DNA sequence for cloned cDNA for murine amelogenin reveal the amino acid sequence for enamel-specific protein

Vol. 129, No. 3, 1985 June

28,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

1985

Pages

DNA

Malcolm Alan

SEQUENCE THE AMINO

FOR CLONED cDNA FOR MURINE AMELOGENIN REVEAL ACID SEQUENCE FOR ENAMEL-SPECIFIC PROTEIN

L. Snead (l*l, Eduardo C. Lau (11, Margarita Zeichner-David G. Fincham (11, Savio L. C. Woo (2) and Harold C. Slavkin

(11 The University of Southern California, Graduate Program in Craniofacial Biology, Andrus University Park MC-0191, Los Angeles, California (21 The Howard Hughes Medical Institute, Baylor College of Medicine, Received

May 15,

812-818

(11, (1)

Gerontology, 90089-0191

Department of Cell Houston Texas 77030

Biology,

1985

Enamel is the unique and highly mineralized extracellular matrix that covers vertebrate teeth. Amelogenin proteins represent the predominate subfamily of gene products found in developing mammalian enamel, and are implicated in the regulation of the formation of the largest hydroxyapatite crystals in the vertebrate body. Previous attempts to isolate, purify and characterize amelogenins extracted from developing matrix have proven difficult. We now have determined the DNA sequence for a cDNA for the 26-kDa class of murine amelogenin and deduced its corresponding amino acid sequence. The murine amino acid sequence is homologous to bovine or porcine amelogenins extracted from developing enamel matrices. However, an additional lo-residues were found at the carboxy terminus of the murine amelogenin. This is the most complete sequence database for amelogenin peptides and the only DNA sequence for enamel specific fD 1985 Academic Press, Inc. genes.

Enamel

is a unique

vertebrate

teeth.

specialized

cells

of ameloblasts epithelial the

dependent

and polypeptides:

abundant

gel

amelogenins

is

organ:

upon

extracellular synthesized,

instructive

neural

enamelin,

crest

RNA followed

electrophoresis

identified

correspondence

26-

and 22-kDa

should

0006-291X/85 $1.50 Cowright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

ectomesenchyme

cells

be addressed.

812

oral

(l-41;

results

in

of enamel-specific (5-7).

by imnunodetection

(51.

by

differentiation

between

amelogenin

a 62-kDa

of

and organized

Terminal

interactions

and (ii1

covering

secreted,

of two families

messenger

of 28-,

matrix

the ameloblasts.

by ameloblasts (il

of murine

whom all

matrix

and cranial expression

polyacrylamide

mineralized

of the enamel is

coordinated

translation

(*ITo

The enamel

cells

genes

and highly

and analysis

enamelin,

The calcium

In vitro

and three

hydroxyapatite

by more

Vol.

129,

No. 3, 1985

crystalites in the

that

are

vertebrate

maturation,

all

Enamel

for

maturation

encode

the

changes the that

however,

enamel

forming

enamel

(5).

largest

phase amelogenins)

of enamelins

in association

are

class for

of murine the carboxy

than

first

of hydrophobic

approach

obviates

is:

the

and 4) similar

acid

amelogenin

to other

and hydrophilic

of our

2) divergent

3) 180 amino

MATERIALS

species

1) homologous

100 residues;

parameters obstacle

during

(3,6-7,11-13).

inherent

RNAs which dynamic

participating efforts

to porcine

isolated with

in

indicate

to porcine

residues

amelogenins

mineral

by proteolytic

DNA to the messenger

The results

analyzed

organ

area

from

A major

followed

for

problem

isolated

are degraded

of the molecular

terminus;

to this

(8-12).

secretion

complementary

any previously

matrix);

phase

any one tooth

amelogenin

and

mechanism(s)

proteins

the proteins

formation.

growth

the physico-chemical

of both

This

initiation, although

for

mineral

that

image

of enamel

at their longer

become the

mineral

One approach

to elucidate

within

a static

phase

residues

COMMUNICATIONS

(essentially

the

sequence

a continuum

proteins

amelogenins

maintenance

is

simultaneously

by providing

amelogenins

acid

has been to construct

the i!6-kDa

bovine

(8-10).

