Advances in Dental Research
Advanceshttp://adr.sagepub.com/ in Dental Research
Isolation of Two Bovine Amelogenin Peptides and Their Amino Acid Sequences J.-H. Yeh, T. Takagi and S. Sasaki ADR 1987 1: 276 DOI: 10.1177/08959374870010021701 The online version of this article can be found at: http://adr.sagepub.com/content/1/2/276
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Advances in Dental Research can be found at: Email Alerts: http://adr.sagepub.com/cgi/alerts Subscriptions: http://adr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
Downloaded from adr.sagepub.com by guest on June 9, 2011 For personal use only. No other uses without permission.
ISOLATION OF TWO BOVINE AMELOGENIN PEPTIDES AND THEIR AMINO ACID SEQUENCES J.-H. YEH, T. TAKAGI, AND S. SASAKI1 Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan
Adv Dent Res 1(2): 276-281, December, 1987 ABSTRACT
wo peptide fractions of bovine amelogenin having a highly aggregative property to form polymers were purified by chromatography, SDS-polyacrylamide gel electrophoresis, and HPLC. Amino acid sequences of purified peptides were determined by automated Edman degradation. One peptide was found to be composed of 63 amino acid residues having a molecular weight of 7105, and the other of 86 residues having that of 9683. The sequence of the smaller peptide was identical to the C-terminal 63 residues of the amelogenin molecule of 170 residues previously reported, but the larger contained eight residues which are absent in the amelogenin sequence. There is a possibility that the latter peptide might be synthesized independently from mRNA spliced at different positions.
T
INTRODUCTION In the early developmental stages of dental enamel, amelogenin is a predominant constituent and consists of several sub-components (Eastoe, 1960; Sasaki and Shimokawa, 1979). In our previous paper, we succeeded in determining the primary structure of a principal species of bovine amelogenin which is composed of 170 amino acids having a molecular weight of 19,340 daltons (Takagi et aL, 1984). Most of the low-molecular-weight peptides present in developing enamel were thought to be derived from the degradation of this principal amelogenin molecule after being secreted into the enamel (Shimokawa and Sasaki, 1978; Sasaki and Shimokawa, 1979; Sasaki et aL, 1982,1983). However, the existence of a peptide named "leucine-rich amelogenin peptide" (Fincham et aL, 1981) cannot be explained as a degradation product, because it has a composition derived by connection of the amino-terminal 33 residues to the carboxy-terminal 13 without the central sequence of the principal amelogenin molecule. It may be a translaPresented at the Second Carolina Conference on Tooth Enamel Formation, March 16-17,1987, at the University of North Carolina, Chapel Hill Address correspondence and reprint requests to Dr. S. Sasaki, Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo 113, Japan. 276
tion product of a different mRNA from that of the abovementioned amelogenin. The purpose of the present study is to investigate the origin and nature of lower-molecular-weight amelogenin peptides present in developing enamel. Here we report two peptide fractions from bovine enamel at the maturation stage; one had a sequence identical to that of the C-terminal 63 residues of the principal amelogenin, and the other, longer, peptide was found to contain an amino acid sequence of eight residues which was not present in the principal amelogenin molecule reported by Takagi et aL (1984).
MATERIALS AND METHODS Preparation of Bovine Amelogenin Peptides Developing bovine incisors were obtained, and enamel was collected at the maturation stage, with chalky consistency. Organic components were extracted from the enamel with 0.5 mol/L acetic acid and lyophilized. The extract (100 mg) was fractionated with a column (2.5 x 190 cm) of Bio-Gel P-30 (Bio-Rad, Richmond, CA) and eluted with 0.1 mol/L formic acid. Two fractions from the P-30 column were lyophilized and applied to a DEAE-Sephacel ion-exchange column of 1.5 x 10 cm (Fincham et aL, 1983). Elution was performed with 7 mol/L urea, 0.05 mol/ L Tris HC1 (pH 7.4) with a linear gradient of 0 to 0.5 mol/L NaCl. The unretained proteins (50 mg) from
Downloaded from adr.sagepub.com by guest on June 9, 2011 For personal use only. No other uses without permission.
TWO BOVINE AMELOGENIN PEPTIDES
Vol. 1 No. 2
277
0.6 -
3.5-
Secretory Stage E
c
• 0.5-
Maturation Stage
Ai A2 B i » t 50
90
110
130
40
150
Fraction Number
120
80
Fraction Number
Fig. 1—Bio-Gel P-30 ckromatography of acetic acid extracts from bovine enamel at secretory and maturation stages eluted with 0.1 mol/L formic acid. Fractions of 5 mL each were collected. Peaks A2 and Bl were further purified. The second peak "Al" is the main amelogenin peak.
