Neurochemical Research, Vol. 17, No. 3, 1992, pp. 239-246
Isolation and Characterization of Two Novel Peptide Amides Originating from Myelin Basic Protein in Bovine Brain Ken Takamatsu 1,2,3 and Kazuhiko Tatemoto I
(Accepted August 5, 1991)
During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-SerGln-Arg-Ser-Lys-Tyr-amideand Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-AlaSer-Ala-amide. These 12 and 16 residues peptides had the primary structure identical to the Nterminal fragment of myelin basic protein (MBP). The peptides were therefore designated myelin peptide amide-12 (MPA-12) and -16 (MPA-16). Unlike other amidated peptides, MPA might be generated from MBP by hydroxyl radicals produced via a Fenton reaction in situ. However, this unique amidation seems to occur exclusively to MBP in a site specific manner in the brain. KEY WORDS: Peptide amide; amidation; myelin basic protein; oxygen radicals; bovine brain.
carried out and a number of novel neural and hormonal peptides were isolated (4-8). Recently we performed a similar search in the bovine brain acid extracts, and isolated two novel peptide amides, one with a tyrosine amide and the other with an alanine amide at their C-termini. These peptides were found to have the primary structures identical to the N-terminal fragments of myelin basic protein. Unlike other amidated peptide, these peptides were not produced by the treatment of the precursor peptides with peptidylglycine alpha-amidating enzyme. This report describes the isolation, complete amino acid sequence and the generation mechanism of these novel peptide amides.
INTRODUCTION The C-terminal amide structure is a characteristic feature of many biologically active secretary peptides (1,2). Peptides with such a structure can be assayed by using the chemical detection method in which the Cterminus of the peptide is cleaved off by a proteolytic enzyme, converted into N-dansyl derivative, extracted selectively and identified by thin-layer chromatography (3). Using this chemical detection method, a search for previously unknown peptides that possess the C-terminal amide structure in the extracts of brain and intestine was 1 Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305. 2 Department of Physiology, Toho University School of Medicine, Oomort-nishi, Oota-ku, Tokyo 143, Japan. 3 Address reprint requests to Dr. Ken Takamatsu, Department of Physiology, Toho University School of Medicine, 5-21-16, Oomori-nishi, Oota-ku, Tokyo 143, Japan. Phone: 81-3-3762-4151 ext. 2345, Fax: 81-3-3761-0546
EXPERIMENTAL PROCEDURE Extraction Procedure. Bovine brain (50 kg) were boiled for 20 rain in 5 times tissue volume of distilled water, cooled and then homogenized. Acetic acid was added to 0.5 M and the homogenate stored
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240 at 4~ overnight with stirring. After filtration to remove tissue debris, the peptides in the extract were adsorbed onto alginic acid at pH 2.7, and eluted with 0.2 M HC1. The eluate was then lyophilized after pH adjusted to 7.2. ChromatographicPurification. The lyophilizate was dissolved with 0.2 M acetic acid and loaded onto a Sephadex G25F column (10 • 150 cm) and then eluted with 0.2 M acetic acid. Fractions containing peptides with low molecular weight (1000-3000 Daltons) were collected and lyophilized. An aliquot (200 rag) of the lyophilizate was loaded directly onto a MClgel ODS-10U column (20 • 250 ram, Mitsubishi Kasei, Tokyo, Japan), then eluted with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 10 ml/min. A total of 2 g of the preparations was purified by repeating the HPLC separation. Fractions were collected at 1 rain intervals and assayed for the chemical detection of a C-terminal amide. Fractions containing a C-terminal amide were then loaded onto a TSKgel ODS-120T column (4.6 x 250 mm, Tosoh, Tokyo, Japan), and developed at a flow rate of 1 ml/ rain with a linear gradient of acetonitrile in 5 mM sodium phosphate buffer pH 6.5. Fractions from second HPLC containing a C-terminal amide were loaded onto a Chemcosorb 5-ODS-H column (4.6 x 250 ram, Chemco, Osaka, Japan), eluted with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 1 ml/min. Each peaks were collected and assayed for a