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Molecular Microbiology Sep 2006Type-1 fimbriae are important virulence factors for the establishment of Escherichia coli urinary tract infections. Bacterial adhesion to the high-mannosylated uroplakin...
Type-1 fimbriae are important virulence factors for the establishment of Escherichia coli urinary tract infections. Bacterial adhesion to the high-mannosylated uroplakin Ia glycoprotein receptors of bladder epithelium is mediated by the FimH adhesin. Previous studies have attributed differences in mannose-sensitive adhesion phenotypes between faecal and uropathogenic E. coli to sequence variation in the FimH receptor-binding domain. We find that FimH variants from uropathogenic, faecal and enterohaemorrhagic isolates express the same specificities and affinities for high-mannose structures. The only exceptions are FimHs from O157 strains that carry a mutation (Asn135Lys) in the mannose-binding pocket that abolishes all binding. A high-mannose microarray shows that all substructures are bound by FimH and that the largest oligomannose is not necessarily the best binder. Affinity measurements demonstrate a strong preference towards oligomannosides exposing Manalpha1-3Man at their non-reducing end. Binding is further enhanced by the beta1-4-linkage to GlcNAc, where binding is 100-fold better than that of alpha-d-mannose. Manalpha1-3Manbeta1-4GlcNAc, a major oligosaccharide present in the urine of alpha-mannosidosis patients, thus constitutes a well-defined FimH epitope. Differences in affinities for high-mannose structures are at least 10-fold larger than differences in numbers of adherent bacteria between faecal and uropathogenic strains. Our results imply that the carbohydrate expression profile of targeted host tissues and of natural inhibitors in urine, such as Tamm-Horsfall protein, are stronger determinants of adhesion than FimH variation.
Topics: Adhesins, Escherichia coli; Amino Acid Sequence; Bacterial Adhesion; Carbohydrate Sequence; Escherichia coli; Fimbriae Proteins; Fimbriae, Bacterial; Hemagglutination; Mannose; Mannosides; Microarray Analysis; Molecular Sequence Data; Mutation; Protein Conformation
PubMed: 16930149
DOI: 10.1111/j.1365-2958.2006.05352.x -
The Journal of Neuroscience : the... Jul 2005Mice with alpha-mannosidase gene inactivation provide an experimental model for alpha-mannosidosis, a lysosomal storage disease with severe neuropsychological and...
Neurocognitive and psychotiform behavioral alterations and enhanced hippocampal long-term potentiation in transgenic mice displaying neuropathological features of human alpha-mannosidosis.
Mice with alpha-mannosidase gene inactivation provide an experimental model for alpha-mannosidosis, a lysosomal storage disease with severe neuropsychological and psychopathological complications. Neurohistological alterations in these mice were similar to those in patients and included vacuolations and axonal spheroids in the CNS and peripheral nervous system. Vacuolation was most prominent and evenly distributed in neuronal perikarya of the hippocampal CA2 and CA3 regions, whereas CA1 and dentate gyrus were weakly or not affected. Field potential recordings from CA1 region in hippocampal slices showed enhanced theta burst-induced long-term potentiation (LTP) in alpha-mannosidase-deficient mice. Longitudinal assessment in age-matched alpha-mannosidase-deficient and wild-type littermates, using an extended test battery, demonstrated a neurocognitive and psychotiform profile that may relate to the psychopathological alterations in clinical alpha-mannosidosis. Brainstem auditory-evoked potentials and basic neuromotor abilities were not impaired and did not deteriorate with age. Exploratory and conflict tests revealed consistent decreases in exploratory activity and emotional blunting in the knock-out group. alpha-Mannosidosis mice were also impaired in aversively motivated learning and acquisition of signal-shock associations. Acquisition and reversal learning in the water maze task, passive avoidance learning in the step-through procedure, as well as emotional response conditioning in an operant procedure were all impaired. Acquisition or shaping of an appetitive instrumental conditioning task was unchanged. Appetitive odor discrimination learning was only marginally impaired during shaping, whereas both the discrimination and reversal subtasks were normal. We propose that prominent storage and enhanced LTP in hippocampus have contributed to these specific behavioral alterations in alpha-mannosidase-deficient mice.
