An Insight Into Biological Function Of A Highly Conserved Carboxyl-terminal Domain Of Indian Hedgehog N-fragment

Indian hedgehog(Ihh) is a highly conserved secreted signaling protein involved in regulating chondrogenic differention and proliferation. Ihh is synthesized as a precursor that undergoesautoprocessing to generate a highly conserved N-terminal domain(IhhN), which is responsible for the signaling activities of themolecule and aC-terminal domain(IhhC)implicated in the autoproteolysis reaction. During this cleavage, the carboxyl-terminal domain of IhhN is modified by covalent addition of cholestrol(IhhNp) followed by a second lipid,palmitate to its amino terminus. These two lipids play important roles in localizing Ihh to membrane and the freely difusible multimeric formation and biological potency. The long-range effectsof Ihhare facilitated by large, stable multimers(s-Ihh Np). The IhhN product triggers Ihh pathway activation bybinding to Patched(Ptc), primary receptor of the Ihh signaling pathway.Ihh has two structurally and functionally related hedgehog homologous(Sonic Hedgehog and Desert Hedgehog) which are highly conserved in aminoacid sequence. The N-terminal domain even exhibits ~80% conservation across various homologs. Recently, there is much data to support mechanisms and functions of lipid modification, functions of the highly conserved N-terminal, structure of Hh protein as well as multimeric form in long-range signaling. However, there has been little evidence to date for the functions of the carboxyl terminus of HhN which is quite near the autoprocessing site and highly conserved cross various homologs. Taken together, it indicates that the carboxyl terminus of HhN plays a key role in biological functions.In this study, eukaryotic constructs carrying different truncations on the carboxyl terminus of HhN were generated and then transfected into ECHO cell line. Consequently, western blot analysis for the whole cell lysate indicated that IhhN was unstable and easily degraded when six amino acids,labeled from S195 to T200,were truncated. In addition, autoprocessing was disrupted when ten amino acids including from K191 to T200 was deleted. On this basis, we further constructed various site mutations in the domain mentioned above and demonstrated that T200 contributes to the stability of IhhN fragment. Furthermore, we validatedthat four amino acids including K191, S192, E193 and H194, acting as a domain, were essential for Ihh autoprocessing. We found that the influence on Shh washighly conserved. These new findings may provide a fresh point of view to perceive the mechanisms of Hedgehog autoprocessing.Three-dimensional structure of IhhN protein showed that at least three amino acids(S195,A196 and A197) on the highly conserved carboxyl terminal domain of IhhN interacted with the other IhhN molecule which indicated that this domain were important for s-IhhNp(multimeric Ihh form) formation. Consequently, we screened stable ECHO cell line that expressed Ihh-SAA gene( delete S195,A196 and A197) to study s-IhhNp formation of this mutant. Gel filtration chromatography of supernatant collected from stable ECHO line above showed a significant reduction in s-IhhNp formation when S195,A196 and A197 were deleted. Thus, we proved that S195, A196 and A197 were important for the s-IhhNp formation.Taken together, we may safely come to a conclusion that the highly conserved carboxyl terminus of HhN play a key role in biological function of Ihh signaling pathway.