in order

and that

synthetic

remnants

crystal;

in the forming

occurs

Our approach

of enamel

to control

an enigma

matrix,

by the model,

degradation

only

hydroxyapatite

the amino

participation

imposed

matrix

constituents

leaving

has remained

enamel

their

phase

of the protein

have been postulated

has been to determine developing

RESEARCH

extracellular

At the conclusion

of the calcium

actijon

BIOPHYSICAL

(6-7).

pro,t.eins

terminatioin

AND

in this

by the ameloblasts

HAP crystals

their

initiated

body (8).

almost

resorbed with

BIOCHEMICAL

long from respect

and or bovine (10 the to the

domains.

AND METHODS

The construction and identification of the murine amelogenin cDNA clone, pMa 5-5, has been previouly reported (5). The DNA sequence of pMa 5-5 was determined using the enzymatic dideoxy technique of Sanger (14) after subcloning the cDNA insert into the filamentous phage Ml3 (15). The nested radiolabeled fragments were resolved in polyacrylamide gels cast and electrophoresed using the methods of Sanger and Coulson (16). The base end-to-end

sequences overlaps

for the DNA were read directly from radiograms, melded by and converted to their encoding amino acid residues using 813

Vol. 129, No. 3, 1985

BIOCHEMICALAND

BIOPHYSICALRESEARCH

COMMUNICATIONS

the software programs of Larson and Messing (17). The relative hydropathy leg hydrophilicity versus hydropathyl of the resulting amelogenin was computer determined using the program of Hopp and Wood (181, optimized with the free-energy values for amino acid side chains as suggested by Kyte and Doolittle (19). RESULTS AND DISCUSSION We have,

for

enamel

specific

clone,

pMa 5-5

prior

to the

untranslated messenger

the first

time,

gene. is

experimentally

The deoxynucleotide

shown in Figure

homopolymer region

determined

presence

for

the

that

RNA. As a consequence

the sequence the deduced

of the protein.

amelogenin

of a polyadenylic

ends of the cDNA, a "stop-codon"

indicate

to the carboxy-terminus

l.The

sequence

the DNA sequence

is

amino

The encoded

cDNA acid-tail

to the 3' end of the

sequence

must

corresponding

be complete

153 amino

CTG CAG GGG GGG GGG GGG GGC CAG AGC ATG ATA AGG CAG CCG TAT GLN SER MET ILE ARG GLN PRO TYR CCT TCC TAT GGT TAC GAA CCC ATG GGT GGA TGG CTG CAC CAC CAA PRO SER TYR GLY TYR GLU PRO MET GLY GLY TRP LEU HIS HIS GLN ATC ATC CCT GTG CTG TCT CAA CAG CAT CCC CCG AGT CAC ACC CTT ILE ILE PRO VAL LEU SER GLN GLN HIS PRO PRO SER HIS THR LEU CAG CCT CAT CAC CAC CTT CCC GTG GTG CCA GCT CAA CAG CCC GTG GLN PRO HIS HIS HIS LEU PRO VAL VAL PRO ALA GLN GLN PRO VAL GCC GCC CAG CAA CCA ATG ATG CCA GTT CCT GGC CAC CAC TCC ATG ALA PRO GLN GLN PRO MET MET PRO VAL PRO GLY HIS HIS SER MET ACT CCA ACC CAA CAC CAT CAG CCA AX ATC CCT CCA TCC GCC CAG THR PRO THR GLN HIS HIS GLN PRO ASN ILE PRO PRO SER ALA GLN CAG CCC TTC CAG CAG CCC TTC CAG CCC CAG GCC ATT CCA CCC CAG GLN PRO PHE GLN GLN PRO PHE GLN PRO GLN ALA ILE PRO PRO GLN TCT CAT CAG CCC ATG CAG CCC CAG TCA CCT CTG CAT CCC ATG CAG SER HIS GLN PRO MET GLN PRO GLN SER PRO LEU HIS PRO MET GLN CCC CTG GCA CCA CAG CCA CCT CTG CCT CCA CTG TTC TCC ATG CAG PRO LEU ALA PRO GLN PRO PRO LEU PRO PRO LEU PHE SER MET GLN CCC CTG TCC CCC ATT CTT CCT GAG CTG CCT CTG GAA GCT TGG CCA PRO LEU SER PRO ILE LEU PRO GLU LEU PRO LEU GLU ALA TRP PRO GCG ACA GAC AAG ACC AAG CGG GAA GAA GTG GAT TAA ALA THR ASP LYS THR LYS ARG GLU GLU VAL ASP stop AAAATTCAGAAAATGAGAGAACCGAAGTGGATACTTTGGTTGTTTTTAGGAATAACTCAA CACAATGATTTGTGCCTACAATCACTTAGTAAATTCTGTAACTAAAAATAAGTATCATTA GCAGATAATAAAATGTTTGAAAAATCAAAAAAAAAAAAAACCCCCCCCCCCCCTGCA (adenylation FIGURE