Topics: Animals; Appetitive Behavior; Avoidance Learning; Behavior, Animal; Conditioning, Operant; Discrimination, Psychological; Disease Models, Animal; Emotions; Evoked Potentials, Auditory, Brain Stem; Excitatory Postsynaptic Potentials; Exploratory Behavior; Female; Hand Strength; Hippocampus; Humans; Learning Disabilities; Long-Term Potentiation; Lysosomes; Maze Learning; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Nervous System; Neurons; Psychomotor Performance; Smell; Vacuoles; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 16014715
DOI: 10.1523/JNEUROSCI.0283-05.2005 -
Intracellular transport of human lysosomal alpha-mannosidase and alpha-mannosidosis-related mutants.The Biochemical Journal Jul 2004Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a...
Human LAMAN (lysosomal a-mannosidase) was synthesized as a 120 kDa precursor in transfected COS cells [African-green-monkey kidney cells], which was partly secreted as a single-chain form and partly sorted to the lysosomes being subsequently cleaved into three peptides of 70, 40 and 15 kDa respectively. Both the secreted and the lysosomal forms contained endo H (endoglucosidase H)-resistant glycans, suggesting a common pathway through the trans-Golgi network. A fraction of LAMAN was retained intracellularly as a single-chain endo H-sensitive form, probably in the ER (endoplasmic reticulum). The inherited lack of LAMAN causes the autosomal recessive storage disease a-mannosidosis. To understand the biochemical consequences of the disease-causing mutations, 11 missense mutations and two in-frame deletions were introduced into human LAMAN cDNA by in vitro mutagenesis and the resulting proteins were expressed in COS cells. Some selected mutants were also expressed in Chinese-hamster ovary cells. T355P (Thr355Pro), P356R, W714R, R750W and L809P LAMANs as well as both deletion mutants were misfolded and arrested in the ER as inactive single-chain forms. Six of the mutants were transported to the lysosomes, either with less than 5% of normal specific activity (H72L, D196E/N and R220H LAMANs) or with more than 30% of normal specific activity (E402K LAMAN). F320L LAMAN resulted in much lower activity in Chinese-hamster ovary cells when compared with COS cells. Modelling into the three-dimensional structure revealed that the mutants with highly reduced specific activities contained substitutions of amino acids involved in the catalysis, either co-ordinating Zn2+ (His72 and Asp196), stabilizing the active-site nucleophile (Arg220) or positioning the active-site residue Asp319 (Phe320).
Topics: Animals; CHO Cells; COS Cells; Cattle; Cell Line; Chlorocebus aethiops; Cricetinae; Genotype; Isoenzymes; Lysosomes; Mannosidases; Models, Molecular; Mutagenesis, Site-Directed; Phenotype; Protein Structure, Quaternary; Protein Transport; Transfection; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 15035660
DOI: 10.1042/BJ20031499 -
Biochimica Et Biophysica Acta Mar 2002alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of the lysosomal alpha-mannosidase. We report here the sequencing and expression of the... (Comparative Study)
Comparative Study
alpha-Mannosidosis is a lysosomal storage disorder caused by deficient activity of the lysosomal alpha-mannosidase. We report here the sequencing and expression of the lysosomal alpha-mannosidase cDNA from normal and alpha-mannosidosis guinea pigs. The amino acid sequence of the guinea pig enzyme displayed 82-85% identity to the lysosomal alpha-mannosidase in other mammals. The cDNA of the alpha-mannosidosis guinea pig contained a missense mutation, 679C>T, leading to substitution of arginine by tryptophan at amino acid position 227 (R227W). The R227W allele segregated with the alpha-mannosidosis genotype in the guinea pig colony and introduction of R227W into the wild-type sequence eliminated the production of recombinant alpha-mannosidase activity in heterologous expression studies. Furthermore, the guinea pig mutation has been found in human patients. Our results strongly indicate that the 679C>T mutation causes alpha-mannosidosis and suggest that the guinea pig will be an excellent model for investigation of pathogenesis and evaluation of therapeutic strategies for human alpha-mannosidosis.