Fig. 3 —SP-Sephadex C-25 chromatography of "Dl" fraction in Fig. 2 eluted with a linear gradient of 0 to 0.5 mol/L NaCl in 0.05 mol/ L sodium acetate buffer, pH 3.8. Fractions of 4.6 mL each were collected.
the DEAE-Sephacel column were chromatographed further with an SP-Sephadex C-25 column (2.5 x 12 cm) in 0.05 mol/L. sodium acetate buffer, pH 3.8, with a linear gradient of 0 to 0.5 mol/L NaCl over a total volume of 500 mL. SDS-polyacrylamide gradient gel (15-30%) electrophoresis at an acrylamide/bis ratio of 56:1 was carried out with 0.05 mol/L Tris/glycine buffer, pH 8.3, containing 0.1% SDS. Gels were stained with Coomassie Brilliant Blue R-250 or by being silverstained (Wray et al., 1981). Electrophoresis with polyacrylamide gels (7.5%) containing 6 mol/L urea was carried out at an acrylamide/bis ratio of 37.5:1 with 0.5 mol/L Tris/borate buffer, pH 8.6 (Fincham et al., 1972). After electrophoresis, the desired protein band was excised and recovered by means of an apparatus for protein recovery from the gel (Maxyield Protein Recoverer, Type AE-3590, Atto Co., Tokyo, Japan) according to Hanaoka et al. (1979).
The protein band recovered from the electrophoretic gel was further purified with an HPLC system equipped with a solvent programmer Model 660 and a Data Module 730 (Waters Assoc, Inc., Milford, MA) with a C4 (Hi-Pore RP-304) column (Bio-Rad) eluted with 0.1% trifluoroacetic acid (TFA) solution in a convex gradient (No.3, 0-100%) of 0.1% TFA/acetonitrile.
i.o-
D1
c
0.5
CO
^0.5
\
E
-a
0.3
c O
00 CJ
12
t
E o
Atnino Acid Analysis Samples were hydrolyzed in 5.7 mol/L HC1 containing 0.01% 2-mercaptoethanol in vacuo at 110°C for
/
/
O ra
/D2 ' • ' • "• ' : '• ' • •• / •' '•
A \ \ \ \
' • '• • ••
10
20 30 Fraction Number
0.1
40
Fig. 2-DEAE-Sephacel chromatography of "A2" fraction shown in Fig. 1. Elution was performed with 7 mol/L urea, 0.05 mol/L Fig. 4 —Electrophoresis of "Peak 3" in Fig. 3 on 7.5% polyacrylTris HC1, with a linear gradient of 0 to 0.5 mol/L NaCl and finally amide gel containing 6 mol/L urea in Tris/borate buffer, pH 8.6. with 1 mol/L NaCl in the same buffer. Fractions of 5 mL each were Lane 1: Total enamel extract from the maturation stage. Lane 2: Downloaded from adr.sagepub.com by guest on June 9, 2011 For"Peak personal use other uses 3"only. inNoFig. 3. without permission. collected.
27a
Adv Dent Res December 1987
YEH et al.
TABLE AMINO ACID COMPOSITIONS OF PURIFIED FRACTIONS FROM "A2" AND "Bl" PEAKS
24 hr (Fincham et al., 1983). The hydrolyzates were analyzed with an amino acid analyzer (Model 6300, Beckman Instruments, Inc., Palo Alto, CA). Sequence Determination Automated Edman degradation was performed with an amino acid sequencer (Model 890C, Beckman, Inc., or Model 470A, Applied Biosystems, Foster City, CA) in the presence of polybrene (Takagi et al., 1984). Phenylthiohydantoin derivatives of amino acids were identified with an HPLC system (Model 8100, Spectra Physics, San Jose, CA). Carboxy-terminal Determination Digestion with carboxypeptidase W (Pentel Co., Tokyo, Japan) was carried out according to the method of Umetsu et al. (1981) in 0.02 mol/L sodium citrate buffer, pH 4.0, at 40°C with an enzyme/substrate ratio of 1:50 (w/w). The samples were periodically removed from the digestion mixture, and the free amino acids liberated were determined with an amino acid analyzer.
