Ig. The patient 1 presented with a multisystemic disorder with microcephaly, developmental retardation, convulsions and dysmorphic signs. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains. In skin fibroblasts from the patient, the activity of Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase was severely reduced leading to the accumulation of Man(7)GlcNAc(2)-PP-Dol, which was transferred to newly synthesized glycoproteins. Sequencing of the Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase cDNA revealed a compound heterozygosity for two point mutations, leading to the exchange of leucine(158) for a proline residue and a premature translation stop with loss of the C-terminal 74 amino acids. The parents were heterozygous for one of the two mutations. Retroviral expression of the wild-type Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase cDNA in patient's fibroblasts normalized the mannosyltransferase activity

Identification of the Gene Encoding the α1,3-Mannosyltransferase (ALG3) in Arabidopsis and Characterization of Downstream N-Glycan Processing[W]

Glycosyltransferases are involved in the biosynthesis of lipid-linked N-glycans. Here, we identify and characterize a mannosyltransferase gene from Arabidopsis thaliana, which is the functional homolog of the ALG3 (Dol-P-Man:Man5GlcNAc2-PP-Dol α1,3-mannosyl transferase) gene in yeast. The At ALG3 protein can complement a Δalg3 yeast mutant and is localized to the endoplasmic reticulum in yeast and in plants. A homozygous T-DNA insertion mutant, alg3-2, was identified in Arabidopsis with residual levels of wild-type ALG3, derived from incidental splicing of the 11th intron carrying the T-DNAs. N-glycan analysis of alg3-2 and alg3-2 in the complex-glycan-less mutant background, which lacks N-acetylglucosaminyl-transferase I activity, reveals that when ALG3 activity is strongly reduced, almost all N-glycans transferred to proteins are aberrant, indicating that the Arabidopsis oligosaccharide transferase complex is remarkably substrate tolerant. In alg3-2 plants, the aberrant glycans on glycoproteins are recognized by endogenous mannosidase I and N-acetylglucosaminyltransferase I and efficiently processed into complex-type glycans. Although no high-mannose-type glycoproteins are detected in alg3-2 plants, these plants do not show a growth phenotype under normal growth conditions. However, the glycosylation abnormalities result in activation of marker genes diagnostic of the unfolded protein response

Deficiency of GDP-Man:GlcNAc(2)-PP-Dolichol Mannosyltransferase Causes Congenital Disorder of Glycosylation Type Ik

The molecular nature of a severe multisystemic disorder with a recurrent nonimmune hydrops fetalis was identified as deficiency of GDP-Man:GlcNAc(2)-PP-dolichol mannosyltransferase, the human orthologue of the yeast ALG1 gene (MIM 605907). The disease belongs to the group of congenital disorders of glycosylation (CDG) and is designated as subtype CDG-Ik. In patient-derived serum, the total amount of the glycoprotein transferrin was reduced. Moreover, a partial loss of N-glycan chains was observed, a characteristic feature of CDG type I forms. Metabolic labeling with [6-(3)H]glucosamine revealed an accumulation of GlcNAc(2)-PP-dolichol and GlcNAc(1)-PP-dolichol in skin fibroblasts of the patient. Incubation of fibroblast extracts with [(14)C]GlcNAc(2)-PP-dolichol and GDP-mannose indicated a severely reduced activity of the β1,4-mannosyltransferase, elongating GlcNAc(2)-PP-dolichol to Man(1)GlcNAc(2)-PP-dolichol at the cytosolic side of the endoplasmic reticulum. Genetic analysis of the patient’s hALG1 gene identified a homozygous mutation leading to the exchange of a serine residue to leucine at position 258 in the hALG1 protein. The disease-causing nature of the hALG1 mutation for the glycosylation defect was verified by a retroviral complementation approach in patient-derived primary fibroblasts and was confirmed by the expression of wild-type and mutant hALG1 in the Saccharomyces cerevisiae alg1-1 strain

Congenital Disorder of Glycosylation Type Ik (CDG-Ik): A Defect of Mannosyltransferase I

This study describes the discovery of a new inherited disorder of glycosylation named “CDG-Ik.” CDG-Ik (congenital disorder of glycoslyation type Ik) is based on a defect of human mannosyltransferase I (MT-I [MIM 605907]), an enzyme necessary for the elongation of dolichol-linked chitobiose during N-glycan biosynthesis. Mutations in semiconserved regions in the corresponding gene, HMT-1 (yeast homologue, Alg1), in two patients caused drastically reduced enzyme activity, leading to a severe disease with death in early infancy. One patient had a homozygous point mutation (c.773C→T, S258L), whereas the other patient was compound heterozygous for the mutations c.773C→T and c.1025A→C (E342P). Glycosylation and growth of Alg1-deficient PRY56 yeast cells, showing a temperature-sensitive phenotype, could be restored by the human wild-type allele, whereas only slight restoration was observed after transformation with the patients' alleles