Bioscience Reports i, 771-778 (1981)
Printed in Great Britain
771
D e n t a l e n a m e l m a t r i x : s e q u e n c e s of t w o
amelogenin polypeptides
A.G. FINCHAM,*% A.B. BELCOURT,**# 3.D. TERMINE,#
W.T. BUTLER~w and W.C. COTHRANw
#Skeletal Matrix Biochemistry Section, Laboratory of
Biological Structure, National Institute of Dental
Research, National Institutes of Health, Bethesda,
Maryland 20205, U.S.A.; and w
of Dental Research,
School of Dentistry, University of Alabama in Birmingham,
Birmingham, Alabama 35294, U.S.A.
(Received 15 September 1981)
The amino acid sequences of a leucine-rich amelogenin
p o l y p e p t i d e ( L R A P ) and a tyrosine-rich amelogenin
polypeptide ( T R A P ) , isolated from foetal bovine enamel
matrix, were determined.
Both LRAP and TRAP
o c c u r r e d in two f o r m s ; in e a c h case, one of the
m o l e c u l a r s p e c i e s a p p e a r e d to be shortened at the
COOH terminus by 2 and t~ residues, respectively.
A
striking finding was that LRAP and TRAP had identical
sequences for the first 33 residues but were almost
completely different for the remaining 12 amino acids.
Developing dental enamel matrix contains a complex mixture of
proteins of unusual composition ( 1 - # ) .
The proline-rich amelogenins
c o n s t i t u t e the major protein group of the extracellular matrix and
have been postulated to play a role in the mineralization and struct u r a l o r g a n i z a t i o n of the foetal enamel (1, #-6).
With increasing
enamel mineralization, there is a reduction in the proportion of the
h i g h e r - m o l e c u l a r - w e i g h t (M r = 25 000-30 000) amelogenins and an
increase in the proportion of smaller-sized (M r = 5000) polypeptides
(2J-9).
It has been suggested (g-10) that this change in proportions
r e f l e c t s a discrete degradation process, functionally related to enamel
maturation.
In this paper, we describe the isolation and primary
s t r u c t u r e of two of the principal amelogenin polypeptides of the bovine
e n a m e l m a t r i x , d e s i g n a t e d the leucine-rich amelogenin polypeptide
(LRAP) and the tyrosine-rich amelogenin polypeptide (TRAP).
M a t e r i a l s and M e t h o d s
Enamel matrix isolation and chromatographic fractionation
B o v in e e n a m e l matrix protein was obtained from foetuse% 5-7
months in utero, as previously described (2).
Primary fractionation
Present address: *Faculty of Medicine, University of West Indies,
Mona, Kingston 7, Jamaica, West Indies.
**Groupe de Recherches
INSERM U 157, Centre de Recherches Odontologiques, Facult~ de
Chirurgie Dentaire, Universit~ Louis Pasteur, 1 Place de l'H6pital, 67000 Strasbourg, France.
9 1981 The Biochemical Society
772
FINCHAM
ET AL.
was c a r r i e d out on columns of Bio-Gel P30 e l u t e d with 0.1 M f o r m i c
acid at 10~
(11).
C o l u m n f r a c t i o n s w e r e f u r t h e r purified by
r e c h r o m a t o g r a p h y in the s a m e s y s t e m and then by c h r o m a t o g r a p h y on
calibrated
c o l u m n s of S e p h a c r y l $200 ( P h a r m a c i a ) , as d e s c r i b e d
elsewhere (2).
Analytical and preparative electrophoresis
Chromatographic fractions were monitored using a Tris-borate-urea
p o l y a c r y l a m i d e - g e l system (12).
Preparative electrophoretic fractionation was conducted using cylindrical gels (14 x 150 mm) buffered
with the same system.
Preparative gels were each loaded with 2-4
mg of protein and electrophoresed for 20-24 h at 2 mA per gel at
10~
The positions of the separated protein bands were detected in
a reference gel by precipitation in 10% (w/v) trichloroacetic acid.
Protein was isolated from the remaining gels by slicing the appropriate
regions from partially frozen gels, homogenizing the slices in 0.5 M
formic acid, and extracting the homogenates for 24 h at 4~ in this
solvent.
The extracts were freed of polyacrylamide particles by
c e n t r i f u g a t i o n and then lyophilized.
The freeze-dried preparations
were desalted by chromatography on columns of Bio-Gel P2 (2.5 x 30
cm) Muted with 0.1 M formic acid, and the protein fractions were
further purified by chromatography on a column (1.6 x 90 cm) of
Bio-Gel P l 0 eluted w i t h the same solvent.