Amino Acid
"A2" peptides d c b
Asx (D/N) Thr (T) Ser (S) Glx (E/Q) Pro (P) Ala (A) Val (V) Met (M) lie (I) Leu (L) Phe (F) His(H) Trp(W)»
2 2 2 20 30 1 1 3 3 13 2 4
2 2 2 20 30 2 1 3 3 13 2 5 (1)
Total
83
86
2 2 3 20 30 2 1 3 3 13 2 4
"Bl" peptide Peak 2 1 1 14 24 1 1 2 3 11 1 3 (1)
85 63 (Residues/Molecule) *Tryptophan was not determined by amino acid analysis, estimated from the sequence.
RESULTS
The P-30 gel filtration pattern of the acetic acid extract from bovine enamel at the maturation stage is shown in Fig. 1. Two small peaks, "A2" and "BY', were separated after the main amelogenin peak. Peaks "A2" and "BY' were chromatographed on a DEAESephacel column (Fig. 2). In both cases, there were unretained and retained fractions. The unretained fraction from peak "A2" was further chromatographed on an SP-Sephadex C-25 column (Fig. 3). There were several fractions on the profile. The peak 3 fraction was collected and subjected to 7.5% PAGE (Tris-urea system), and was separated into four bands (a, b, c, and d in Fig. 4). Bands b, c, and d were excised from the gel and extracted by means of the protein recovery apparatus. Amino acid analyses indicated that all bands had an almost identical composition, rich in glutamic acid, proline, and leucine (Table). The extract from the third band (band "c") was purified by the HPLC system, and a single peak was obtained (Fig. 5). The amino acid sequence of the peptide purified by HPLC was determined, and the result is shown in Fig. 7. Thirty-four residues of the peptide from the aminoterminal were directly determined by Edman degradation. The carboxy-terminal sequence of the peptide was determined by digestion with carboxy-peptidase W and was found to be Pro-Leu-Gln-Ala-Trp-COOH, which is identical to that of the principal amelogenin molecule having 170 residues (Fig. 7). Its primary structure was finally determined to be composed of 86 amino acid residues having a molecular weight of 9683. The DEAE-Sephacel column chromatographic pattern of peak "BY' was almost identical to that of peak
% z 100^,
o 0.04-
50^ 0.02-
I
Isolation of Two Bovine Amelogenin Peptides and Their Amino Acid Sequences J.-H. Yeh, T. Takagi and S. Sasaki ADR 1987 1: 276 DOI: 10.1177/08959374870010021701 The online version of this article can be found at: http://adr.sagepub.com/content/1/2/276
Published by: http://www.sagepublications.com
On behalf of: International and American Associations for Dental Research
Additional services and information for Advances in Dental Research can be found at: Email Alerts: http://adr.sagepub.com/cgi/alerts Subscriptions: http://adr.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
Downloaded from adr.sagepub.com by guest on June 9, 2011 For personal use only. No other uses without permission.
ISOLATION OF TWO BOVINE AMELOGENIN PEPTIDES AND THEIR AMINO ACID SEQUENCES J.-H. YEH, T. TAKAGI, AND S. SASAKI1 Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University,
1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan
Adv Dent Res 1(2): 276-281, December, 1987 ABSTRACT
wo peptide fractions of bovine amelogenin having a highly aggregative property to form polymers were purified by chromatography, SDS-polyacrylamide gel electrophoresis, and HPLC. Amino acid sequences of purified peptides were determined by automated Edman degradation. One peptide was found to be composed of 63 amino acid residues having a molecular weight of 7105, and the other of 86 residues having that of 9683. The sequence of the smaller peptide was identical to the C-terminal 63 residues of the amelogenin molecule of 170 residues previously reported, but the larger contained eight residues which are absent in the amelogenin sequence. There is a possibility that the latter peptide might be synthesized independently from mRNA spliced at different positions.
