Molecular modelling and experimental studies of mutation and cell-adhesionsites in the fibronectin type III and whey acidic protein domains of humananosmin-1

Anosmin-1, the gene product of the KAL gene, is implicated in the pathogene sis of X-linked Kallmann's syndrome. Anosmin-1 protein expression is restri cted to the basement membrane and interstitial matrix of tissues affected i n this syndrome during development. The anosmin-1 sequence indicates an N-t erminal cysteine-rich domain, a whey acidic protein (WAP) domain, four fibr onectin type III (FnIII) domains and a C-terminal histidine-rich region, an d shows similarity with cell-adhesion molecules, such as neural cell-adhesi on molecule, TAG-1 and Ll. We investigated the structural and functional si gnificance 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 s ubstitutions, N267K, E514K and F517L, were mapped to structurally defined p ositions on the GFCC ' beta -sheet face of the first and third FnIII domain s. 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 motif s that may constitute potential physiological cleavage sites for anosmin-1 had no effect on cleavage. Peptides based on the anosmin-1 sequences R254-K 285 and P504-K527 were then synthesized in order to assess the effect of th e three mutations on cellular adhesion, using cell lines that represented p otential functional targets of anosmin-1. Peptides (10 mug/ml) incorporatin g 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 t o 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 sulpha te binding, and that this critically affects the function of anosmin-1.