signal)

(poly-d(A)

tail)

1:

Nucleotide sequence predicted translation. as in Materials and

for

the The Methods.

murine sequence

amelogenin of cDNA

814

pMa

cDNA 5-5

clone was

an

and a short

complete acid

for

pMa 5-5 determined

and and

computer analyzed

acid

Vol.

129,

residues

(Figure

analyzed. acid

BIOCHEMICAL

No. 3, 1985

1)were

that

non-homologous

to the 5

amelogenin residues

were

(2)

which

murine

amelogenin

weight

of 23,532

sequences

Although

overlap

the deduced

Da.

The size

bovine

in Figure

stop

signals,

the cDNA sequence

lacks

of extracted

the amino

residues

the

with

from 81 coding

26 amino

intact

extracellular molecular

amelogenin

has been

of mRNA followed

by in vitro

electrophoresis

(5).

(20 II and porcine

(21)

amino

s striking

homology,

2) reveal

amelogenin as well

acid

as some

RESIDUE NUMBER ( 1)MPLPPHPGHPG"INFSYEt:TPLKWYQNMIRHPYTSYG;: (~)MPLPPHPGHPGYINFSYQVLTPLKWYQSMIRHPYPSYGYQ (3)-------- ---______________ Q$,,fIRQPYp$,‘G,‘E (4)MPLPPHPGHPGYINFSYEVLTPLKWY ******************************* WC*****

(I)PMGGNLHHQI~PVVSQQT~&HALQPHHH~PMYPAQQP~~ (2)PMGGWLHHQIIPVVSQQTPQDHALQPPHHIPMVPAQQPVV (3)PMGGWLHHQIIPVLSQQHPPSHTLQPHHHLPVVPAQQPVA ************* *** * ** ****** * *******

100

120

(1)PQQPMMPLPGQHSMTPTQHHQPNLPLPAQQPFQPQPVQPQ (2)PQQPMMPVPGQHQPNLPLPAQTPFQPQSIQPQPHQPLQPH (3)PQQPMMPVPGHHSMTPTQHHQPNIPPSAQQPFQQPFQPQA ********** ************ ** ******

140 (l)PHQPLQPQPSMHPIQPLQPPPPQLPLPMFS---------(2)QPLQPMQPLQPLQPLQPLHPPQPIVQPIPFPPLPPQPLPP (3)IPPQSHQPMQPQSPLHPMQPLAPQPPLPPLFSMQPLSPIL * * ** ** ** * * * * *

*

160

*

180 (l)---------------------------------------(2)MLP,,LPLQAW-------- ____________________ (3)PELPLEAWPATOKTKREEVD-------------------* FIGURE ~-

Comparison residues indicates

200

2:

of

20) to computer

amino

acid

predicted

(4) for the murine homology between

residues

murine

from

porcine

(3) amelogenin

amelogenin murine and

are either

815

(l)(

Ref.

acid

a calculated

of murine

gel

or amino

murine

terminal

Hence,

long

class

by SDS-polyacrylamide

the cDNA were

omitted

sequence.

this

COMMUNICATIONS

were

by cDNA hybrid-selection

of murine, (shown

for

for

in premature

has provided

acid

frames

determinations

class

as 26-kDa

A comparison

shown).

is 180 amino

reading

RESEARCH

to amelogenins,

of this

determined

translation

not

BIOPHYSICAL

resulted

end of the molecule,

protein

previously

all

which

analys i s (data

bases

after

Frame orientations

sequences

further

deduced

AND

21);

polypeptides.

taken from reference porcine or bovine

bovine

(E)(Ref.

