287
Biochimica et Biophysics Acta, 1160 (1992) 287-292 0 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34341
Purification and amino-acid sequence of a nerve growth factor from the venom of Viper0 russelli russelli Jun-ichi Koyama a, Seiji Inoue a, Kiyoshi Ikeda a and Kyozo Hayashi b a Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Kawai, Matsubara, Osaka (Japan) and ’ Department of Molecular Biology, Gifu Pharmaceutical University, Gifu (Japan)
(Received 13 May 1992)
Key words: Amino-acid sequence; Nerve growth factor; Snake venom; (I’. r. russelli)
Nerve growth factor (NGF) was purified from the venom of Vipera russellirusselli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The K r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13 280 Da.
Introduction
Materials
Nerve growth factor (NGF) is a polypeptide hormone which is necessary for the survival and growth of developing sympathetic and sensory neurons, as well as basal forebrain cholinergic neurons in the brain [1,2]. Previously, we determined the amino-acid sequence of NGFs from the venoms of Formosan cobra Nuju naju atru [3], Indian cobra Nuju nuju and Thailand cobra Nuju naju siumensis [4]. These cobra NGFs have shown to give lower biological activities than mouse NGF with respect to the increase in length of neurites that grow out from chicken dorsal root ganglia [5,6]. On the contrary, it has been demonstrated that the biological activity of NGF isolated from the venom of l+eru russelli is virtually the same as that of mouse NGF [7]. Unlike cobra and mammalian NGFs, the Qeridae venom NGFs have been reported to be glycoproteins [8,9]. In this study, we purified NGF from Kperu russelli russelli venom by simpler methods than those described previously [8] and determined its amino-acid sequence. This is the first report of the entire sequence of glycosylated NGF, although about 2% of the mouse NGF molecules have been reported to be glycosylated at Asn-45 [lo].
Isolation of NGF from the venom of Vipera russelli russelli. About 1 g of the lyophilized venom of I/. r. russelli (Latoxan, France) was dissolved in 1% acetic
Correspondence to: S. Inoue, Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Kawai, Matsubara, Osaka 580, Japan. Abbreviations: NGF, nerve growth factor; PE-NGF, S-pyridylethylated derivative of NGF, CM-NGF, S-carboxymethylated derivative of NGF.
and Methods
acid. After centrifugation, the supernatant was fractionated on a Sephadex G-50 column (3.2 x 113 cm) which had been equilibrated with the same solvent. The fractions exhibiting NGF activity were dialyzed. against 50 mM sodium acetate buffer (pH 5.0) and applied to an S-Sepharose (Pharmacia LKB Biotechnology, Sweden) column (1 x 30 cm) which had been equilibrated with the same buffer. After the column was washed with the same buffer, the adsorbed proteins were eluted stepwise with the buffer containing 0.15, 0.35 and 2 M NaCl. The fractions eluting with the buffer containing 0.35 M NaCl were loaded on a Blue Sepharose CL-6B (Pharmacia LKB Biotechnology) column (1.9 x 20.5 cm) which had been equilibrated with 0.05 M sodium acetate buffer (pH 5.0) containing 0.35 M NaCl. After the column was washed stepwise with the same buffer containing 1 M NaCl and 0.05 M Tris-HCl buffer (pH 8.5) containing 1 M NaCl, NGF was eluted with 0.05 M sodium acetate buffer (pH 4.5) containing 1 M NaCl and 6 M urea. Finally, in order to remove a small amount of inpurities and some low molecular mass compounds, NGF was further purified by reverse-phase HPLC on a Vydac C, column (The Separation Group) with 0.05% trifluoroacetic acid containing a gradient from 0 to 45% acetonitrile. During the course of purification, NGF activity was estimated by the increase in length and density of
