Molecular cloning, functional expression and characterization of p15, a novel fungal protein with potent neurite-inducing activity in PC12 cells.
p15 is a novel fungal protein which induces neurite outgrowth and neuronal differentiation of PC12 cells. In the present study, we report molecular cloning, functional expression and characterization of the gene encoding p15. The deduced amino acid sequence suggested that p15 is synthesized as a precursor with 31 extra amino-terminal amino acids including a putative signal sequence, and 20 carboxy-terminal amino acids, in addition to the 118 amino acids-long mature region with neurite-inducing activity. From the poly(A)(+) RNA prepared from the producing fungal strain, a cDNA fragment encoding the mature region of p15 was amplified and His(6)-tagged recombinant p15 was produced in Escherichia coli. The recombinant protein purified by a single step on Ni(2+) agarose column chromatography exhibited comparable specific activity as native p15 in the PC12 neurite extension assay. The effect of His(6)-p15 was blocked by nicardipine, suggesting that Ca(2+) influx through the L-type Ca(2+) channels is essential for its neurite-inducing activity. In addition, mutational analysis of His(6)-p15 demonstrated that both intramolecular disulfide bonds are essential for its biological activity.
Wakatsuki
S
S
Department of Biotechnology, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, 113-8657, Tokyo, Japan.
Yokoyama
T
T
Nakashima
S
S
Nishimura
A
A
Arioka
M
M
Kitamoto
K
K
D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000494:pharmacology; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000378:metabolism; D016900:Neurofilament Proteins / Q000378:metabolism; D016716:PC12 Cells / Q000187:drug effects; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics*; D051381:Rats; D011994:Recombinant Proteins / Q000494:pharmacology
D015220:Calcium Channels; D005656:Fungal Proteins; D009414:Nerve Growth Factors; D016900:Neurofilament Proteins; D011498:Protein Precursors; D011994:Recombinant Proteins; C047805:neurite-inducing factor; C445673:p15 precursor protein, Helicosporium; C061195:neurofilament protein M