Five to ten mg of
electrophoreticaIly purified protein was obtained in this manner.
Amino acid compositions
The amino acid and h e x o s a m i n e c o m p o s i t i o n s w e r e d e t e r m i n e d as
p r e v i o u s l y d e s c r i b e d (2).
T r y p t o p h a n was not d e t e r m i n e d in the
hydrolysates,
COOH-terminal analysis
S a m p l e s of the purified a m e l o g e n i n s (0.5-1 rag) w e r e dissolved in 5
ml os the 0.2 M N - e t h y l m o r p h o l i n e a c e t a t e b u f f e r (pH g.5) c o n t a i n i n g
norleucine as an i n t e r n a l s t a n d a r d ,
The p r o t e i n was digested with
c a r b o x y p e p t i d a s e - A ( S i g m a a g a r o s e - i n s o l u b l e e n z y m e , 3 units) at 37~
f o r t i m e periods up to ig0 rain.
S a m p l e s of t h e digest w e r e i n a c t i v a t e d by addition of 20% f o r m i c acid and w e r e c l e a r e d f r o m the
agarose-enzyme
by c e n t r i f u g a t i o n , and the s u p e r n a t a n t s w e r e e v a p orated.
The residues w e r e t a k e n up in pH-2.2 sodium c i t r a t e b u f f e r
and s u b j e c t e d to amino acid analysis. Amino acid c o n c e n t r a t i o n s w e r e
n o r m a l i z e d to the norleucine c o n c e n t r a t i o n s .
Sequence determinations
Sequences were determined by automated Edman degradation with a
Model g90c Beckman automatic sequencer operated at 56~
The
procedure utilized was the Slow Protein-Quadrol Program (No. 042772,
Beckman Sequencer Manual) using 0.5 M Quadrol and 2.0 mg Polubrene
(13).
P h e n y l t h i o h y d a n t o i n (Pth) amino acids were identified by
high-performance liquid chromatography (HPCL) (14) and by thin-layer
ENAMEL
POLYPEPTIDE SEQUENCES
773
chromatography (15). Pth-arginine and Pth-histidine were identified in
the water layer with the phenanthrenequinone spot test and the Pauly
diazo reagent, respectively.
Cyanogen bromide cleavage
A sample of TRAP (1.3 mg) was dissolved in 1.5 ml of 70%
f o r mic acid and incubated with 10 mg of CNBr at 2#~ for # h. The
mixture was diluted with 20 ml of water and lyophilized to remove
formic: acid and CNBr.
The products were separated on a 1 . 5 - x
150-cm column of 5ephadex G-50 (Superfine) eluted in 0.1 M acet i c
acid.
Two peptides, representing residues 2-29 (CB2) and 30-#2
(CB3) of TRAP (see Fig. 2) were well resolved by this procedure.
Peptide CB2 was used for studies of organic phosphate whilst CB3 was
submitted to aut om a t e d Edman degradation.
Assessment of the organic phosphate content of TRAP
In order to determine the presence of phosphorylated serines in
T R A P , t h e CNBr f r a g m e n t CB2 (residues 2-29; see Fig. 2) was
s u b j e c t e d to t h e f o l l o w i n g alkaline elimination experiment.
One
aliquot of peptide CB2 was hydrolysed with 6 N HCI at I03~ for 2r
h in an N 2 atmosphere and analysed for inorganic phosphate and amino
acids (16).
A second aliquot was t r e a t e d with 1.0 ml of 0.2 M NaOH
at 37~ for 6 h. The sample was neutralized while in an ice bath by
the cautious addition of 0.2 ml of 1.0 N HCI.
One portion of this
sampJe was analysed directly for inorganic phosphate eliminated by the
alkaline t r e a t m e n t .
Another portion was hydrolysed with 6 N HCI as
above prior to amino acid analysis in order to assess the loss of serine
(if the phosphate were indeed at t ached to the 6 - h y d r o x y group of a
seryl residue. )
Results
Chromatographic and electrophoretic characterization
The LRAP was isolated from P30 columns (10,11) and rechrom at ographed as described above. LRAP was obtained from Sephacryl $200
# M guanidine hydrochloride columns as a single peak with a Kav of
0.47 and an apparent M[ of 6500. The TRAP was isolated from P30
columns as two peaks ( T R A P - I and T R A P - 2 ) which were found to
rechromatograph distinctly in this system but appeared at the same
elution position ( K a v = 0.59) on the $200 columns with an apparent
M r of 5000 (2,10)11~.