288 neurites that grow out from chicken dorsal root ganglia [11]. Isoelectrofocussing gel electrophoresis of the purified NGF preparation was carried out in the presence of 8 M urea [12]. SDS-PAGE of deglycosylated NGF. Deglycosylation of NGF was performed by N-glycanase digestion. About 1 mg of the NGF was incubated with 1.5 unit of N-glycanase (Genzyme) at 37°C for 60 h in 0.25 M sodium phosphate buffer (pH 8.6) containing 10 mM EDTA. SDS-PAGE of the intact and deglycosylated NGFs was carried out with a Phast System (Pharmacia LKB Biotechnology) using Phast Gel Homogeneous 20. The samples were dissolved in 10 mM Tris-HCl buffer (pH 8.0) containing 2.5% (w/v) SDS, 1 mM EDTA and 50 mM dithiothreitol. Protein bands were visualized by silver staining. Studies on the amino-acid sequence of NGF. S-pyridylethylated (PE) and S-carboxymethylated (CM) derivatives of the NGF were prepared by the method described previously [13,14] in the presence of 6 M guanidine hydrochloride (Nacalai Tesque, Japan). PE-NGF was cleaved by cyanogen bromide in 70% formic acid at 4°C for 24 h. The PE-NGF and CM-NGF were digested by lysylendopeptidase (Wako, Japan) in 50 mM Tris-HC1 buffer (pH 9.0) containing 4 M urea at 37°C for 6 h (enzyme/substrate mass ratio of 1 : 200). The peptides, thus, obtained were separated by reverse.phase HPLC on a Capcell pak Cts (Shiseido, Japan) column with 0.05% trifluoroacetic acid containing a linear gradient from 0 to 54% acetonitrile. Amino-acid sequences of the PE-NGF and its fragments were determined with a protein/peptide sequencer (Applied Biosystems, model 477A) equipped with an on-line phenylthiohydantoin (PTH) analyzer (model 120A). The C-terminal sequences of NGF were determined by carboxypeptidase Y (Cooper Biomedical) digestion at room temperature for 60 rain in 0.1 M pyridine acetate buffer (pH 5.6) containing 1% SDS [15]. The released amino acids were determined with an aminoacid ~_nalyzer (Hitachi model L-8500). Amino-acid analysis. The protein and peptide samples were hydrolyzed in 6 M HC1 containing 0.2% phenol at ll0°C for 24 h in evacuated sealed tubes. The amino-acid compositions were determined with the amino-acid analyzer.
-.
I
I
I
I 40
60
I
I
I
4-
80
100
120
140
5
g
1
o
..........
i- ....... 20
FRACTION
NUMBER
Fig. 1. Gel filtration of V. r. russelli venom on a Sephadex G-50 column (3.2× 113 cm) with 1% acetic acid.
activity were applied to a S-Sepharose column which had been equilibrated with 0.05 M acetate buffer (pH 5.0). The NGF activity was recovered in the fractions eluted with the same buffer containing 0.35 M NaC1. The fractions were then applied to a Blue-Sepharose CL-6B column. The NGF was adsorbed so tightly on the Blue-Sepharose column that the elution needed a buffer containing 6 M urea (Fig. 2). The NGF, thus, purified gave a single peak on a reverse-phase HPLC pattern (Fig. 3). Finally, 2.6 mg of the purified NGF was obtained from 1.0 g of the lyophilized venom. From the isoelectrofocussing gel electrophoresis, the isoelectric point of the V. r. russelli NGF was found to be around 9.3 in the presence of 8 M urea. As previously reported [7], the purified V. r. russelli NGF showed almost the same biological activity as the mouse NGF toward chicken dorsal ganglia at the same concentration of 10 ng/ml (data not shown).
