Anosmin-1, a protein defective in X-linked Kallmann's syndrome (X-KS), presents a structure comprising a cysteine rich N terminus (CR), a whey acid protein (WAP) domain followed by four fibronectin-like III domains (FnIII); and its function is heparan sulphate (HS) dependent. However, anosmin-1 and HS binding affinity is unknown. Although the WAP domain belongs to a protein family demonstrating serine protease inhibitory activity, the interacting serine protease remains to be identified. This study aimed to investigate: a direct interaction between anosmin-1 and HS; and to screen anosmin-1's function on urokinase-type plasminogen activator (uPA) and thrombin amidolytic activity. We have used as a model a human prostatic carcinoma (PC-3) cell line .
Proteins containing N-terminus backbone (PIW) alone and variable numbers of FnIII repeats (PIWF1, PIWF2, PIWF3 and PIWF4) were generated. HS competition assay, ELISA and surface plasmon resonance were used for anosmin-1-HS interaction. Thrombin and uPA amydolityc activities were measured using specific chromogenic substrates after PIWF1, PIWF4 and HS incubation. PC-3 proliferation was quantified by MTT assay. Immunoprecipitation (IP) was performed by incubation of PC-3 lysate with PIWF1, using anti-uPA or anti-PIWF1 antibodies.
Co-incubation with HS showed higher displacement efficiency for PIW protein, suggesting a low HS binding affinity for the N-terminus. PIWF1 and PIWF4 showed dose-dependent HS-binding, the highest affinity demonstrable with PIWF4. These observations are consistent with the dissociation constant (Kd<10nM). PIWF1 and PIWF4 treatments induced a significant enhancement on uPA but not on thrombin activity, independent of HS. PIWF1 and PIWF4 produced a biphasic effect on PC-3 proliferation; effects neutralized by heparinase III, HS and anti-uPA-Ab. IP confirmed the binding complex formed by PIWF1 and uPA from PC-3 lysate.
FnIII mediate high affinity HS binding, whereas N-terminus is likely to regulate uPA activity. The demonstration of cross-talk between anosmin-1, HS and uPA have shed novel insights into the molecular pathogenesis of X-KS.
22 - 24 Mar 2004
British Endocrine Societies