Functional and structural studies of anosmin-1, the protein implicated in X-linked Kallmann's syndrome
Kallmann's syndrome (KS) is characterised by the association of anosmia (a loss of sense of smell) and hypogonadotrophic hypogonadism. Defects of anosmin-1, a protein encoded by KAL-1, underlie the X-linked inherited form of KS. Anosmin-1 comprises an N-terminal cysteine rich domain and a whey acidic protein like (WAP) domain, followed by four consecutive fibronectin type HI (Fnlll) like repeats. In vivo and in vitro studies revealed that anosmin-1 functions as an adherent extracellular matrix protein involved in cell adhesion and neurite outgrowth, actions dependent on an interaction of Fnin domains with heparan sulphate (HS). However, the binding characteristics and kinetics between C-terminal Fnin domains of anosmin-1 and HS have not hitherto been systematically investigated. Although anosmin-1's N-terminal WAP domain shares homology with serine protease inhibitors, its putative protease ligand is still unknown. In this thesis: 1. A Drosophila S2 cell expression system was used to express full length and truncated domain specific versions of anosmin-1. The full-length (PIWF4), truncated (PIWF1, with single FnlH-1 domain) protein, and their C172R WAP domain mutants were purified using Ni-NTA affinity chromatography. The high yield of PIWF1 protein enables the generation of an anti-anosmin-1 antibody. 2. A HS competitive assay showed that anosmin-1 was S2 cell surface associated through a Fnin domain-HS interaction. A solid phase ELISA assay indicated purified anosmin-1 bound to HS in a dose-dependent manner. Surface plasmon resonance (SPR) revealed real-time kinetic interactions with K& = 2 nM.