0.05M AcONa buffer - 1M NaCI - 6 M Urea (pH 4.5)
0.2
J
L
0 0 5 M Tris-HCI buffel 1M NaCt (pH 8 5 )
L 0.05M ACONa buffer - fM NaCI (pH 5.0)
O,1 z
Biochimica et Biophysics Acta, 1160 (1992) 287-292 0 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4838/92/$05.00
BBAPRO 34341
Purification and amino-acid sequence of a nerve growth factor from the venom of Viper0 russelli russelli Jun-ichi Koyama a, Seiji Inoue a, Kiyoshi Ikeda a and Kyozo Hayashi b a Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Kawai, Matsubara, Osaka (Japan) and ’ Department of Molecular Biology, Gifu Pharmaceutical University, Gifu (Japan)
(Received 13 May 1992)
Key words: Amino-acid sequence; Nerve growth factor; Snake venom; (I’. r. russelli)
Nerve growth factor (NGF) was purified from the venom of Vipera russellirusselli by Sephadex G-50 gel filtration, S-Sepharose column chromatography and Blue-Sepharose CL-6B column chromatography. The purified NGF was found to be a glycoprotein, whose apparent molecular mass was estimated to be about 17.5 kDa by SDS-PAGE. The amino-acid sequence was determined by a combination of conventional methods. The K r. russelli NGF was composed of 117 amino-acid residues with one residue, Asn-21, being N-linked glycosylated and the molecular mass of its protein portion was calculated to be 13 280 Da.
Introduction
Materials
Nerve growth factor (NGF) is a polypeptide hormone which is necessary for the survival and growth of developing sympathetic and sensory neurons, as well as basal forebrain cholinergic neurons in the brain [1,2]. Previously, we determined the amino-acid sequence of NGFs from the venoms of Formosan cobra Nuju naju atru [3], Indian cobra Nuju nuju and Thailand cobra Nuju naju siumensis [4]. These cobra NGFs have shown to give lower biological activities than mouse NGF with respect to the increase in length of neurites that grow out from chicken dorsal root ganglia [5,6]. On the contrary, it has been demonstrated that the biological activity of NGF isolated from the venom of l+eru russelli is virtually the same as that of mouse NGF [7]. Unlike cobra and mammalian NGFs, the Qeridae venom NGFs have been reported to be glycoproteins [8,9]. In this study, we purified NGF from Kperu russelli russelli venom by simpler methods than those described previously [8] and determined its amino-acid sequence. This is the first report of the entire sequence of glycosylated NGF, although about 2% of the mouse NGF molecules have been reported to be glycosylated at Asn-45 [lo].
Isolation of NGF from the venom of Vipera russelli russelli. About 1 g of the lyophilized venom of I/. r. russelli (Latoxan, France) was dissolved in 1% acetic
Correspondence to: S. Inoue, Department of Biochemistry, Osaka University of Pharmaceutical Sciences, Kawai, Matsubara, Osaka 580, Japan. Abbreviations: NGF, nerve growth factor; PE-NGF, S-pyridylethylated derivative of NGF, CM-NGF, S-carboxymethylated derivative of NGF.
and Methods
acid. After centrifugation, the supernatant was fractionated on a Sephadex G-50 column (3.2 x 113 cm) which had been equilibrated with the same solvent. The fractions exhibiting NGF activity were dialyzed. against 50 mM sodium acetate buffer (pH 5.0) and applied to an S-Sepharose (Pharmacia LKB Biotechnology, Sweden) column (1 x 30 cm) which had been equilibrated with the same buffer. After the column was washed with the same buffer, the adsorbed proteins were eluted stepwise with the buffer containing 0.15, 0.35 and 2 M NaCl. The fractions eluting with the buffer containing 0.35 M NaCl were loaded on a Blue Sepharose CL-6B (Pharmacia LKB Biotechnology) column (1.9 x 20.5 cm) which had been equilibrated with 0.05 M sodium acetate buffer (pH 5.0) containing 0.35 M NaCl. After the column was washed stepwise with the same buffer containing 1 M NaCl and 0.05 M Tris-HCl buffer (pH 8.5) containing 1 M NaCl, NGF was eluted with 0.05 M sodium acetate buffer (pH 4.5) containing 1 M NaCl and 6 M urea. Finally, in order to remove a small amount of inpurities and some low molecular mass compounds, NGF was further purified by reverse-phase HPLC on a Vydac C, column (The Separation Group) with 0.05% trifluoroacetic acid containing a gradient from 0 to 45% acetonitrile. During the course of purification, NGF activity was estimated by the increase in length and density of
