Vol. 188, No. 3, 1992 November
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ALTERNATIVE SPLICING OF THE AllOUSE AMELOGENIN WCMARY RNA TRANSCRIPT CONTRIBUTES TO AMELOGENIN HETEROGENEITY Eduardo C. Lau; James P. Simmer, Pablo Bringas, Jr., Dora D.-J. Hsu, Ching-Chun Hu, Margarita Zeichner-David, Flavia Thiemann, Malcolm L. Snead, Harold C. Slavkin and Alan G. Fincham University of Southern California, Center for Craniofacial Molecular Biology, 2250 Alcazar Street, CSA 1st Floor, Los Angeles, California 90033 Received
September
28,
1992
m: A heterogeneous population of amelogenin proteins is derived from a single copy of the mouse amelogenin gene. To investigate the one gene-multiple protein enigma, we designed a study to distinguish between alternative splicing and proteolytic cleavage models. A pulse of [35S]methionine labeling demonstrated that multiple amelogenins are synthesized concurrently, a result consistent with an alternative splicing mechanism. Using reverse transcription and polymerase chain reaction we cloned a segment from the 5’ end of a mouse amelogenin mRNA and connected it to a previously isolated abbreviated cDNA clone. Four additional cDNAs derived from alternatively spliced amelogenin mRNAs have been cloned and characterized. The five transcripts encode amelogenins 180,156,141,74, and 59 amino acids in length. 0 1992 Academic Press, Inc.
Amelogenins
are the principal
protein components of the extracellular
matrix of
developing dental enamel (1,2). In man (3-5), and in cow (6), copies of the amelogenin gene (AMEL) have been identified on both the X and Y-chromosomes, but in mice the amelogenins are expressed from a single gene located on the X-chromosome (3,7). Despite being expressed from one gene, multiple amelogenins are observed in the developing mouse enamel matrix (810). The observed heterogeneity of amelogenins in enamel protein preparations has historically been accounted for by proteolysis of a single proamelogenin (1 l), by translational start codon skipping (12), and recently by alternative splicing. In this report we present data obtained from a metabolic labeling study coupled to the isolation of four additional amelogenin cDNA clones that suggest alternative splicing is a major mechanism for the generation of amelogenin heterogeneity in mice. MATERIALS
AND METHODS
Metabolic Labeling of Amelogenin with [35S]Methionine. Maxillary and mandibular molars from 23 four-day postnatal mice were extracted and incubated at 37°C in BGJb medium (GIBCO BRL, Gaithersburg, MD) for 30 min as previously described (13). The explants were *To whom correspondence should be addressed.
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0006-291X/92 $4.00 Copyright 0 1992 by Academic Press, Im. All rights of reproduction in an! form reserved
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transferred to BGJb methionine-depleted medium for 30 min. [3sS]Methionine was added to a final concentration of 50 mCi/ml and the cultures incubated for another 15 min. The molars were divided into four groups and chased by incubation in unlabeled BGJb medium for 0,20,40, and 60 min. Each group was rinsed in phosphate-buffered saline and the pulp and follicle tissue removed. The mineralized tissue was then extracted in 10% (v/v) acetic acid for 3 h at 4”C, desalted on a Bio-Gel P2 (Bio-Rad Laboratories, Richmond, CA) column and the soluble proteins lyophilized. The enamel protein extract was fractionated by reversed phase high performance liquid chromatography (HPLC) as previously described (10). This procedure concentrates the hydrophobic amelogenin fraction in the retarded region of the chromatogram, while the more hydrophilic components (serum proteins, enamelins, etc.) are eluted in the column void. Fractions corresponding to the principal chromatographic peaks were individually collected and the radioactivity in each fraction quantified by liquid scintillation spectrometry. RNA Isolation. Total RNA was extracted from tooth organs dissected from 4-day postnatal mice according to a standard protocol (14) with adjustments made for preparation of smaller quantities. Extension of the Abbreviated Mouse cDNA Clone pMa5-5. The amelogenin cDNA insert of pMa5-5 plasmid was excised by PstI digestion, and subcloned into the PsrI site of pGEM1 plasmid vector (Promega Corp., Madison, WI) to produce a construct named pGEM17-2 (9). For RT-PCR amplification of amelogenin mRNA, the upstream amplimer was 