Topics: Amino Acid Sequence; Animals; Base Sequence; Binding Sites; COS Cells; Cloning, Molecular; DNA, Complementary; Disease Models, Animal; Genetic Therapy; Guinea Pigs; Mannosidases; Molecular Sequence Data; Mutation, Missense; Sequence Alignment; Species Specificity; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 11959458
DOI: 10.1016/s0925-4439(01)00081-3 -
Biochimica Et Biophysica Acta Oct 1999Glycoproteinoses belong to the lysosomal storage disorders group. The common feature of these diseases is the deficiency of a lysosomal protein that is part of glycan... (Review)
Review
Glycoproteinoses belong to the lysosomal storage disorders group. The common feature of these diseases is the deficiency of a lysosomal protein that is part of glycan catabolism. Most of the lysosomal enzymes involved in the hydrolysis of glycoprotein carbohydrate chains are exo-glycosidases, which stepwise remove terminal monosaccharides. Thus, the deficiency of a single enzyme causes the blockage of the entire pathway and induces a storage of incompletely degraded substances inside the lysosome. Different mutations may be observed in a single disease and in all cases account for the nonexpression of lysosomal glycosidase activity. Different clinical phenotypes generally characterize a specific disorder, which rather must be described as a continuum in severity, suggesting that other biochemical or environmental factors influence the course of the disease. This review provides details on clinical features, genotype-phenotype correlations, enzymology and biochemical storage of four human glycoprotein lysosomal storage disorders, respectively alpha- and beta-mannosidosis, fucosidosis and alpha-N-acetylgalactosaminidase deficiency. Moreover, several animal disorders of glycoprotein metabolism have been found and constitute valuable models for the understanding of their human counterparts.
Topics: Animals; Carbohydrate Sequence; Congenital Disorders of Glycosylation; Disease Models, Animal; Fucosidosis; Glycoside Hydrolases; Hexosaminidases; Humans; Molecular Sequence Data; Phenotype; alpha-Mannosidosis; alpha-N-Acetylgalactosaminidase
PubMed: 10571005
DOI: 10.1016/s0925-4439(99)00077-0 -
American Journal of Human Genetics Jan 1999alpha-Mannosidosis is an autosomal recessive disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). The resulting intracellular accumulation of...
alpha-Mannosidosis is an autosomal recessive disorder caused by deficiency of lysosomal alpha-mannosidase (LAMAN). The resulting intracellular accumulation of mannose-containing oligosaccharides leads to mental retardation, hearing impairment, skeletal changes, and immunodeficiency. Recently, we reported the first alpha-mannosidosis-causing mutation affecting two Palestinian siblings. In the present study 21 novel mutations and four polymorphic amino acid positions were identified by the screening of 43 patients, from 39 families, mainly of European origin. Disease-causing mutations were identified in 72% of the alleles and included eight splicing, six missense, and three nonsense mutations, as well as two small insertions and two small deletions. In addition, Southern blot analysis indicated rearrangements in some alleles. Most mutations were private or occurred in two or three families, except for a missense mutation resulting in an R750W substitution. This mutation was found in 13 patients, from different European countries, and accounted for 21% of the disease alleles. Although there were clinical variations among the patients, no significant LAMAN activity could be detected in any of the fibroblast cultures. In addition, no correlation between the types of mutations and the clinical manifestations was evident.
