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Publication Detail
Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1
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Publication Type:Journal article
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Publication Sub Type:Article
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Authors:Robertson A, MacColl GS, Nash JAB, Boehm MK, Perkins SJ, Bouloux PMG
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Publication date:2001
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Pagination:647, 659
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Journal:Biochemical Journal
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Volume:357
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Issue:Pt 3
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Print ISSN:0264-6021
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Keywords:Anosmin-1, Fibronectin type III, molecular modelling, ACID, Adhesion, adhesion molecule, Adhesion molecules, ADJACENT, Affect, amino acid, Amino Acid Motifs, Amino Acid Sequence, Amino Acid Substitution, Anosmin-1, As, Basement Membrane, BASEMENT-MEMBRANE, BINDING, C terminal, Canine, cell, Cell Adhesion, Cell Adhesion Molecule, cell adhesion molecules, Cell Line, Cell lines, CELL-ADHESION, CELL-LINE, CELL-LINES, CELLS, Cells, Cultured, Cellular, chemistry, CLEAVAGE, CLUSTER, CLUSTERS, CRYSTAL, CRYSTAL STRUCTURE, crystal structures, CRYSTAL-STRUCTURE, CRYSTAL-STRUCTURES, development, DNA Mutational Analysis, domain, DOMAINS, enhanced, epithelial, epithelial cell, Epithelial Cells, EPITHELIAL-CELLS, experimental, experimental study, expression, extracellular, face, Fibronectin, Fibronectins, function, functional, GENE, GENE PRODUCT, GENE-PRODUCT, genetics, Heparitin Sulfate, IM, INCREASE, INDICATE, insect, Interstitial, Kallmann's syndrome, Kidney, L1, LA, LINE, LINES, lysine, maps, MATRIX, May, membrane, metabolism, milk, Milk Proteins, MISSENSE MUTATION, missense mutations, modelling, Models, Molecular, Molecular, Molecular Sequence Data, Molecule, MOLECULES, MOTIFS, Mutant, MUTANTS, Mutation, Missense, MUTATIONS, N-terminal, nerve, Nerve Tissue Proteins, neural, neurons, NMR, ORDER, PATHOGENESIS, peptide, Peptides, PHYSIOLOGICAL, physiology, POSITION, product, PROTEIN, Protein Conformation, Protein Expression, Protein Structure, Tertiary, Proteins, rat, recombinant, REGION, Removal, Result, secretion, SEQUENCE, Sequence Homology, Amino Acid, SEQUENCES, SITE, SITES, structural, Structure, SUBSTITUTION, SULFATE, Support, Non-U.S.Gov't, SURFACE, surfaces, Syndrome, target, targets, Tissue, Tissues, WILD-TYPE, X-linked, 2.0 ANGSTROM, ACID, ANGSTROM RESOLUTION, BASIC RESIDUES, beta, Candidate Gene, CELL-ADHESION MOLECULES, CRYSTAL- STRUCTURE, DROSOPHILA-MELANOGASTER, electrostatic maps, English, EXTRACELLULAR DOMAIN, heparan sulphate, HEPARIN-BINDING, HUMAN TISSUE FACTOR, INSECT CELLS, LINKED KALLMANN-SYNDROME, multidomain proteins, PLACE, PRESS, Sequence Alignment
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Author URL:
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Notes:Journal English Article PORTLAND PRESS AUG 1 462DQ LONDON BIOCHEM J 59 PORTLAND PLACE, LONDON W1N 3AJ, ENGLAND
Abstract
Anosmin-1, the gene product of the KAL gene, is implicated in the pathogenesis of X-linked Kallmann's syndrome. Anosmin-1 protein expression is restricted to the basement membrane and interstitial matrix of tissues affected in this syndrome during development. The anosmin-1 sequence indicates an N-terminal cysteine-rich domain, a whey acidic protein (WAP) domain, four fibronectin type III (FnIII) domains and a C-terminal histidine-rich region, and shows similarity with cell-adhesion molecules, such as neural cell-adhesion molecule, TAG-1 and Ll. We investigated the structural and functional significance of three loss-of-function missense mutations of anosmin-1 using comparative modelling of the four FnIII and the WA-P domains based on known NMR and crystal structures. Three missense mutation-encoded ani-ino acid substitutions, N267K, E514K and F517L, were mapped to structurally defined positions on the GFCC ' beta -sheet face of the first and third FnIII domains. Electrostatic maps demonstrated large basic surfaces containing clusters of conserved predicted heparan sulphate-binding residues adjacent to these mutation sites. To examine these modelling results anosmin-1 was expressed in insect cells. The incorporation of the three mutations into recombinant anosmin-1 had no effect on its secretion. The removal of two dibasic motifs that may constitute potential physiological cleavage sites for anosmin-1 had no effect on cleavage. Peptides based on the anosmin-1 sequences R254-K285 and P504-K527 were then synthesized in order to assess the effect of the three mutations on cellular adhesion, using cell lines that represented potential functional targets of anosmin-1. Peptides (10 mug/ml) incorporating the N267K and E514K substitutions promoted enhanced adhesion to 13.S.1.24 rat olfactory epithelial cells and canine MDCK1 kidney epithelial cells (P < 0.01) compared with the wild-type peptides. This result was attributed to the introduction of a lysine residue adjacent to the large basic surfaces. We predict that two of the three missense mutants increase the binding of anosmin-1 to an extracellular target, possibly by enhancing heparan sulphate binding, and that this critically affects the function of anosmin-1
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