With analytical electrophoresis the LRAP polypeptides yielded two
principal bands, whereas the two TRAP polypeptides each appeared as
a single band, and had identical mobilities,
The LRAP chromato=
g r a p h i c p r e p a r a t i o n was fractioned into its two principal el ect rop h o r e t i c components by the use of preparative gel electrophoresis.
The two LRAP components were designated as LRAP-1 and L RA P-2
(Fig. 1), the l a t t e r having the gr e a ter e l e c t r o p h o r e t i c mobility.
77t~
FINCHAM
ET
AL.
Fig. i. Analytical Tris-borate-urea polyacrylamidegel electrophoresis
of leucine-rich amelogenin
polypeptides
(LRAP), LRAP-I and LRAP-2, isolated
from bovine enamel matrix P30 column fraction F2.
(i) Total bovine enamel matrix; (2) P30 LRAP
fraction; (3) LRAP-I; (4) LRAP-2.
Amino acid compositions
The a m i n o acid c o m p o s i t i o n s , e x p r e s s e d as residues per m o l e c u l e ,
a r e s h o w n in T a b l e 1.
It was noted t h a t the L R A P c o m p o n e n t
L R A P - I d i f f e r e d f r o m L R A P - 2 only in the a b s e n c e of one residue
e a c h of g l u t a m i c acid, proline, alanine, and leucine, whilst the two
T R A P p r e p a r a t i o n s a p p e a r e d to d i f f e r only by one additional glycine
residue in the T R A P - 2 .
H e x o s a m i n e s w e r e not d e t e c t e d in any of the
preparations.
Amino acid sequences
The s e q u e n c e s for T R A P and L R A P shown in Fig. 2 w e r e d e t e r mined in the following m a n n e r .
E d m a n d e g r a d a t i o n of the T R A P - 1
s p e c i e s i n d i c a t e d the first 34 residues.
The s e q u e n c e of residues
ENAMEL
POLYPEPTIDE SEQUENCES
Table i.
775
Amino acid composition of purified bovine
amelogenins
(Residues/molecule*)
Leucine-rich
Aspartic acid
Threonine
Serine
Glutamic acid
Proline
Glycine
Alanine
Valine
Methioninet
!soleucine
Leucine
Tyrosine
Phenylalanine
Histidine
Lysine
Arginine
Tryptophanw
Totals
(LRAP)
LRAP-I
LRAP-2
1.9
1.0
1.9
2.2
10.5
2.2
0.2
1.0
2.1
1.7
5.6
2.9
1.0
2.8
1.0
1.0
1.9
1.0
1.8
2.8
12.0
2.1
0.9
1.0
2.1
1.8
6.8
2.9
1.0
2.8
1.0
1.0
42
(2)
(I)
(2)
(2)
(Ii)
(2)
(0)
(i)
(3)
(2)
(6)
(3)
(i)
(3)
(i)
(i)
(I)
46
(2)
(i)
(2)
(3)
(12)
(2)
(i)
(i)
(3)
(2)
(7)
(3)
(I)
(3)
(!)
(i)
(i)
Tyrosine-rich
TRAP-I
1.3
1.0
2.7
3.4
8.5
4.0
0.0
i.I
2.2
1.7
3.2
5.8
i.i
2.8
1.0
i.i
43
(i)
(i)
(3)
(3)
(9)
(4)
(0)
(i)
(3)
(2)
(3)
(6)
(i)
(3)
(i)
(I)
(I)
(TRAP)
TRAP-2
1.3 (i)
1.0 (i)
2.8 (3)
3.4 (3)
8.5 (9)
4.9 (5)
0.0 (0)
1.0 (1)
2.2 (3)
1.7 (2)
3.1 (3)
5.8.(6)
1.2 (I)
2.9 (3)
1.0 (i)
1.2 (I)
(2)
45
*Integer values are given in parentheses.
Average of three
analyses.
~Values corrected from sequence data.
w
sequence
and carboxypeptidase data.
3 0 - 4 2 was d e t e r m i n e d by E d m a n d e g r a d a t i o n of p e p t i d e CB3.
Carboxypeptidase A experiments showed that glycine was the COOHterminal residue.
The T R A P- 2 preparation was sequenced by identif i c a t i o n of t h e f i r s t 44 r e s i d u e s by Edman degradation, and by
carboxypeptidase A digestion that showed a COOH-terminal tryptophan.
F o r t h e s t r u c t u r e of L R A P, all 46 residues were determined by
a u t o m a t e d Edman degradation of LRAP-2.