The first 2. (*) amelogenins.

26

Vol.

129,

No. 3, 1985

intriguing

BIOCHEMICAL

differences.

amelogenins

determined)

excellent

homology

throughout

different

vertebrate

species

bovine

the

homology

compared

78 residues declines

is

beta-turns

amino high

the mechanism

mineral

phase

maintain

is

the

sequence

for

matches

For residues

data.

bovine

requiring

is these

three

85 residues

101 through as

feature perhaps

and 180,

amelogenins

An intriguing

content

vertebrate

there

residues

porcine

and 21% for

shell

expect

parameters

still

different

The relative

hydropathy

of the

as many as 44

for

either

despite

acid

substitution

maintenance

of these

amelogenins

in preserving

functional

domain gene,

and that

domains

Moreover,

for

amelogenins, resides

to

true,

as

one

physico-chemical

acid

sequence

interest

is

sequence

required

to bring

about

residues

51-180

acid

as well

should

that

the observed

of these

816

for

reflect

that

the

observed

of

mineral

phase

amongst

selected

suggests

that

the

deoxynucleotide

sequence

of the protein. changes

in the DNA coding

substitutions

or tranposition changes

are maintained

to the function

as conserved terminus

shown in Figure

may indicate

residues,

the observation

transition

This

homology

is

domains

requisite

in the amino

of greater

the majority

the

100 amino

Perhaps

Rather,

is

behavior

were

proteins

important

a microenvironment

not

in amino

species.

is intrinsically

the first

are

this similar

or hydrophobic

between

for

probably

of the

of calcium

If

display

changes

function

physico-chemical

crystal.

would

hydrophilic

and maturation. species

by means of their

each of the amelogenin

that

initiation

and growth

formation

property.

and indicates amino

the initation

amelogenins

this

to the

one postulated

the growing

such as hydropathy, distort

contribute

in dispute,

perhaps

surrounding

that

grossly

mammalian

amelogenins

for

crystals,

a hydrophobic

codon.

Moreover, acid

amelogenin.

proline

by which

a microenvironment

in the

100 amino

porcine

acid

(24%)

of enamel

hydroxyapatite

not

first

COMMUNICATIONS

common to all

(2,11-12).

of amelogenins:

to 15% for

their

residues

present

the

RESEARCH

in the molecule.

Although

would

is

of the murine

to the murine

amelogenins

BIOPHYSICAL

The 33 amino-terminal

(as yet

the

AND

in the

in amino third

alteration

acid

base of the in either

or

3

Vol.

129,

BIOCHEMICAL

No. 3, 1985

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Murine

1

-

$0

,46

,60

,80

,I043

,129

,146

,160

,100

,120

,146

,160

Porcine

. -3

,4d

,20

,80

,60

RESIDUE --FIGURE

NUMBER

3:

Computer predicted hydropathy for selected amelogenins. The program of Hopp and Wood (18), optimized with the free-energy values for amino acid side chains of Kyte and Doolittle (19) were used to predict the relative linear average hydropathy of selected amelogenins over seven residue segments.

both

of the

first

substitutions

and second

tend

resulting

chain, (Figure While

to be similar in similar

of the codon.

In these

in the hydropathy

hydropathy

charts

for

instances

character these

for

different

amino their

acid

side

amelogenins

3). the merits

of these

corroboration,

they

of the

for

the

bases

first

gene(s)

33 amino

observations

may be of particular

acid

residues

are common to all

(22-24).

