JOURNAL OF BONE AND MINERAL RESEARCH Volume 6, Number 9, 1991 Mary Ann Liebert, Inc., Publishers

Rapid Publication cDNA and Deduced Amino Acid Sequence of Mouse Matrix gla Protein: One of Five Glutamic Acid Residues Potentially Modified to gla Is Not Conserved in the Mouse Sequence TOHRU IKEDA,' AKIRA YAMAGUCHI,' TATEO ICHO,' NOBUO TSUCHIDA,' and SHUSAKU YOSHIKI'

ABSTRACT A cDNA library was constructed using the mouse osteoblastic cell line MC3T3-El treated with la,25-dihydroxyvitamin D3, based on the finding that the treatment increased ninefold the expression of 0.7 kb matrix gla protein (MGP) mRNA. cDNA clones encoding mouse MGP were isolated from the library. The nucleotide sequence showed an open reading frame of 312 nucleotides encoding 104 amino acids. Murine MGP shared 84-89% amino acid sequence homology with bovine, rat, and human MGP. However, there are five glutamic acid residues potentially modified to y-carboxyglutamic acid (gla) in those species: in murine MGP, lysine replaced glutamic acid 37. Also, an extra tyrosine was added at the carboxyl terminus. The significance of the substitution is discussed in relation to the y-carboxylation sites in MGP protein.

INTRODUCTION (MGP) is a low-molecular-weight protein containing y-carboxyglutamic acid (gla) residues. It was initially isolated from demineralized bovine bone matrix"' and also found to be synthesized in many noncalcified tissues.") The C-terminal region of bovine M G P has 20% sequence identity to bone gla protein (BGP), the only other gla protein isolated from bone. These two proteins may thus have diverged from a common a n c e ~ t o r . ' ~In) addition to these bone-associated proteins, seven blood coagulation proteins contain gla residues.") Gla residues are highly conserved among mammalian species and blood coagulation proteins.'J.4) The conserved gla residues of BGP and blood coagulation proteins perform the important function of binding to calc i ~ m 'and ~ ) cell membrane.l6+) Five glutamic acid residues potentially modified to gla are conserved in bovine, rat,

M

ATRIX GLA PROTEIN

and human MGPs.") Although each of the gla residues may be functionally important, whether they are all essential for the function of M G P is as yet unclear. In the present study, mouse M G P cDNA was cloned and sequenced for comparison with MGPs from other mammalian species. Four glutamic acid residues were conserved, but glutamic acid 37 was replaced by lysine in mouse MGP.

MATERIALS AND METHODS

Northern blot analysis The mouse osteoblastic cell line MC3T3-El ('')I was treated with la,25-dihydroxyvitamin D, [ Iu,~S-(OH),D,] (2 x lo-' M) for 24 h. Total RNA was isolated by the guanidine thiocyanate-cesium chloride method,'lI) electrophoresed on a 1.2% agarose-formaldehyde gel, and blotted onto a Hybond-N membrane (Amersham, UK). The membrane was subsequently hybridized as recom-

'Department of Oral Pathology, School of Dentistry, Showa University, Tokyo, Japan. 'Department of Molecular and Cellular Oncology, School of Dentistry, Tokyo Medical and Dental University, Tokyo, Japan.

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mended by the supplier with a 3'P-labeled mixed oligonucleotide probe (33 bases) corresponding to amino acids 1-11 of rat,(3) bovine,('2) and human'") MGPs. The sequence of this probe was 5'-TTCATAG/AGATTCCAT/ GGCTTTCG/AT-GAGATTCATA-3'. The membrane was washed in SSC containing 0.1070 SDS (sodium dodecyl sulfate) at 52°C and exposed to Kodak XAR-5 films with duPont Cronex lightning plus intensifying screens.

1

2

28Sm

Isolation of cDNA clone Poly(A)+ RNA was purified from total RNA with an oligo(dT)cellulose column (Collaborative Res., Bedford, MA).('3) A pcDNA I1 phagemid library was constructed from the RNA using the Librarian I1 system (Invitrogen, San Diego, CA). About 1.5 x lo4 colonies on Hybond-N membranes (Amersham, UK) were screened using the 32Plabeled oligonucleotide probe.

18s-

DNA sequencing One cloned cDNA was sequenced by the method of dideoxynucleotide chain termination' 14) using Sequenase version 2.0 enzyme (United States Biochemical Corp., Cleveland, OH) with an ABI 370 A sequencer (Applied Biosysterns, Foster City, CA). Single-stranded phagemid DNAs were rescued with R408 helper Phage and used as ternplates. Fluorescent -21M13 (Applied Biosystems) was used as a primer. For double-stranded sequencing, alkalinedenatured double-stranded phagemid DNAs were used as templates and fluorescent M13RP1 (Applied Biosystems) as a primer.

RESULTS

Expression of MGP mRNA in MC3T3-EI cells As shown in Fig. 1, mRNA for MGP was detected in MC3T3-El cells. The length of the mouse mRNA was about 700 bases, essentially the same as that of rat mRNA.(') The level of MGP mRNA was increased ninefold in the MC3T3-El cells by treatment with la,25(OH),D,. This was estimated by densitometry from the intensities of the band.