T
INTRODUCTION In the early developmental stages of dental enamel, amelogenin is a predominant constituent and consists of several sub-components (Eastoe, 1960; Sasaki and Shimokawa, 1979). In our previous paper, we succeeded in determining the primary structure of a principal species of bovine amelogenin which is composed of 170 amino acids having a molecular weight of 19,340 daltons (Takagi et aL, 1984). Most of the low-molecular-weight peptides present in developing enamel were thought to be derived from the degradation of this principal amelogenin molecule after being secreted into the enamel (Shimokawa and Sasaki, 1978; Sasaki and Shimokawa, 1979; Sasaki et aL, 1982,1983). However, the existence of a peptide named "leucine-rich amelogenin peptide" (Fincham et aL, 1981) cannot be explained as a degradation product, because it has a composition derived by connection of the amino-terminal 33 residues to the carboxy-terminal 13 without the central sequence of the principal amelogenin molecule. It may be a translaPresented at the Second Carolina Conference on Tooth Enamel Formation, March 16-17,1987, at the University of North Carolina, Chapel Hill Address correspondence and reprint requests to Dr. S. Sasaki, Department of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-Ku, Tokyo 113, Japan. 276
tion product of a different mRNA from that of the abovementioned amelogenin. The purpose of the present study is to investigate the origin and nature of lower-molecular-weight amelogenin peptides present in developing enamel. Here we report two peptide fractions from bovine enamel at the maturation stage; one had a sequence identical to that of the C-terminal 63 residues of the principal amelogenin, and the other, longer, peptide was found to contain an amino acid sequence of eight residues which was not present in the principal amelogenin molecule reported by Takagi et aL (1984).
MATERIALS AND METHODS Preparation of Bovine Amelogenin Peptides Developing bovine incisors were obtained, and enamel was collected at the maturation stage, with chalky consistency. Organic components were extracted from the enamel with 0.5 mol/L acetic acid and lyophilized. The extract (100 mg) was fractionated with a column (2.5 x 190 cm) of Bio-Gel P-30 (Bio-Rad, Richmond, CA) and eluted with 0.1 mol/L formic acid. Two fractions from the P-30 column were lyophilized and applied to a DEAE-Sephacel ion-exchange column of 1.5 x 10 cm (Fincham et aL, 1983). Elution was performed with 7 mol/L urea, 0.05 mol/ L Tris HC1 (pH 7.4) with a linear gradient of 0 to 0.5 mol/L NaCl. The unretained proteins (50 mg) from
Downloaded from adr.sagepub.com by guest on June 9, 2011 For personal use only. No other uses without permission.
TWO BOVINE AMELOGENIN PEPTIDES
Vol. 1 No. 2
277
0.6 -
3.5-
Secretory Stage E
c
• 0.5-
Maturation Stage
Ai A2 B i » t 50
90
110
130
40
150
Fraction Number
120
80
Fraction Number
Fig. 1—Bio-Gel P-30 ckromatography of acetic acid extracts from bovine enamel at secretory and maturation stages eluted with 0.1 mol/L formic acid. Fractions of 5 mL each were collected. Peaks A2 and Bl were further purified. The second peak "Al" is the main amelogenin peak.
Fig. 3 —SP-Sephadex C-25 chromatography of "Dl" fraction in Fig. 2 eluted with a linear gradient of 0 to 0.5 mol/L NaCl in 0.05 mol/ L sodium acetate buffer, pH 3.8. Fractions of 4.6 mL each were collected.
the DEAE-Sephacel column were chromatographed further with an SP-Sephadex C-25 column (2.5 x 12 cm) in 0.05 mol/L. sodium acetate buffer, pH 3.8, with a linear gradient of 0 to 0.5 mol/L NaCl over a total volume of 500 mL. SDS-polyacrylamide gradient gel (15-30%) electrophoresis at an acrylamide/bis ratio of 56:1 was carried out with 0.05 mol/L Tris/glycine buffer, pH 8.3, containing 0.1% SDS. Gels were stained with Coomassie Brilliant Blue R-250 or by being silverstained (Wray et al., 1981). Electrophoresis with polyacrylamide gels (7.5%) containing 6 mol/L urea was carried out at an acrylamide/bis ratio of 37.5:1 with 0.5 mol/L Tris/borate buffer, pH 8.6 (Fincham et al., 1972). After electrophoresis, the desired protein band was excised and recovered by means of an apparatus for protein recovery from the gel (Maxyield Protein Recoverer, Type AE-3590, Atto Co., Tokyo, Japan) according to Hanaoka et al. (1979).
