Endocrine Abstracts

Introduction: Loss of anosmin-1 function underlies the pathogenesis of X-linked Kallmann's syndrome (X-KS), a disorder characterized by anosmia (loss of smell) and hypogonadotrophic hypogonadism, due to olfactory bulb (OB) dysgenesis and failed migration of gonadotrophin releasing hormone (GnRH) neurons. Additional phenotypic features include bimanual synkinesis and unilateral renal agenesis. Anosmin-1 contains a whey acidic protein-like (WAP) domain and four contiguous fibronectin-like type III (FnIII) repeats; its WAP domain contains eight highly conserved cysteine residues forming four intramolecular disulphide bonds. This four-disulphide core motif is common to WAP protein family mermbers, which generally function as serine protease inhibitors. Anosmin-1 and urokinase-type plasminogen activator (uPA) expression is detected in developing OB. Furthermore, uPA has been reported to influence cell proliferation, tissue remodelling, and neurite outgrowth. In this study, uPA was thus investigated as a putative serine protease candidate ligand for anosmin-1.

Methods:

The effect of anosmin-1 on thrombin and uPA amidolytic activity was investigated using a specific chromogenic substrate. The direct binding of anosmin-1 to uPA and their kinetic interactions were investigated by surface plasmon resonance (SPR). The functional consequences of co-operation of anosmin-1/uPA were investigated on PC-3 cell proliferation. Co-immunoprecipitation was performed in PC-3 cell lysates.

Results:

Anosmin-1 unexpectedly and significantly enhanced uPA amidolytic activity, but was without effect on thrombin. Direct high affinity binding of these molecules (Kd = 6.91 nM) was demonstrated by SPR. In PC-3 cells, anosmin-1/uPA cooperation induced cell proliferation, and complex formation between these two proteins was demonstrated by co-immunoprecipitation from PC-3 cell lysates.

Conclusion:

uPA constitutes a novel ligand for anosmin-1. Together with previous observations that C-terminal FnIII domain of anosmin-1 binds to heparan sulphate with a high affinity (Kd = 2nM), we can propose that anosmin-1 modulates uPA's catalytic activity and its signaling pathway, whereas HS determines cell surface localization of the anosmin-1/uPA complex.