Biomedical subjects

Ludwig Lehle

Publications and source records attributed to Ludwig Lehle.

11 recordsLinked to original sources

PubMed2006

Protein glycosylation, conserved from yeast to man: a model organism helps elucidate congenital human diseases.

Proteins can be modified by a large variety of covalently linked saccharides. The present review concentrates on two types, protein N-glycosylation and protein O-mannosylation, which, with only a few exceptions, are evolutionary conserved from yeast to man. They are also distinguished by some special features: The corresponding glycosylation processes start in the endoplasmatic reticulum, are continued in the Golgi apparatus, and require dolichol-activated precursors for the initial biosynthetic steps. With respect to the molecular biology of both types of protein glycosylation, the pathways and the genetic background of the reactions have most successfully been studied with the genetically easy-to-handle baker's yeast, Saccharomyces cerevisae. Many of the severe developmental disturbances in children are related to protein glycosylation, for example, the CDG syndrome (congenital disorders of glycosylation) as well as congenital muscular dystrophies with neuronal-cell-migration defects have been elucidated with the help of yeast.

Evolution, Molecular↗

PubMed2006

Pir proteins of Saccharomyces cerevisiae are attached to beta-1,3-glucan by a new protein-carbohydrate linkage.

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the gamma-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of beta-1,3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

Amino Acid Sequence↗

PubMed2006

Defects in N-glycosylation induce apoptosis in yeast.

N-glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Defects of N-glycosylation in humans lead to congenital disorders. The pivotal step of this pathway is the transfer of the evolutionarily conserved lipid-linked core-oligosaccharide to the nascent polypeptide chain, catalysed by the oligosaccharyltransferase. One of its nine subunits, Ost2, has homology to DAD1, originally characterized in hamster cells as a defender against apoptotic death. Here we show that ost mutants, such as ost2 and wbp1-1, display morphological and biochemical features of apoptosis upon induction of the glycosylation defect. We observe nuclear condensation, DNA fragmentation as well as externalization of phosphatidylserine. We also demonstrate induction of caspase-like activity, both determined by flow cytometric analysis and in cell-free extracts. Similarly, the N-glycosylation inhibitor tunicamycin in combination with elevated temperature is able to challenge the apoptotic cascade. Heterologous expression of anti-apoptotic human Bcl-2 diminishes caspase activation, improves survival of cells and suppresses the temperature-sensitive growth defect of wbp1-1. Furthermore, accumulation of reactive oxygen species occurs in response to defective glycosylation. As deletion of the metacaspase YCA1 does not seem to abrogate glycosylation-induced apoptosis, we postulate a different proteolytic process to be involved in this death pathway.

Anti-Bacterial Agents↗

PubMed2005

Yeast oligosaccharyltransferase consists of two functionally distinct sub-complexes, specified by either the Ost3p or Ost6p subunit.

The key step of N-glycosylation of proteins in the endoplasmic reticulum is catalyzed by the hetero-oligomeric protein complex oligosaccharyltransferase (OST). It transfers the lipid-linked core-oligosaccharide to selected Asn-X-Ser/Thr-sequences of nascent polypeptide chains. Biochemical and genetic approaches have revealed that OST from Saccharomyces cerevisiae consists of nine subunits: Wbp1p, Swp1p, Stt3p, Ost1p, Ost2p, Ost4p, Ost5p, Ostp3 and Ost6p. By blue native polyacrylamide electrophoresis we show that yeast OST consists of two isoforms with distinct functions differing only in the presence of the two related Ost3 and Ost6p proteins. The OST6-complex was found to be important for cell wall integrity and temperature stress. Ost3p and Ost6p are not essential for OST activity, and can in part displace each other in the complex when overexpressed, suggesting a dynamic regulation of the complex formation.

Cell Wall↗

PubMed2005

Biosynthesis of lipid-linked oligosaccharides in Saccharomyces cerevisiae: Alg13p and Alg14p form a complex required for the formation of GlcNAc(2)-PP-dolichol.

N-Glycosylation in the endoplasmic reticulum is an essential protein modification and highly conserved in evolution from yeast to man. Here we identify and characterize two essential yeast proteins having homology to bacterial glycosyltransferases, designated Alg13p and Alg14p, as being required for the formation of GlcNAc(2)-PP-dolichol (Dol), the second step in the biosynthesis of the unique lipid-linked core oligosaccharide. Down-regulation of each gene led to a defect in protein N-glycosylation and an accumulation of GlcNAc(1)-PP-Dol in vivo as revealed by metabolic labeling with [(3)H]glucosamine. Microsomal membranes from cells repressed for ALG13 or ALG14, as well as detergent-solubilized extracts thereof, were unable to catalyze the transfer of N-acetylglucosamine from UDP-GlcNAc to [(14)C]GlcNAc(1)-PP-Dol, but did not impair the formation of GlcNAc(1)-PP-Dol or GlcNAc-GPI. Immunoprecipitating Alg13p from solubilized extracts resulted in the formation of GlcNAc(2)-PP-Dol but required Alg14p for activity, because an Alg13p immunoprecipitate obtained from cells in which ALG14 was down-regulated lacked this activity. In Western blot analysis it was demonstrated that Alg13p, for which no well defined transmembrane segment has been predicted, localizes both to the membrane and cytosol; the latter form, however, is enzymatically inactive. In contrast, Alg14p is exclusively membrane-bound. Repression of the ALG14 gene causes a depletion of Alg13p from the membrane. By affinity chromatography on IgG-Sepharose using Alg14-ZZ as bait, we demonstrate that Alg13-myc co-fractionates with Alg14-ZZ. The data suggest that Alg13p associates with Alg14p to a complex forming the active transferase catalyzing the biosynthesis of GlcNAc(2)-PP-Dol.