The protein band recovered from the electrophoretic gel was further purified with an HPLC system equipped with a solvent programmer Model 660 and a Data Module 730 (Waters Assoc, Inc., Milford, MA) with a C4 (Hi-Pore RP-304) column (Bio-Rad) eluted with 0.1% trifluoroacetic acid (TFA) solution in a convex gradient (No.3, 0-100%) of 0.1% TFA/acetonitrile.
i.o-
D1
c
0.5
CO
^0.5
\
E
-a
0.3
c O
00 CJ
12
t
E o
Atnino Acid Analysis Samples were hydrolyzed in 5.7 mol/L HC1 containing 0.01% 2-mercaptoethanol in vacuo at 110°C for
/
/
O ra
/D2 ' • ' • "• ' : '• ' • •• / •' '•
A \ \ \ \
' • '• • ••
10
20 30 Fraction Number
0.1
40
Fig. 2-DEAE-Sephacel chromatography of "A2" fraction shown in Fig. 1. Elution was performed with 7 mol/L urea, 0.05 mol/L Fig. 4 —Electrophoresis of "Peak 3" in Fig. 3 on 7.5% polyacrylTris HC1, with a linear gradient of 0 to 0.5 mol/L NaCl and finally amide gel containing 6 mol/L urea in Tris/borate buffer, pH 8.6. with 1 mol/L NaCl in the same buffer. Fractions of 5 mL each were Lane 1: Total enamel extract from the maturation stage. Lane 2: Downloaded from adr.sagepub.com by guest on June 9, 2011 For"Peak personal use other uses 3"only. inNoFig. 3. without permission. collected.
27a
Adv Dent Res December 1987
YEH et al.
TABLE AMINO ACID COMPOSITIONS OF PURIFIED FRACTIONS FROM "A2" AND "Bl" PEAKS
24 hr (Fincham et al., 1983). The hydrolyzates were analyzed with an amino acid analyzer (Model 6300, Beckman Instruments, Inc., Palo Alto, CA). Sequence Determination Automated Edman degradation was performed with an amino acid sequencer (Model 890C, Beckman, Inc., or Model 470A, Applied Biosystems, Foster City, CA) in the presence of polybrene (Takagi et al., 1984). Phenylthiohydantoin derivatives of amino acids were identified with an HPLC system (Model 8100, Spectra Physics, San Jose, CA). Carboxy-terminal Determination Digestion with carboxypeptidase W (Pentel Co., Tokyo, Japan) was carried out according to the method of Umetsu et al. (1981) in 0.02 mol/L sodium citrate buffer, pH 4.0, at 40°C with an enzyme/substrate ratio of 1:50 (w/w). The samples were periodically removed from the digestion mixture, and the free amino acids liberated were determined with an amino acid analyzer.
Amino Acid
"A2" peptides d c b
Asx (D/N) Thr (T) Ser (S) Glx (E/Q) Pro (P) Ala (A) Val (V) Met (M) lie (I) Leu (L) Phe (F) His(H) Trp(W)»
2 2 2 20 30 1 1 3 3 13 2 4
2 2 2 20 30 2 1 3 3 13 2 5 (1)
Total
83
86
2 2 3 20 30 2 1 3 3 13 2 4
"Bl" peptide Peak 2 1 1 14 24 1 1 2 3 11 1 3 (1)
85 63 (Residues/Molecule) *Tryptophan was not determined by amino acid analysis, estimated from the sequence.
RESULTS
The P-30 gel filtration pattern of the acetic acid extract from bovine enamel at the maturation stage is shown in Fig. 1. Two small peaks, "A2" and "BY', were separated after the main amelogenin peak. Peaks "A2" and "BY' were chromatographed on a DEAESephacel column (Fig. 2). In both cases, there were unretained and retained fractions. The unretained fraction from peak "A2" was further chromatographed on an SP-Sephadex C-25 column (Fig. 3). There were several fractions on the profile. The peak 3 fraction was collected and subjected to 7.5% PAGE (Tris-urea system), and was separated into four bands (a, b, c, and d in Fig. 4). Bands b, c, and d were excised from the gel and extracted by means of the protein recovery apparatus. Amino acid analyses indicated that all bands had an almost identical composition, rich in glutamic acid, proline, and leucine (Table). The extract from the third band (band "c") was purified by the HPLC system, and a single peak was obtained (Fig. 5). The amino acid sequence of the peptide purified by HPLC was determined, and the result is shown in Fig. 7. Thirty-four residues of the peptide from the aminoterminal were directly determined by Edman degradation. The carboxy-terminal sequence of the peptide was determined by digestion with carboxy-peptidase W and was found to be Pro-Leu-Gln-Ala-Trp-COOH, which is identical to that of the principal amelogenin molecule having 170 residues (Fig. 7). Its primary structure was finally determined to be composed of 86 amino acid residues having a molecular weight of 9683. The DEAE-Sephacel column chromatographic pattern of peak "BY' was almost identical to that of peak
% z 100^,
o 0.04-
50^ 0.02-
I