The sequence of the first
39 residues of LRAP-1 was identical to that of LRAP-2.
Organic phosphate
In order to assess the presence and nature of organic phosphate in
T R A P - l , CNBr peptide CB2 was subjected to B-elimination.
A ft er
mild a l k a l i n e t r e a t m e n t , about 1 mol phosphate/mol peptide was
liberated from CB2. Accompanying the phosphate elimination was the
loss of 0.33 mol threonine and 0.73 tool serine.
Discussion
Low-molecular-weight amelogenins of a distinctive composition were
first described by Seyer and Glimcher (17), who isolated four such
FINCHAM
776
5
ET AL.
i0
15
TRAP :
Met-Pro-Leu-Pro-Pro-His-Pro-Gly-His-Pro-Gly-Tyr-lle-Asn-Phe-
LRAP :
Met-Pro-Leu-Pro-Pro-His-Pro-Gly-His-Pro-Gly-Tyr-Ile-Asn-Phe-
TRAP :
20
25
30
Ser-Tyr-Glu-Val-Leu-Thr-Pro-Leu-Lys-Trp-Tyr-Gln-Ser-Met-Ile-
LRAP :
Ser-Tyr-Glu-Val-Leu-Thr-Pro-Leu-Lys-Trp-Tyr-Gln-Ser-Met-Ile-
TRAP :
35
40
Arg-His-Pro~ProITyr-Gly-Tyr-Glu-Pro-Met-Gly-Gly-Trp
LRAP :
Arg-His-Pro~Pro-Le~ProtPro-Met-Leu-Pro-Asp-Leu-Pro-Leu-GlutAla
45
I
Fig. 2.
Sequences of bovine enamel matrix amelogenins;
TRAP and LRAP.
TRAP, tyrosine-rich
amelogenin
polypeptide.
LRAP, leucine-rich
amelogenin polypeptide.
Regions of difference are
indicated by the enclosed sections.
fractions (designated ' E l - E # ' ) from the foetal bovine enamel matrix.
The fraction E4 was noted to contain 140 res/1000 of tyrosine, whilst
E3 had a similar proportion of leucine. Fractions E l - E 2 had a lower
p r o p o r t i o n of leucine and tyrosine but showed enhanced levels of
glycine (100 res/1000).
These amelogenin polypeptides had Mr values
of about 5000, and the E3 and E# components t oget her constituted
approximately 15-20% of the matrix protein. Partial sequence studies
of t h e s e polypeptides (5,17-19) led to the postulate that specific
phosphoseryl-containing sequences might act as mineral nucleating sites
(5).
More recently (l 1), it has been shown that polypeptides of
similar compositions to the bovine LRAP and TRAP can be isolated
from other mammalian foetal enamels.
Extensive fractionation and analytical studies of the bovine enamel
matrix (2,#,8,10,11) have shown that the P30 column LRAP comprises
the principal leucine-rich matrix component and that T R A P - I and -2
fractions are the tyrosine-rich components.
The amino acid compositions for TRAP and LRAP (Table 1) are in close a g r e e m e n t with
the Et~ data of Seyer and Glimcher ( l g ) and of Fincham (20), and of
the E3 data reported by Papas et al. (19).
We conclude that the
LRAP fraction sequenced in this study can be equated with the E3
poJypeptide and that the TRAP fraction represents the Et~ polypeptide.
From the compositional and sequence data it appears that the two
forms of TRAP are identical except that the T RA P-2 species has an
additional Gly-Trp sequence at the COOH terminus. Similarly the two
LRAP forms are identical except at the COOH terminus, where the
P r o - L e u - G l u - A l a sequence is missing from the LRAP-1 form.
These
d a t a s u g g e s t t h e p o s s i b i l i t y of specific cleavage of Gly-Gly and
L e u - P r o bonds as a p a r t of t h e natural formation of the lowmolecular-weight amelogenins.
ENAMEL
POLYPEPTIDE SEQUENCES
777
The sequence for the TRAP (Fig. 2) is in a g r e e m e n t with the
partial sequence of E4 reported by Zalut et al. (21) but is quite
d i f f e r e n t from other sequence determinations (18,19).
In particular
the G l u - P S e r - T y r and G l u - P S e r - L e u sequences (19) postulated to play
an i m p o r t a n t role in enamel mineralization were not found.
The
B-elimination experiments did suggest that Set 16 and/or Set 35 and
even Thr 21 could be partially phosphorylated.