Blotting, Western↗

PubMed2004

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.

Genetic Diseases, Inborn↗

PubMed2004

Deficiency of GDP-Man:GlcNAc2-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 beta 1,4-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.

DNA Mutational Analysis↗

PubMed2003

The auxin-induced maize gene ZmSAUR2 encodes a short-lived nuclear protein expressed in elongating tissues.

By differential screening of a cDNA library from auxin-induced maize coleoptiles we have isolated and characterized a SAUR gene, designated ZmSAUR2, belonging to a not yet characterized subtype of the SAUR family. ZmSAUR2 encodes a 15.3-kDa protein and is specifically induced by auxin in elongating coleoptile tissue but not in primary leaves or in roots. The transcript level rapidly increased within minutes and preceded auxin-stimulated elongation of coleoptile segments. Cycloheximide also induced ZmSAUR2 transcription, as has been shown for other early auxin-induced genes, whereas abscisic acid, brassinolide, ethylene, gibberellic acid, kinetin, and methyl jasmonate did not provoke an increase in ZmSAUR2 mRNA abundance. In pulse-chase experiments using auxin-induced coleoptiles and an anti-ZmSAUR2 antibody we were able to precipitate a protein of the expected molecular mass and to determine a half-life of about 7 min, which is among the shortest known in eukaryotes. In gel shift assays binding of calmodulin to ZmSAUR2 was demonstrated, suggesting the possibility of post-transcriptional regulation. Upon transformation of onion epidermal cells with a ZmSAUR2::GUS construct the corresponding chimeric protein was detected in the nucleus. The results suggest that ZmSAUR2 encodes a short-lived nuclear protein that might be involved in auxin-mediated cell elongation.

Base Sequence↗

PubMed2003

A new type of congenital disorders of glycosylation (CDG-Ii) provides new insights into the early steps of dolichol-linked oligosaccharide biosynthesis.

Deficiency of GDP-Man:Man1GlcNAc2-PP-dolichol mannosyltransferase (hALG2), is the cause of a new type of congenital disorders of glycosylation (CDG) designated CDG-Ii. The patient presented normal at birth but developed in the 1st year of life a multisystemic disorder with mental retardation, seizures, coloboma of the iris, hypomyelination, hepatomegaly, and coagulation abnormalities. An accumulation of Man1GlcNAc2-PP-dolichol and Man2GlcNAc2-PP-dolichol was observed in skin fibroblasts of the patient. Incubation of patient fibroblast extracts with Man1GlcNAc2-PP-dolichol and GDP-mannose revealed a severely reduced activity of the mannosyltransferase elongating Man1GlcNAc2-PP dolichol. Because the Saccharomyces cerevisiae mutant alg2-1 was known to accumulate the same shortened dolichol-linked oligosaccharides as the patient, the yeast ALG2 sequence was used to identify the human ortholog. Genetic analysis revealed that the patient was heterozygous for a single nucleotide deletion and a single nucleotide substitution in the human ortholog of yeast ALG2. Expression of wild type but not of mutant hALG2 cDNA restored the mannosyltransferase activity and the biosynthesis of dolichol-linked oligosaccharides both in patient fibroblasts and in the alg2-1 yeast cells. hALG2 was shown to act as an alpha1,3-mannosyltransferase. The resulting Manalpha1,3-ManGlcNAc2-PP dolichol is further elongated by a yet unknown alpha1,6-mannosyltransferase.

Base Sequence↗

PubMed2003

Overexpression of GDP-mannose pyrophosphorylase in Saccharomyces cerevisiae corrects defects in dolichol-linked saccharide formation and protein glycosylation.

Thermosensitive mutants of Saccharomyces cerevisiae, affected in the endoplasmic reticulum (ER) located glycosylation, i.e. in Dol-P-Man synthase (dpm1), in beta-1,4 mannosyl transferase (alg1) and in alpha-1,3 mannosyltransferase (alg2), were used to assess the role of GDP-Man availability for the synthesis of dolichol-linked saccharides. The mutants were transformed with the yeast gene MPG1 (PSA1/VIG9) encoding GDP-Man pyrophosphorylase catalyzing the final step of GDP-Man formation. We found that overexpression of MPG1 allows growth at non-permissive temperature and leads to an increase in the cellular content of GDP-Man. In the alg1 and alg2 mutants, complemented with MPG1 gene, N-glycosylation of invertase was in part restored, to a degree comparable to that of the wild-type control. In the dpm1 mutant, the glycosylation reactions that depend on the formation of Dol-P-Man, i.e. elongation of Man(5)GlcNAc(2)-PP-Dol, O-mannosylation of chitinase and synthesis of GPI anchor were normal when MPG1 was overexpressed. Our data indicate that an increased level of GDP-Man is able to correct defects in mannosylation reactions ascribed to the ER and to the Golgi.

Blotting, Northern↗

PubMed2002

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.

Chromatography, High Pressure Liquid↗