Topics: DNA Mutational Analysis; Fibroblasts; Humans; Mannosidases; Molecular Sequence Data; Mutagenesis; Mutagenesis, Insertional; Mutation, Missense; Polymerase Chain Reaction; Polymorphism, Genetic; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 9915946
DOI: 10.1086/302183 -
American Journal of Human Genetics Oct 1998alpha-Mannosidosis is an autosomal recessive lysosomal-storage disorder caused by a deficiency of lysosomal alpha-mannosidase activity. This disease shows a wide range...
alpha-Mannosidosis is an autosomal recessive lysosomal-storage disorder caused by a deficiency of lysosomal alpha-mannosidase activity. This disease shows a wide range of clinical phenotypes, from a severe, infantile form (type I), which is fatal at <3-8 years of age, to a less severe, late-onset form (type II), which ultimately may involve hearing loss, coarse face, mental retardation, and hepatosplenomegaly. To elucidate the molecular mechanism underlying this disease in both types of patients, we have used PCR, followed by either SSCP analysis or direct sequencing, to analyze the 24 exons and intron/exon boundaries of the alpha-mannosidase gene (MANB) from five patients. Two amino acid substitutions-H72L and R750W, in exons 2 and 18, respectively-and two nonsense mutations-Q639X and R760X, in exons 15 and 19, respectively-were identified in four type II patients. One amino acid substitution, P356R, was identified in exon 8 from a type I patient. This patient and three of the type II patients were homozygous for their mutations (H72L, P356R, R750W, and R760X) and one type II patient was heterozygous for the Q639X and R750W mutations. Transfection experiments of COS 7 cells, using the alpha-mannosidase cDNA containing one of the missense mutations-H72L, P356R, or R750W-revealed that each of these mutations dramatically reduces the enzymatic activity of alpha-mannosidase. These data demonstrate that widely heterogeneous missense or nonsense mutations of the MANB gene are the molecular basis underlying alpha-mannosidosis.
Topics: Child; Child, Preschool; Codon, Nonsense; DNA Primers; Female; Genetic Heterogeneity; Humans; Infant; Lysosomes; Male; Mannosidases; Middle Aged; Mutation; Mutation, Missense; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Recombinant Proteins; Sequence Analysis, DNA; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 9758606
DOI: 10.1086/302048 -
The Biochemical Journal Dec 1997alpha-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal alpha-mannosidase. Feline alpha-mannosidosis is a well-characterized...
alpha-Mannosidosis is a lysosomal storage disorder that is caused by the deficiency of lysosomal alpha-mannosidase. Feline alpha-mannosidosis is a well-characterized animal model used for studying pathological and therapeutic aspects of lysosomal storage disorders. We here report the purification of feline liver lysosomal alpha-mannosidase and determination of its cDNA sequence. The active enzyme consisted of three polypeptides, with molecular masses of 72, 41 and 12 kDa, joined by non-covalent forces. The cDNA sequence of feline lysosomal alpha-mannosidase was determined from reverse transcriptase PCR products obtained from skin fibroblast mRNA. The deduced amino acid sequence contained the N-terminal sequences of the 72 and 41 kDa peptides. This indicated that the enzyme is synthesized as a single-chain precursor with a putative signal peptide of 50 amino acids followed by a polypeptide chain of 957 amino acids, which is cleaved into the three polypeptides of the mature enzyme. The deduced amino acid sequence was 81.1 and 83.2% identical with the human and bovine lysosomal alpha-mannosidases sequences respectively. A 4 bp deletion was identified in an alpha-mannosidosis-affected Persian cat by DNA sequencing of reverse transcriptase PCR products. The deletion resulted in a frame shift from codon 583 and premature termination at codon 645. No lysosomal alpha-mannosidase activity could be detected in the liver of this cat. A domestic long-haired cat expressing a milder alpha-mannosidosis phenotype than the Persian cat had a lysosomal alpha-mannosidase activity of 2% of normal. This domestic long-haired cat did not possess the 4 bp deletion, proving molecular heterogeneity for feline alpha-mannosidosis.
Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Cats; Cattle; Cloning, Molecular; Codon; DNA Mutational Analysis; DNA, Complementary; Disease Models, Animal; Frameshift Mutation; Humans; Liver; Lysosomes; Mannosidases; Molecular Sequence Data; Mutation; Protein Conformation; Protein Processing, Post-Translational; Sequence Deletion; Sequence Homology, Nucleic Acid; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 9396732
DOI: 10.1042/bj3280863 -
European Journal of Biochemistry Jun 1997Bovine kidney lysosomal alpha-mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 16 kb and its...