C-terminal amide. ChemicalAssay for a C-TerminalAmide. Peptide amide was assayed by the chemical method based on the detection of a C-terminal amino acid amide which was released by the treatment of sample with a thermolysin (Boehringer, Mannheim, F.R.G.). The released amino acid amide was then converted into N-dansyl derivative, extracted selectively and identified by thin layer chromatography (3). Fragmentation of the Peptides. Approximately 5 nmol of intact peptides were digested with TPCK-treated trypsin (enzyme/substrate ratio; 1/400, Sigma, St. Louis, U.S.A.), or thermolysin (enzyme/substrate ratio; 1/100) in 10 ~1 of 50 mM 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (HEPES)-NaOH pH 8.0, 1 mM CaCIz for 6 hours at 37~ After adding 100 ~1 of 2% formic acid, the proteolytic digests were directly fractionated on a Chemcosorb 5-ODS-H reversed phase column (4.6 • 250 ram) using a linear gradient over 45 rain of 0-15% acetonitrile in 0.1% TFA at flow rate of 1 ml/min. Removal of Acyl Blocking Group. The blocked N-terminal tryptic fragment was treated with acylamino acid releasing enzyme (AARE, EC 3.4.19.3.) (Takara Shuzo, Kyoto, Japan) as described (9). In brief, approximately 5 nmol of the N-terminal tryptic fragment was dissolved in 20 ixl of 20 mM sodium phosphate buffer pH 7.2, and added 20 ~1 of.AARE solution (0.01 unit/p.l of the same buffer). The reaction was carried out at 37~ for 6 hours and stopped by addition of 100 ILl of 2% formic acid. The reaction mixture were fractionated on a Chemcosorb 5-ODS-H column (4.6 x 250 ram) using a linear gradient over 45 min of 0-15% acetonitrile in 0.1% TFA at a flow rate of 1 ml/min. The peaks of the newly appearing reaction product were collected in a single tube. These materials were taken for amino acid and sequence analyses. Amino Acid Analysis. Approximately I00 pmol of purified peptides were hydrolyzed by vapor HCI containing 0.5% phenol at ll0~ for 20 hours, and were applied to an automated amino acid analyzer (Japan Spectroscopic Co., Tokyo, Japan). Amino Acid SequenceAnalysis. Approximately 500 pmol of peptides or their proteolytic fragments were subjected to an automated gas phase sequencer (A 470, Applied Biosystems, CA, USA), and phenylthiohydantoin derivatives of amino acids were analyzed by HPLC as described (10). Mass SpectralAnalysis. Approximately 5 nmol of intact peptide was dissolved in 2 Ixl of 5% (v/v) aqueous acetic acid and was added
Takamatsu and Tatemoto to I Izl of glycerol on a stainless steel sample stage. Positive ion spectra of intact peptide was obtained using a fast atom bombardment (FAB) mass spectrometer (JMS-AX505H, Japan Electron Optics Laboratory, Tokyo, Japan). The mass value for the intact peptide is the monoisotopic mass of the protonated molecular ion. Peptide Synthes&. Peptide were synthesized manually using the method of Fmoc-polyamide synthesis (11). Fmoc-PAL-resin, Fmocamino acid-resins and Fmoc-amino acid with conventional protection groups (MilliGen/Biosearch, CA, USA) were used. Enzymatic Amidation. Five nmol of N-dansyl synthetic peptides was dissolved with 100 Izl of 100 mM HEPES-KOH pH 6.4, followed by addition of KI (to 10 mM), catalase (to 2.5 mg/ml), CuSO4 (to 0.25 p.M), 5% acetonitrile. After preincubation for 10 min at 37~ ascorbic acid (to 3 raM) and peptidylglycine alpha-amidating enzyme (EC 1.14.17.3.) (5 m unit, Takara Shuzo, Kyoto, Japan) were added to the reaction mixtures. The reaction was carried out at 37~ for 1 hour and was stopped by addition of 100 ~1 of 2% formic acid. Then, the reaction mixture was fractionated on a Chemcosorb 5-ODS-H using a linear gradient over 45 rain of 0-80% acetonitrile in 0.1% TFA or 5 mM sodium phosphate pH 6.5 at a flow rate of i ml/min. ChemicalAmidation. Ten nmol of N-dansyl synthetic peptide was dissolved with 200 I~1 of 20 mM sodium phosphate buffer pH 7.0 followed by addition of cupric chloride (0.2-5 I~M) or ferric chloride (0.1-2 mM), ascorbic acid (0.5-10 raM) and hydrogen peroxide (0.15 raM). Where appropriate oxygen radical scavenger was added before the cupric or ferric chloride. The reaction mixtures were incubated at 37~ for 1 hour and then examined for the formation of C-terminal amide using reversed phase HPLC as described above.