5’TACTCGAGATGCCGAAATGGGGACCTTG-3’ containing an XhoI site linked to the previously published bovine amelogenin leader sequence (15). The downstream amplimer was 5’-TGCAGCCATCCACCCATGGGTT-3’. The polymerase chain reaction (PCR) product was then digested with XhoI and NcoI, and ligated to pGEM17-2 which had been cut with 5afI and NcoI. The resulting construct was named pMa16, which contained the coding region and 3’ untranslated sequence for the 26-kDa mouse amelogenin. Cloning of Alternatively Spliced Amelogenin cDNA Clones. For PCR amplification of amelogenin cDNAs, a different downstream amplimer (5’- ATCCAC!ITCTICCCGC’ITGGTCT TGTC -3’) which annealed to the 3’ end of the amelogenin coding region was used. The PCRamplified DNAs were cloned into pUCl18 plasmid vector using the Amp-N-Clone kit from Gold BioTechnology, Inc. (St. Louis, MO). The ligation mix was used to transform XLl-Blue cells (Stratagene, La Jolla, CA) and recombinants were screened by in situ hybridization of bacterial colonies according to standard procedures (16). Plasmid DNA from colonies giving the strongest hybridization signal was grouped by the pattern produced by Hue111 restriction and selected clones sequenced using the Sequenase Version 2.0 kit (US Biochemicals, Cleveland, OH).
RESULTS Metabolic
Labeling
of Amelogenins.
Our initial hypothesis was that a single
proamelogenin is secreted into the enamel matrix and subsequently cleaved by proteases into a complex mixture of proteins. To examine this hypothesis, cultured tooth organs were labeled with [ssS]methionine and the acid-soluble material was fractionated by reversed phase HPLC. We anticipated that a 15-minute exposure of tooth explants to the radiolabel would concentrate radioactivity in a single chromatographic peak. This prediction was not met. The results (Figure 1) showed that the metabolic label was distributed among multiple chromatographic fractions, previously characterized as being amelogenins (10). Furthermore, the number and intensity of radioactive peaks depicted in the reversed phase HPLC separation (Figure 1) did not change during a subsequent chase for up to 75 min after initiation of labeling (data not shown).
The S-End Sequence of Mouse Amelogenin cDNA. The major amelogenin protein product is 180 amino acid residues in length (M180) which migrates as a 25-26 kDa band on SDS-polyacrylamide gels (10). An abbreviated cDNA clone, pMa5-5, encoding residues 28-180 of this mouse amelogenin, had previously been isolated and the DNA sequence determined (17,18). Using specific oligodeoxynucleotide primers coupled to reverse transcription and 1254
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6000
1
Figure
2
3
4
5 6 7 6 HPLC Peak No.
9 10
1. Metabolic labeling and HPLC fractionation of amelogenins in mouse molars cultured in vitro in the presence of [3?3]methionine for 15 minutes. (A) The absorption profile of reversed phase HPLC separation of acid extracted enamel proteins. (B) Bar graph of the radioactivity (cpm) determined in each fraction corresponding to a HPLC chromatographic peak. The numbers beneath the bars correspond to the major peaks of the above profile.
chain reaction, we amplified the coding region for the missing amino-terminal region and linked it to pMa5-5 by means of an overlapping restriction site. The resulting construct, polymerase
pMa16, contains all of the coding region for the M180. The nucleotide and deduced amino acid sequences of the coding region are shown in Figure 2. This corrects two previously published amino acid assignments (19): His(9) changes to Ser(9), and Phe(15) changes to Leu(15). The published nucleotide sequence for pMa5-5 (18) contained a typographical error: nucleotide 241 should be changed from a G to a C. Alternatively
Spliced Amelogenin cDNAs. Alternatively spliced mRNAs from 4-day
postnatal mouse molars were identified using a strategy based upon reverse transcriptionpolymerase chain reaction (RT-PCR) employing a 5’ amplimer containing the previously published sequence for the bovine amelogenin leader sequence (15), and a 3’ amplimer complementary to the coding region for the carboxyl-terminal amino acid residues of the Ml 80. The resulting products were cloned into pUCll8
plasmid. Over 90% of the cDNA clones (e.g.
clone # 17) contained the coding region for a 59-amino acid amelogenin. This clone corresponds to the mRNA for the leucine-rich amelogenin polypeptide (LRAP) previously isolated from cow as a 48-amino
acid cleavage product (20).