Bovine kidney lysosomal alpha-mannosidase was purified to homogeneity and the gene was cloned. The gene was organized in 24 exons that spanned 16 kb and its corresponding cDNA contained an open reading frame of 2997 bp beginning from a putative ATG start codon. The deduced amino acid sequence contained a signal peptide of 50 amino acids adjacent to a protein sequence of 949 amino acids that was cleaved into five peptides in the mature enzyme; starting with the peptide derived from the N-terminal part of this precursor, their molecular masses were 35/38 (peptide a), 11/13 (peptide b), 22 (peptide c), 38 (peptide d) and 13/15 kDa (peptide e). Variation in the degree of N-glycosylation accounts for molecular mass heterogeneities of peptides a, b and e. Peptides a, b and c were disulphide-linked. A T961-->C transition, resulting in Phe321-->Leu substitution, was identified in the cDNA of alpha-mannosidosis-affected Angus cattle. In affected Galloway cattle, a G662-->A transition that causes Arg221-->His substitution was identified. Phe321 and Arg221 are conserved among the alpha-mannosidase class-2 family, indicating that the substitutions resulted from disease-causing mutations in these breeds.
Topics: Amino Acid Sequence; Animals; Base Sequence; Cattle; Chromatography, Gel; Chromatography, Ion Exchange; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Exons; Mannosidases; Molecular Sequence Data; Mutation; Sequence Homology, Amino Acid; Species Specificity; alpha-Mannosidase; alpha-Mannosidosis
PubMed: 9208932
DOI: 10.1111/j.1432-1033.1997.00410.x -
The Journal of Biological Chemistry Nov 1996We have cloned and expressed two cDNAs encoding the human lysosomal alpha-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the...
We have cloned and expressed two cDNAs encoding the human lysosomal alpha-mannosidase (EC 3.2.1.24) by RT-PCR of human spleen mRNA. This enzyme is required for the degradation of N-linked carbohydrates during glycoprotein catabolism in eucaryotic cells. The shorter of the two cDNAs (3 kilobases (kb)) was found to encode an open reading frame of 2964 base pairs and, when expressed in Pichia pastoris, was found to encode an enzyme that could cleave high mannose oligosaccharides, oligosaccharides isolated from alpha-mannosidosis fibroblasts, and p-nitrophenyl-alpha-D-mannopyranoside substrates. In addition, the Pichia-expressed enzyme was inhibited by swainsonine, and had a pH optimum, Km, and Vmax characteristic of the enzyme purified previously from human liver. The second, larger RT-PCR product (3.6 kb) was found to contain an insertion and a deletion relative to the 3-kb spleen amplimer and encoded a truncated coding region, indicating that it resulted from alternate transcript splicing. No alpha-mannosidase activity could be detected in Pichia transformants containing this coding region, indicating that it did not encode a functional enzyme. Antiserum raised to the recombinant product of the 3-kb alpha-mannosidase cDNA immunoprecipitated lysosomal alpha-mannosidase activity from human fibroblast extracts. Northern blots identified a 3-kb RNA transcript in all human tissues tested, including alpha-mannosidosis fibroblasts, while minor transcripts of 3.6 kb were also present in several adult tissues. Human chromosome mapping of the mannosidase gene confirmed that the functional gene maps to the MANB locus on chromosome 19. Sequence comparisons were made to previously published human cDNA sequences encoding a putative lysosomal alpha-mannosidase (Nebes, V. L., and Schmidt, M. C. (1994) Biochem. Biophys. Res. Commun. 200, 239-245) and several differences were found relative to the functional lysosomal alpha-mannosidase encoded by the 3-kb spleen cDNA.
Topics: Adult; Amino Acid Sequence; Animals; Cloning, Molecular; Cricetinae; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation, Enzymologic; Humans; Lysosomes; Mannosidases; Molecular Sequence Data; Pichia; Polymerase Chain Reaction; RNA, Messenger; Sequence Alignment; Spleen; alpha-Mannosidase
PubMed: 8910458
DOI: 10.1074/jbc.271.45.28348