RESULTS
Purification of Myelin Peptide Amides. The peptides were extracted from bovine brain (50 kg) in 0.5 M acetic acid and partially purified by adsorption onto alginic acid and Sephadex G25F gel filtration as described. An aliquot (200 mg) of the fractions of molecular weight 1000-3000 Daltons from Sephadex G25F were loaded directly onto a MClgel ODS-10U column, then eluted with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 10 ml/min (Figure l-a). A total of 2 g of the preparation was purified by repeating the HPLC separation. Two fractions, one containing a tyrosine amide (shaded area Y) and the other containing an alanine amide (shaded area A), which had different elution profiles from those of known peptide amides, were found by the chemical detection method. Then, these fractions were further purified by successive reversed phase HPLC on a TSKgel ODS120T column and on a Chemcosorb 5ODS-H column (Figure l-b, c). The final HPLC preparations obtained (24 ~g for tyrosine amide and 60 ~g for alanine amide) were found to be homogeneous and were subjected to structural analysis. Structural Studies. Amino acid analysis of these highly purified materials suggested that the peptide with a tyrosine amide consisted of 12 amino acid residues and
Two Novel Peptide Amides Originating from Myelin Basic Protein
241
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Isolation and Characterization of Two Novel Peptide Amides Originating from Myelin Basic Protein in Bovine Brain Ken Takamatsu 1,2,3 and Kazuhiko Tatemoto I
(Accepted August 5, 1991)
During a systematic search for peptides that possess the C-terminal amide structure, two novel peptide amides, one with a tyrosine amide and the other with an alanine amide were isolated from bovine brain by acid extraction and sequential steps of reversed phase HPLC. Microsequence, amino acid and mass spectral analyses revealed the structures: Ac-Ala-Ala-Gln-Lys-Arg-Pro-SerGln-Arg-Ser-Lys-Tyr-amideand Ac-Ala-Ala-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-AlaSer-Ala-amide. These 12 and 16 residues peptides had the primary structure identical to the Nterminal fragment of myelin basic protein (MBP). The peptides were therefore designated myelin peptide amide-12 (MPA-12) and -16 (MPA-16). Unlike other amidated peptides, MPA might be generated from MBP by hydroxyl radicals produced via a Fenton reaction in situ. However, this unique amidation seems to occur exclusively to MBP in a site specific manner in the brain. KEY WORDS: Peptide amide; amidation; myelin basic protein; oxygen radicals; bovine brain.
carried out and a number of novel neural and hormonal peptides were isolated (4-8). Recently we performed a similar search in the bovine brain acid extracts, and isolated two novel peptide amides, one with a tyrosine amide and the other with an alanine amide at their C-termini. These peptides were found to have the primary structures identical to the N-terminal fragments of myelin basic protein. Unlike other amidated peptide, these peptides were not produced by the treatment of the precursor peptides with peptidylglycine alpha-amidating enzyme. This report describes the isolation, complete amino acid sequence and the generation mechanism of these novel peptide amides.
