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.
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 L1. 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 WAP domains based on known NMR and crystal structures. Three missense mutation-encoded amino 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 microg/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.
Robertson
A
A
Department of Medicine, Royal Free Campus, Royal Free and University College Medical School, University College London, Rowland Hill Street, London NW3 2PF, UK.
MacColl
G S
GS
Nash
J A
JA
Boehm
M K
MK
Perkins
S J
SJ
Bouloux
P M
PM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002448:Cell Adhesion / Q000502:physiology; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D016326:Extracellular Matrix Proteins; D005353:Fibronectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism; D006801:Humans; D008894:Milk Proteins / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
D016326:Extracellular Matrix Proteins; D005353:Fibronectins; D006497:Heparan Sulfate; D008894:Milk Proteins; D009419:Nerve Tissue Proteins; D010455:Peptides; C076266:ANOS1 protein, human; C024471:whey acidic proteins