Robertson, Alexis; (2000) Structural and functional studies of anosmin-1, the protein disrupted in x-linked Kallmann's syndrome. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

X-linked Kallmann's syndrome, a disease characterised by anosmia (a consequence of hypoplastic/absent olfactory bulbs) and hypogonadotrophic hypogonadism (due to a defect of GnRH neuron migration into the hypothalamus) is caused by mutations in the KAL gene. Anosmin-1, the protein product of KAL, is extracellular and consists of a cysteine rich C-terminal region, followed by four fibronectin type III (Fnlll) domains. It has been observed in vivo as a 100 kDa glycosylated protein and as additional cleaved 55 kDa and 45 kDa fragments in vitro. Its overall function and domain structure is unknown, but current data suggests a role in neuronal and kidney development. In this thesis: 1. A Two Hybrid assay using a foetal brain cDNA library was utilised to study potential protein/protein interactions involving anosmin-1. Although recombinant anosmin-1 was produced, no positive interactors were identified. 2. Computer comparative modelling methodologies were used to study the potential three dimensional conformation of domains of the mature protein and the nature of amino acid substitutions encoded for by three known missense mutations. Two were shown to occur at buried locations and are predicted to affect the domain structure, whilst the third occurs at an exposed position, identifying this region as potentially biologically important. 3. A potential cleavage site (KKKRRK) located within the second Fnlll was studied using a mutant anosmin-1 lacking this site. Cleavage of this protein was studied and compared to that of the wild type. The results exclude this protein motif as the site of this cleavage. 4. A cell adhesion bioassay was developed to compare a wild type and mutated peptide (containing the N267K amino acid substitution) generated from the first Fnlll domain sequence. An adhesive property for both peptides was observed, and significantly, the mutant showed no loss of function when compared with the wild type.

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