INTRODUCTION The C-terminal amide structure is a characteristic feature of many biologically active secretary peptides (1,2). Peptides with such a structure can be assayed by using the chemical detection method in which the Cterminus of the peptide is cleaved off by a proteolytic enzyme, converted into N-dansyl derivative, extracted selectively and identified by thin-layer chromatography (3). Using this chemical detection method, a search for previously unknown peptides that possess the C-terminal amide structure in the extracts of brain and intestine was 1 Department of Psychiatry and Behavioral Sciences, Stanford University School of Medicine, Stanford, California 94305. 2 Department of Physiology, Toho University School of Medicine, Oomort-nishi, Oota-ku, Tokyo 143, Japan. 3 Address reprint requests to Dr. Ken Takamatsu, Department of Physiology, Toho University School of Medicine, 5-21-16, Oomori-nishi, Oota-ku, Tokyo 143, Japan. Phone: 81-3-3762-4151 ext. 2345, Fax: 81-3-3761-0546
EXPERIMENTAL PROCEDURE Extraction Procedure. Bovine brain (50 kg) were boiled for 20 rain in 5 times tissue volume of distilled water, cooled and then homogenized. Acetic acid was added to 0.5 M and the homogenate stored
239
0364-3190/92/0300-0239506.50/09 I992PlenumPublishingCorporation
240 at 4~ overnight with stirring. After filtration to remove tissue debris, the peptides in the extract were adsorbed onto alginic acid at pH 2.7, and eluted with 0.2 M HC1. The eluate was then lyophilized after pH adjusted to 7.2. ChromatographicPurification. The lyophilizate was dissolved with 0.2 M acetic acid and loaded onto a Sephadex G25F column (10 • 150 cm) and then eluted with 0.2 M acetic acid. Fractions containing peptides with low molecular weight (1000-3000 Daltons) were collected and lyophilized. An aliquot (200 rag) of the lyophilizate was loaded directly onto a MClgel ODS-10U column (20 • 250 ram, Mitsubishi Kasei, Tokyo, Japan), then eluted with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 10 ml/min. A total of 2 g of the preparations was purified by repeating the HPLC separation. Fractions were collected at 1 rain intervals and assayed for the chemical detection of a C-terminal amide. Fractions containing a C-terminal amide were then loaded onto a TSKgel ODS-120T column (4.6 x 250 mm, Tosoh, Tokyo, Japan), and developed at a flow rate of 1 ml/ rain with a linear gradient of acetonitrile in 5 mM sodium phosphate buffer pH 6.5. Fractions from second HPLC containing a C-terminal amide were loaded onto a Chemcosorb 5-ODS-H column (4.6 x 250 ram, Chemco, Osaka, Japan), eluted with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 1 ml/min. Each peaks were collected and assayed for a C-terminal amide. ChemicalAssay for a C-TerminalAmide. Peptide amide was assayed by the chemical method based on the detection of a C-terminal amino acid amide which was released by the treatment of sample with a thermolysin (Boehringer, Mannheim, F.R.G.). The released amino acid amide was then converted into N-dansyl derivative, extracted selectively and identified by thin layer chromatography (3). Fragmentation of the Peptides. Approximately 5 nmol of intact peptides were digested with TPCK-treated trypsin (enzyme/substrate ratio; 1/400, Sigma, St. Louis, U.S.A.), or thermolysin (enzyme/substrate ratio; 1/100) in 10 ~1 of 50 mM 4-(2-hydroxyethyl)-l-piperazine ethanesulfonic acid (HEPES)-NaOH pH 8.0, 1 mM CaCIz for 6 hours at 37~ After adding 100 ~1 of 2% formic acid, the proteolytic digests were directly fractionated on a Chemcosorb 5-ODS-H reversed phase column (4.6 • 250 ram) using a linear gradient over 45 rain of 0-15% acetonitrile in 0.1% TFA at flow rate of 1 ml/min. Removal of Acyl Blocking Group. The blocked N-terminal tryptic fragment was treated with acylamino acid releasing enzyme (AARE, EC 3.4.19.3.) (Takara Shuzo, Kyoto, Japan) as described (9). In brief, approximately 5 nmol of the N-terminal tryptic fragment was dissolved in 20 ixl of 20 mM sodium phosphate buffer pH 7.2, and added 20 ~1 of.AARE solution (0.01 unit/p.l of the same buffer). The reaction was carried out at 37~ for 6 hours and stopped by addition of 100 ILl of 2% formic acid. The reaction mixture were fractionated on a Chemcosorb 