Sequence analysis revealed that clone # 11
represented a re-isolate of pMa16, which encodes the 180-amino acid amelogenin (M180). 1255
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G~XP~TAGATGCCGAMTOGOCTTG
M
1 1
ACC CCT
21
Thr
41
T
ATG CCC CTA CCA CCT Met Pro Leu Pro Pro
61 121
G
Pro
CCC ATG Pro Met
AND
ATT
TTG TTT Leu Phe
L
-36 -12
Ile
BIOPHYSICAL
RESEARCH
CAT CCT GGA AGC CCT GGT TAT His Pro Gly Ser Pro Gly Tyr
GGT GGA TGG CTG CAC CAC CAA ATC
Gly
Gly
Trp
AGT Ser
241
CCC
CAG CAA CCA ATG
Pro
Gln
Leu
His
His
Gln
Ile
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GCC TGC CTC CTG GGA GCA GCT TTT Ala Cys Leu Leu Gly Ala Ala Phe ATC AAC TTA AGC TAT Ile Am Leu Ser Tyr
TTG AAG TGG TAC CAG AGC ATG ATA AGG CAG CCG TAT Leu Lys Trp Tyr Gin Ser Met Ile Arg Gln Pro Tyr
181 61 81
BIOCHEMICAL
ATC Ile
CCT TCC TAT Pro Ser Tfr
GCT Ala
-1 -1
GAG GTG CTT Glu Val Leu GGT TAC GM Gly Tyr Glu
CCT GTG CTG TCT CAA CAG CAT Pro Val Leu Ser Gln Gln His
CCC CCG Pro Pro
CAC ACC CTT CAG CCT CAT CAC CAC CTT CCC GTG GTG CCA GCT CAA CAG CCC GTG GCC His Thr Leu Gln Pro His His His Leu Pro Val Val Pro Ala Gin Gin Pro Val Ala Gln
Pro
Met
ATG Met
CCA GTT CCT Pro Val Pro
GGC CAC CAC TCC An: Gly His His Ser Met
ACT CCA ACC CAA CAC CAT Thr Pro Thr Gin His His
101
CAG CCA AAC ATC CCT CCA TCC GCC CAG CAG CCC T'K CAG CAG CCC TTC CAG CCC CAG GCC Gln Pro Asn Ile Pro Pro Ser Ala Gln Gln Pro Phe Gln Gin Pro Phe Gln Pro Gln Ala
361 121
ATT Ile
421 141
CTG GCA CCA CAG CCA CCT CTG CCT CCA CTG TTC TCC ATG CAG CCC CTG TCC CCC ATT Leu Ala Pro Gln Pro Pro Leu Pro Pro Leu Phe Ser Met Gln Pro Leu Ser Pro Ile
481 161
CCT GAG CTG CCT CTG GAA GCT TGG CCA GCG ACA GAC AAG ACC MG Pro Glu Leu Pro Leu Glu Ala Trp Pro Ala Thr Asp Lys Thr Lys
301
Figure.
CCA CCC CAG TCT CAT Pro Pro Gln Ser His
CAG CCC An; Gln Pro Met
CAG CCC CAG TCA CCT CTG CAT CCC ATG CAG CCC Gln Pro Gin Ser Pro Leu His Pro Met Gln Pro
CGG GAA GM Arg Glu Glu
CTT Leu
GTG GAT Val Asp
The coding region of the mouse amelogenin cDNA clone pMa16 and its deduced amino acid sequence. The segments corresponding to amplimer sites for RT-PCR reactions are shown in bold. Ser(9), Leu(lS), changes from previous publications.
and the C at nucleotide
241 indicate
Clones # 2, #55 and # 4 coded for novel amelogenins of 156 (M156), 141 (M141) and 74 (M74) amino acid residues, respectively (Figure 3). Each of these clones coded for amelogenins that were collinear to the Ml80 except for an internal deletion. The splice junctions of the deleted
Ml80 Ml56 Ml41 M74 M59
Figure.