The most striking f e a t u r e of the com parat i ve structures for TRAP
and LRAP (Fig. 2) is the occur r ence of an identical sequence for 33
residues followed by separate sequences of 12 residues that are almost
totally d if f e r ent from each other.
Thus, the tyrosine and leucine
enrichment of TRAP and LRAP occur near the COOH terminus. The
conservation of the 33-residue s t r e t c h signifies that it plays a common
and important biological function in the two molecular species.
Another interesting finding is that the sequence of TRAP (bovine)
is almost identical to the NH2-terminal sequence reported by Fukae et
al. (22) for larger {M r = 21 000 and 27 000) amelogenins isolated
from foetal pig enamel.
R ecent data from our laboratories (Fincham
AG, Belcourt AB, Termine 3D, Cothran WC & Butler WT, unpublished
data) also indicate that a principal 30 000-mol.-wt. amelogenin has an
NH2-terminal sequence identical to that of TRAP. These data indicate
that the lower-molecular-weight TRAP and LRAP substances arise by
p r o t e o l y t i c c l e a v a g e of h i g h e r - m o l e c u l a r - w e i g h t amelogenins, as
suggested earlier from developmental biological data (2,g,10).
From the sequence data, TRAP and LRAP have closely similar
sizes (M r ~5000), and yet the LRAP appears with an apparent Mr of
about 6500 when chromatographed on $200 in it M guanidine hydrochloride.
The LRAP is also clearly resolved from the TRAP on the
P30 columns.
The e l e c t r o p h o r e t i c separation of the two forms of
LRAP is explicable in terms of the additional Glu 45, but chromatographic separation of LRAP from TRAP and of the two variants of
T R A P is not r e a d i l y e x p l a i n e d by the compositional data.
The
additional Trp 45 in T R A P- 2 and the appearance of a proline-rich
sequence ( - P r o - P r o - L e u - P r o - P r o - M e t - L e u - P r o - ) in the COOH-terminal
r e g i o n of L R A P s u g g e s t s t h a t hydrophobic and/or conformational
e f f e c t s may be involved in these separations.
References
i. Eastoe JE (1979) J. Dent. Res. 58B, 753-763.
2. Termine JD, Belcourt AB, Christner PJ, Conn KM & Nylen MU
(1980) J. Biol. Chem. 255, 9760-9768.
3. Seyer J & Glimcher MJ (1977) Calc. Tiss. Res. 24, 253-257.
4. Fincham AG (1979) Calc. Tiss. Int. 27, 65-73.
5. Glimcher MJ (1979) J. Dent. Res. 58B, 790-806.
6. Smales FC (1975) Nature (Lond.) 258, 772-774.
7. Robinson C, Briggs HD, Atkinson PJ & Weatherell JA (1979) J.
Dent. Res. 58B, 871-880.
8. Fincham AG, Belcourt AB & Termine JD (1981) Caries Res., in
press.
9. Lyaruu DM, Belcourt AB, Fincham AG & Termine JD (1981) Calcif.
Tiss. Intl., in press.
77g
FINCHAM
ET AL.
I0. Fincham AG, Belcourt AB & Termine JD (in press) in Chemistry
and Biology of Mineralized Connective Tissues (Veis A, ed),
Elsevier.
Ii. Fincham AG, Belcourt AB, Lyaruu DM & Termine JD (1981) Calcif.
Tiss. Intl., in press.
12. Fincham AG, Burkland G & Shapiro IM (1972) Calcif. Tiss. Res.
9, 247-259.
13. Tart GE, Beecher JF, Bell M & McKean DJ (1978) Anal. Biochem.
84, 622-627.
14. Bhown A~ Mole JE & Bennett JC (1981) Anal. Biochem. llO,
355-359.
15. Inagami T & Murakami K (1972) Anal. Biochem. 47, 501-504.
16. Linde A, Bhown M & Butler WT (1980) J. Biol. Chem. 255,
5931-5942.
17. Seyer J & Glimcher MJ (1971) Biochim. Biophys. Acta 236~
279-291.
18. Seyer J & Glimcher MJ (1977) Biochim. Biophys. Acta 493,
441-451.
19. Papas A, Seyer J & Glimcher MJ (1977) FEBS Lett. 79, 276-280.
20. Fincham AG (1979) Biochem. J. 1819 171-175.
21. Zalut C, Henzel WJ & Harris HW (1980) J. Biochem. Biophys.
Meth. 39 11-30.
22. Fukae M~ Tanabe T~ Ijiri H & Shimizu M (1980) Tsurumi Univ.
Dent. J. 6 (2), 87-94.