What remains the

enigmatic

putative

shared

mammalian is

require

further

interest

Current

amelogenin(sl.

epitopes

sequences for

will

in relation

evidence

indicates

(2,11-121, but also

amelogenins

examined

the elucidation

amelogenin prepro-region 817

experimental to the evolution that that

not only

are

immunologic

including

human

of the DNA and amino

(3) characteristic

acid

of

Vol. 129. No. 3, 1985 secretory number

proteins, of amelogenin

extracellular critically

BIOCHEMICAL as well gene(s)

matrix. examine

as the exact which

Experiments these

AND BIOPHYSICAL RESEARCH COMMUNICATIONS experimental

contribute in our

determination

to the forming laboratory

of the

enamel

are currently

underway

to

issues. ACKNOWLEDGEMENT

We thank David Filpula, Jim Nagle, Abdul Ally, Pablo Bringas, Jr., and our colleagues for thoughtful discussion and assistance. This work was supported by NIH grants from the NIDR DE 07006, DE02848. M.L.S. is supported by a Research Career Development Award from the NIDR. S.L.C.W. is an investigator of the Howard Hughes Medical Institute. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Kollar,E.J., and Baird,G.R. (1970) J. Embryol. Exp. Morph. 24, 173-186. Slavkin,H.C., Snead,M.L., Zeichner-David,M., Bringas,P.,and Greenberg,G.L. (1984) Role of the Extracellular Matrix in Development, pp.221-252, Alan Liss, New York. Slavkin,H.C.,Zeichner-David,M., and Siddiqui,M.A.Q. (1981) Molecular Aspects of Medicine, pp.125-188, Pergamon Press, Oxford. Snead, M.L.,Bringas, P.,Bessem, C., and Slavkin, H.C. (1984) Dev. Biol. 104, 255-258 Snead,M.L., Zeichner-David,M., Chandra,T., Robson,K.J.H., Woo,S.L.C,, and Slavkin,H.C. (1983) Proc.Natl.Acad.Sci. 80,7254-7258. Termine,J.D., Belcourt,A.B., Christner,P.J., Conn,K., and Nylen,M.U. (1980) J.Biol.Chem. 255,9760-9768. Termine,J.D., Belcourt,A.B., Miyamoto,M.S., and Conn,K. (1980) J.Biol. Chem. 255,9769-9772. Daculsi,G., Menanteau,J., Kerebel,L.M., and Mitre,D. (1984) Tooth Enamel IV, pp.14-18, Elsevier, Amsterdam. Warshawsky,H., Bai,P., Nanci,A., and Josephsen,K. (1984) Tooth Enamel IV, pp.177-l;zAe;l;e;ier, Amsterdam. Doi,Y., Shlmokawa,H., and Termine,J.D. (1984) Tooth Enamel IV, pp.19-23, El&vie;: Amsterdam. Fincham,A.G., Belcourt,A.B., Termine,J.D., Butler,W.T., and Cothran,W.C. (1983) Bi0chem.J. 211,149-154. Fincham,A.G., Belcourt,A.B., Termine,J.D., Butler,W.T., and Cothran,W.C. (1981) Bioscience Reports 1,771-778. Crenshaw,M.A., and Bawden,J.W. (1984) Tooth Enamel IV, pp.109-114, Elsevier, Amsterdam. Sanger,F., Nicklen,S., and Coulson,A.R. (1977) Proc.Natl.Acad.Sci. 74,5263-5467. Hu,N-T., and Messing,J. (1982) Gene 17,271-277. and Coulson,A.R. (1978) FEBS Letters 87,107-110. Sanger,F., Larson,R., and Messing,J. (19821 Nucleic Acid Res.10,39-49. and Woods,K.R. (1981) Proc.Natl.Acad.Med. 78, 3824-3828. Hopp,T.P., Kyte,J., and Doolittle,R.F. (1982) J.Mol.Biol. 157, 105-132. Takagi,T., Suzuki,M., Baba,T., Minegishi,K., and Sasaki,S. (1984) Biochem.Biophy.Res.Comm. 121,592-597. Fukae,M., and Shimizu,M. (1983) Jap.J.Oral Bio1.25 (supplement),29. Slavkin,H.C., Zeichner-David,M., MacDougall,M., Bringas,P., Bessem,C., and Honig,L.S. (1982) Differentiation 23,73-82. Christner,P.J., Lally,E.T., Ads,A.H., and Herold,R.C. (1983) Arch.oral Biol. 28,773-779. Slavkin,H.C., Zeichner-David,M., Ferguson,M.W.J., Termine,J.D., Graham,E., MacDougall,M., Bringas,P., and Bessem,C.G.M. (1982) Oral Immunogenetics and Tissue Transplantation, pp.241-251, Elsevier, Amsterdam. 818