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J. Biol. Chem., Vol. 283, Issue 7, 4323-4331, February 15, 2008

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Neurotrophic Activity of Neudesin, a Novel Extracellular Heme-binding Protein, Is Dependent on the Binding of Heme to Its Cytochrome b₅-like Heme/Steroid-binding Domain^*

Ikuo Kimura¹, Yoshiaki Nakayama, Hajime Yamauchi, Morichika Konishi, Ayumi Miyake, Masahiro Mori, Mitsuhiro Ohta^¶, Nobuyuki Itoh, and Masafumi Fujimoto

From the Laboratory of Applied Pharmacology, Faculty of Pharmacy, Chiba Institute of Science, Choshi, Chiba 288-0025, Japan, the Department of Genetic Biochemistry, Kyoto University Graduate School of Pharmaceutical Sciences, Sakyo, Kyoto 606-8501, Japan, and the ^¶Department of Medical Biochemistry, Kobe Pharmaceutical University, Higashinada, Kobe 658-8558, Japan

Neudesin is a secreted protein with neurotrophic activity in neurons and undifferentiated neural cells. We report here that neudesin is an extracellular heme-binding protein and that its neurotrophic activity is dependent on the binding of heme to its cytochrome b₅-like heme/steroid-binding domain. At first, we found that at least a portion of the purified recombinant neudesin appeared to bind hemin because the purified neudesin solution was tinged with green and had a sharp absorbance peak at 402 nm. The addition of exogenous hemin extensively increased the amount of hemin-bound neudesin. In contrast, neudesinHBD, a mutant lacking the heme-binding domain, could not bind hemin. The neurotrophic activity of the recombinant neudesin that bound exogenous hemin (neudesin-hemin) was significantly greater than that of the recombinant neudesin in either primary cultured neurons or Neuro2a cells, suggesting that the activity of neudesin depends on hemin. The neurotrophic activity of neudesin was enhanced by the binding of Fe(III)-protoporphyrin IX, but neither Fe(II)-protoporphyrin IX nor protoporphyrin IX alone. The inhibition of endogenous neudesin by RNA interference significantly decreased cell survival in Neuro2a cells. This indicates that endogenous neudesin possibly contains hemin. The experiment with anti-neudesin antibody suggested that the endogenous neudesin detected in the culture medium of Neuro2a cells was associated with hemin because it was not retained on a heme-affinity column at all. Neudesin is the first extracellular heme-binding protein that shows signal transducing activity by itself. The present findings may shed new light on the function of extracellular heme-binding proteins.

Received for publication, August 13, 2007 , and in revised form, November 30, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. S1.

¹ To whom correspondence should be addressed: Laboratory of Applied Pharmacology, Faculty of Pharmacy, Chiba Institute of Science, Shiomi, Choshi, Chiba 288-0025, Japan. Tel.: 81-479-30-4782; Fax: 81-479-30-4740; E-mail: ikimura{at}cis.ac.jp.

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