288 neurites that grow out from chicken dorsal root ganglia [11]. Isoelectrofocussing gel electrophoresis of the purified NGF preparation was carried out in the presence of 8 M urea [12]. SDS-PAGE of deglycosylated NGF. Deglycosylation of NGF was performed by N-glycanase digestion. About 1 mg of the NGF was incubated with 1.5 unit of N-glycanase (Genzyme) at 37°C for 60 h in 0.25 M sodium phosphate buffer (pH 8.6) containing 10 mM EDTA. SDS-PAGE of the intact and deglycosylated NGFs was carried out with a Phast System (Pharmacia LKB Biotechnology) using Phast Gel Homogeneous 20. The samples were dissolved in 10 mM Tris-HCl buffer (pH 8.0) containing 2.5% (w/v) SDS, 1 mM EDTA and 50 mM dithiothreitol. Protein bands were visualized by silver staining. Studies on the amino-acid sequence of NGF. S-pyridylethylated (PE) and S-carboxymethylated (CM) derivatives of the NGF were prepared by the method described previously [13,14] in the presence of 6 M guanidine hydrochloride (Nacalai Tesque, Japan). PE-NGF was cleaved by cyanogen bromide in 70% formic acid at 4°C for 24 h. The PE-NGF and CM-NGF were digested by lysylendopeptidase (Wako, Japan) in 50 mM Tris-HC1 buffer (pH 9.0) containing 4 M urea at 37°C for 6 h (enzyme/substrate mass ratio of 1 : 200). The peptides, thus, obtained were separated by reverse.phase HPLC on a Capcell pak Cts (Shiseido, Japan) column with 0.05% trifluoroacetic acid containing a linear gradient from 0 to 54% acetonitrile. Amino-acid sequences of the PE-NGF and its fragments were determined with a protein/peptide sequencer (Applied Biosystems, model 477A) equipped with an on-line phenylthiohydantoin (PTH) analyzer (model 120A). The C-terminal sequences of NGF were determined by carboxypeptidase Y (Cooper Biomedical) digestion at room temperature for 60 rain in 0.1 M pyridine acetate buffer (pH 5.6) containing 1% SDS [15]. The released amino acids were determined with an aminoacid ~_nalyzer (Hitachi model L-8500). Amino-acid analysis. The protein and peptide samples were hydrolyzed in 6 M HC1 containing 0.2% phenol at ll0°C for 24 h in evacuated sealed tubes. The amino-acid compositions were determined with the amino-acid analyzer.
-.
I
I
I
I 40
60
I
I
I
4-
80
100
120
140
5
g
1
o
..........
i- ....... 20
FRACTION
NUMBER
Fig. 1. Gel filtration of V. r. russelli venom on a Sephadex G-50 column (3.2× 113 cm) with 1% acetic acid.
activity were applied to a S-Sepharose column which had been equilibrated with 0.05 M acetate buffer (pH 5.0). The NGF activity was recovered in the fractions eluted with the same buffer containing 0.35 M NaC1. The fractions were then applied to a Blue-Sepharose CL-6B column. The NGF was adsorbed so tightly on the Blue-Sepharose column that the elution needed a buffer containing 6 M urea (Fig. 2). The NGF, thus, purified gave a single peak on a reverse-phase HPLC pattern (Fig. 3). Finally, 2.6 mg of the purified NGF was obtained from 1.0 g of the lyophilized venom. From the isoelectrofocussing gel electrophoresis, the isoelectric point of the V. r. russelli NGF was found to be around 9.3 in the presence of 8 M urea. As previously reported [7], the purified V. r. russelli NGF showed almost the same biological activity as the mouse NGF toward chicken dorsal ganglia at the same concentration of 10 ng/ml (data not shown).
0.05M AcONa buffer - 1M NaCI - 6 M Urea (pH 4.5)
0.2
J
L
0 0 5 M Tris-HCI buffel 1M NaCt (pH 8 5 )
L 0.05M ACONa buffer - fM NaCI (pH 5.0)
O,1 z