(1)MPLPPHPGSPGYINLSYEVLTPLKWYQSMI (1)MPLPPHPGSPGYINLSYEVLTPLKWYQSMI (l)MPLPPHPCSPGYINLSYE-----------(1)MPLPPHPGSPGYINLSYEVLTPLKWYQSMI (1)MPLPPHPGSPGYINLSYEVLTPLKWYQSMI
Ml80 ,.,I56 Ml41 M74 MS9
(3l)RQPYPSYGYEPMGGWLHHQIIPVLSQQHPP (31) R Q p _ . _ _ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ _ _ H p p (19) . . _ _ _ _ _ _ _ _ _ _ . . . _ _ _ _ _ _ _ _ _ _ ” p p (3l)RQP-----....-------.........~. (3~)RQP..---------......----------
Ml80 Ml56 Ml41 M74 MS9
(6l)SHTLQPHHHLPVVPAQQPVAPQQPMMPVPG (37)SHTLQPHHHLPVVPAQQPVAPQQPMMPVPG (22)SHTLQPHHHLPVVPAQRPVAPQQPMMPVPG (34)-------------..._____________ (34)---------------.-.------------
Ml80 Ml56 Ml41 M74 M59
(9l)HHSMPTTQHHQPNIPPSAQQPFQQPFQPQA (67)HHSMPTTQHHQPNIPPSAQQPFQQPFQPQA (5Z)HYSMPTTQHHQPNIPPSAQQPFQQPFQPQA (34)-----_- - -............._._ (34) - - - - - -________________________
Ml80 Ml56 Ml41 M74 MS9
(12l)IPPQSHQPMQPQSPLHPMQPLAPQPPLPPL (97)IPPQSHQPMQPQSPLHPMQPLAPQPPLPPL (82)IPPQSHQPMQPQSPLHPMQPLAPQPPLPPL (34)-----------------.-PLAPQPPL (34)-------------
Ml80 Ml56 Ml41 M74 MS9
(1Sl)FSMQPLSPILPELPLEAWPATDKTKREEVD (127)FSMQPLSPILPELPLEAWPATDKTKREEVD (112)FSMQPLSPILPELPLEAWPATDKTKREEVD (4S)FSMQPLSPILPELPLEAWPATDKTKREEVD (34)----PLSPILPELPLEAWPATDKTKREEVD
- - -
- - -_-
PPL - - -
Comparison of the deduced amino acid sequences of five mouse amelogenins derived from alternative splicing. 1256
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portions were preceded at their 5’ termini by AG; a characteristic of splice junctions (21). Clone # 55 displayed a possible polymorphism with two single-base substitutions at nucleotides 230 (G to A) and 274 (C to T). These changes resulted in Gln(77) to Arg(77) and His(92) to Tyr(92) substitutions.
JMSCUSSION The alternative processing of primary RNA transcripts (pre-mRNAs) has been the subject of a number of reviews (21-27). The pattern of alternative splicing of amelogenin pre-mRNA may be determined by factors acting in cis (information stored in the sequence of the transcript itself) and in fruns (general or specific splicing factors). The expression of specific trans-splicing factors may be postulated when the pattern of splicing changes in a developmental stage-specific manner, as in the cases of rat rroponin-T (28) and a-TM genes (29). The recent observations of developmental variation in mouse amelogenin mRNA expression (R. I. Couwenhoven and M. L. Snead, manuscript in preparation) suggest that specific trans-splicing factors are participating. We assume the mouse amelogenin gene has a structure similar to that published for humans (4), and propose that alternative splicing of the primary mouse RNA transcript be modeled as the scheme in Figure 4. The four smaller mouse amelogenin cDNAs have been produced by employing three internal 3’ acceptor sites within exon 6 (the largest exon). The clone coding for the Ml41 has the additional feature that exon 5 was skipped. The pattern of alternative splicing suggests that the process may be regulated during 3’ acceptor site selection. Although
mechanisms of amelogenin function are unknown, there is evidence that
amelogenins are critical to enamel biomineralization. The phylogenetic distribution of amelogenins has been examined by immunohistochemical methods which indicated that the evolutionary appearance of amelogenins coincides with the transition from enameloid to enamel (30). A 5-kilobase deletion in an amelogenin gene has been shown to result in a case of human 1
2
3
M,Gv
Fieure.