5-ODS-H column (4.6 x 250 ram) using a linear gradient over 45 min of 0-15% acetonitrile in 0.1% TFA at a flow rate of 1 ml/min. The peaks of the newly appearing reaction product were collected in a single tube. These materials were taken for amino acid and sequence analyses. Amino Acid Analysis. Approximately I00 pmol of purified peptides were hydrolyzed by vapor HCI containing 0.5% phenol at ll0~ for 20 hours, and were applied to an automated amino acid analyzer (Japan Spectroscopic Co., Tokyo, Japan). Amino Acid SequenceAnalysis. Approximately 500 pmol of peptides or their proteolytic fragments were subjected to an automated gas phase sequencer (A 470, Applied Biosystems, CA, USA), and phenylthiohydantoin derivatives of amino acids were analyzed by HPLC as described (10). Mass SpectralAnalysis. Approximately 5 nmol of intact peptide was dissolved in 2 Ixl of 5% (v/v) aqueous acetic acid and was added
Takamatsu and Tatemoto to I Izl of glycerol on a stainless steel sample stage. Positive ion spectra of intact peptide was obtained using a fast atom bombardment (FAB) mass spectrometer (JMS-AX505H, Japan Electron Optics Laboratory, Tokyo, Japan). The mass value for the intact peptide is the monoisotopic mass of the protonated molecular ion. Peptide Synthes&. Peptide were synthesized manually using the method of Fmoc-polyamide synthesis (11). Fmoc-PAL-resin, Fmocamino acid-resins and Fmoc-amino acid with conventional protection groups (MilliGen/Biosearch, CA, USA) were used. Enzymatic Amidation. Five nmol of N-dansyl synthetic peptides was dissolved with 100 Izl of 100 mM HEPES-KOH pH 6.4, followed by addition of KI (to 10 mM), catalase (to 2.5 mg/ml), CuSO4 (to 0.25 p.M), 5% acetonitrile. After preincubation for 10 min at 37~ ascorbic acid (to 3 raM) and peptidylglycine alpha-amidating enzyme (EC 1.14.17.3.) (5 m unit, Takara Shuzo, Kyoto, Japan) were added to the reaction mixtures. The reaction was carried out at 37~ for 1 hour and was stopped by addition of 100 ~1 of 2% formic acid. Then, the reaction mixture was fractionated on a Chemcosorb 5-ODS-H using a linear gradient over 45 rain of 0-80% acetonitrile in 0.1% TFA or 5 mM sodium phosphate pH 6.5 at a flow rate of i ml/min. ChemicalAmidation. Ten nmol of N-dansyl synthetic peptide was dissolved with 200 I~1 of 20 mM sodium phosphate buffer pH 7.0 followed by addition of cupric chloride (0.2-5 I~M) or ferric chloride (0.1-2 mM), ascorbic acid (0.5-10 raM) and hydrogen peroxide (0.15 raM). Where appropriate oxygen radical scavenger was added before the cupric or ferric chloride. The reaction mixtures were incubated at 37~ for 1 hour and then examined for the formation of C-terminal amide using reversed phase HPLC as described above.
RESULTS
Purification of Myelin Peptide Amides. The peptides were extracted from bovine brain (50 kg) in 0.5 M acetic acid and partially purified by adsorption onto alginic acid and Sephadex G25F gel filtration as described. An aliquot (200 mg) of the fractions of molecular weight 1000-3000 Daltons from Sephadex G25F were loaded directly onto a MClgel ODS-10U column, then eluted with a linear gradient of acetonitrile in 0.1% TFA at a flow rate of 10 ml/min (Figure l-a). A total of 2 g of the preparation was purified by repeating the HPLC separation. Two fractions, one containing a tyrosine amide (shaded area Y) and the other containing an alanine amide (shaded area A), which had different elution profiles from those of known peptide amides, were found by the chemical detection method. Then, these fractions were further purified by successive reversed phase HPLC on a TSKgel ODS120T column and on a Chemcosorb 5ODS-H column (Figure l-b, c). The final HPLC preparations obtained (24 ~g for tyrosine amide and 60 ~g for alanine amide) were found to be homogeneous and were subjected to structural analysis. Structural Studies. Amino acid analysis of these highly purified materials suggested that the peptide with a tyrosine amide consisted of 12 amino acid residues and
Two Novel Peptide Amides Originating from Myelin Basic Protein
241
]
2.0
.........
a
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1.5
0
9- ~ ' " ~ " -
1.0E -~"'el 0 j~ 0.5-
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0 < A