4:6
M156
3
M141
3
M74
3
M5g
3
>
alternative splicing. The intron-exon structure of the X-chromosomal copy of the human amelogenin gene is shown at the top of the figure. The lines correspondto introns and the bars to exons. The exons are numbered above while the number of nucleotidesper exon is indicated below each bar. The exonic structuresof five alternatively splicemouse spliced mRNA are diagrammed. Hollow bars indicate a partial exon segment that has been deleted by selection of an internal 3’ acceptor site. Amelogenin
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X-linked
BIOCHEMICAL
amelogenesis
imperfecta
(3 1).
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conserved primary
structures of
amelogenins, their developmental and tissue-specific expression and their dominance as the major matrix protein constituent of developing enamel (32), all support the hypothesis that amelogenins play a central role in enamel formation. Evidence for amelogenin alternative splicing has been found in cow (33-35), pig (36) and human (4) species. We have observed multiple amelogenins, of 28, 26 and 22-kDa apparent sizes, among the cell-free translated products of mouse enamel organ mRNAs (37). The addition of mice to the list of species displaying alternative splicing suggests that this is a general feature of amelogenin synthesis and may be of functional significance.
The cloning of the LRAP
mRNA from cow (33) made the connection that alternatively
spliced transcripts result in
translation products secreted into the enamel matrix (20).
The amino acid compositions,
molecular mass, and isoelectric points of mouse amelogenins have been deduced (Table 1). The significantly lower p1 values of the smallest amelogenins, M74 and M59, could indicate different functional roles.
Furthermore, a 26-kDa amelogenin in developing rat is secreted by pre-
ameloblasts and internalized
by odontoblasts (38). This observation suggested a role of It has been shown that larger amelogenins bind
amelogenin in intercellular communication.
hydroxyapatite, a feature that implies a possible role in the formation and control of crystal size and habit (39,lO). In addition, pig amelogenins of 11 and 13 kDa apparent size are free in the
Table 1. The deduced amino acid compositions, isoelectric points and molecular
weights of the five mouse amelogenins Ml80 amino acid
Res/ Mol
Ala Val Leu Ile
:: 16
pro
474
Met Phe Trp GlY Ser Thr CYS Vr Asn Gln ASP Glu LYS Arg His Total PI Mr
Ml56
Ml41
Res/ Mol
Rest 1000
Resl Mol
Rest 1000
Resl Mol
Res/ 1000
ResJ Mol
Resl
38.9 38.9 88.9 38.9 244.4 50.0 16.7 16.7 33.3 66.7
7
7
4
27.0 40.5
f
33.9
11 3 18 3 :
148.6 40.5 243.2 40.5 27.0 13.5
8
3”9 7 3
49.6 35.5 85.1 28.4 276.6 49.6 21.3
l?2 ii2
135.6 50.8 203.4 33.9 33.9 0.0
:
rli\
2
27.0
2
33.9
29.03
; 0 3 .:
40.5 67.6 0.0 40.5 54.1 13.5
4 i 3 :
67.8 50.8 0.0 50.8 33.9 16.9
4 2 11
27.0 67.6 40.5 27.0 13.5 999.6
2 :
141
2 5 3 2 1 74
33.9 84.7 50.8 33.9 16.9 999.7
: 3 2 22
i 3 2 14
33.3 0.0 33.3 11.1 138.9 11.1 33.3 16.7 11.1 77.8
: 3 2 12
180
1000
156
999.9
: 6 12 : 6 2 25
M59
Resl 1000
44.9 38.5 89.7 32.1 262.8 51.3 19.2 12.8 19.2 64.1 38.5 0.0 19.2 12.8 141.0 12.8 32.1 19.2 12.8 76.9
9
M74
164 4: z 2 3 10
l%
63:8 35.5
z 0 4 19 2
14:2 134.8 14.2 35.5 14.2 14.2 78.0 1000.2
2 1 59
1000
6.563
6.691
6.428
4.909
4.909
20.291 kDa
17.508 kDa
15.720 kDa
8.353 kDa
6.738 kDa
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enamel fluid and appear to regulate Ca2+ concentration (40,41). We hypothesize the amelogenin gene represents a complex transcription unit expressing a heterogeneous mixture of alternatively spliced amelogenins that participate in multiple ways to the development and biomineralization of the tooth enamel matrix.
Acknowledgments. This publication was supported by NIH, NIDR, research grant DE-02848. We thank Mr. Darrin Simmons for his excellent technical assistance.
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