FIG. 1. Northern blot analysis of mouse MGP mRNA. Total RNA (20 fig) prepared from la,25-(OH),D3-treated (lane 1) and untreated (lane 2) MC3T3-El cells were hybridized with the mixed oligonucleotide probe.

rat,(3)and human(12)sequences, respectively. In the mouse sequence, glutamic acid 37, conserved in other mammalian species and potentially modified to gla, is substituted for lysine. The mouse sequence has an extra tyrosine residue at the carboxyl terminus (Fig. 3). To exclude the possibility that these two major differences in mouse MGP may be due to an artefact during cloning, two other clones, MC-11 and MC-12, were analyzed. DNA sequencing indicated that lysine 37 is also present in these clones. An extra tyrosine residue was found to be present based on sensitivity to SspI, since the C-terminal tyrosine codon was within the recognition sequence (data not shown).

Isolation of cDNA clone and DNA sequencing A cDNA library was constructed from the mRNA of MC3T3-EI cells treated with la,25-(OH),D3. From the liDISCUSSION brary, seven positive clones containing a nearly full-length insert were isolated. The cDNA insert of one of the clones, In the present study, we demonstrated that MGP mRNA MC-20, was sequenced. It has an open reading frame of expression was greatly enhanced by treatment with la,25312 nucleotides capable of encoding a protein of 104 (OH),D, in MC3T3-E1, the osteoblastic cell line derived amino acids (Fig. 2). Mouse MGP sequence shares 84.6, from mouse calvariae. The extent of increase is fully con88.5, and 83.7% amino acid homology to b o ~ i n e , ' ' ~ , ~sistent ~) with those previously reported in two rat osteosar-

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cDNA SEQUENCE OF MOUSE MATRIX gla PROTEIN

1

CAGGACACCCGAGACACC

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ATG AAG AGC CTG CTC CCT CTG GCC ATC CTG GCT GCG CTG GCC GTG M K S L L P L A I L A A L A V

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GCA ACC CTG TGC TAC GAA TCT CAC GAA AGC ATG GAG TCC TAT GAA A T L C Y E S E E S M E S Y E

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TTC ATC AAC AGG AGA AAT GCC AAC ACC TTT ATG F I N R R N A N T F M

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CCT CAG CAG AGG TGG CGA GCT AAA GCC CAA AAG AGA GTC CAG GAA P Q Q R W R A K A Q K R V Q E

199

CGC AAC AAG CCT GCC TAC GAG ATC AAC AGA GAG GCC TGC GAT GAC R N K P A Y E I N R E A C D D

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TAC AAG CTG TGT GAG CGC TAC GCC ATG GTC TAC GGC TAC AAC GCT Y K L C E R Y A U V Y G Y N A

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GCC TAC AAC CGC TAC TTC AGG CAG CGC CGA GGA GCC AAA TAT TAG A Y N R Y F R Q R R G A K Ystop

334

CGCGAAGAAACAGTCATTTGGTTGTGGAGTTTCGTTTTATATCTCCTGCAGTAGCATTA

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CTGAAGTATACAGACACGCATGTGTTGCTTGCTCCTTACATGATCTCCTAGCTGGCTGG

452

CCCACTCCTTCCTTCTGCGGGTTGAAAGTAATGAAAGAACAGTATTAAGAAGTGTGTTT

511

ATATATAATAAAATTCTGGCTTGATACGTTAAAAAAAAAAAAAAAA

---a*-!---

FIG. 2. Nucleotide sequence and deduced amino acid sequence of mouse MGP cDNA (DDBJ accession No. D00613). The unique Sspl site is indicated above the C-terminal tyrosine codon.

coma cell lines."61 The great enhancement of MGP mRNA expression was also found in three osteoblastic cell lines, CI1, C20, and C23t87) derived from rat calvariae (manuscript in preparation). These results strongly suggest that the enhancement of MGP mRNA expression with la,25(OH),D, occurs not only in osteosarcoma cells as reported p r e v i o u ~ l y ' 'but ~ ~ also in normal osteoblast and that MGP plays important roles in bone metabolism. In bovine MGP, gla residues were detected at five positions by amino acid sequencing of purified proteintt6'; 80% of the glutamic acid at position 2 and 100% of that at positions 37, 41, 48, and 52 was y-carboxylated.'I6' Although no amino acid sequencing of the proteins was

done, the five glutamic acid residues conserced in rat and human MGP sequences were considered y-carboxylated posttranslationally because they occur in all gla-containing protein^.'^.^ 1 9 ) The substitution of glutamic acid 37 for lysine as observed in mouse MGP thus indicates that the gla 37 in MGPs may not be essential for their function. Such substitution of the gla residue has been reported for a blood coagulation protein, protein C.(*O* I 1 Price et al. aligned all gla-containing proteins, MGP, BGP, and blood coagulation proteins to give maximum homology. Two gla residues, at positions 48 and 52 in MGP, were complately conserved in these proteinst3'; the others were not. This is additional evidence for the nonessential role of gla 37.

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Mouse: Bovine:

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85

FIG. 3. Comparison of the amino acid sequences of mouse, bovine, rat, and human MGPs. The E with an asterisk represents glutamic acid residues potentially modified to gla. The E with asterisk and gla residues in bovine MGP are shaded. The conserved amino acids are boxed. Lysine-37 and tyrosine-85, the two major differences in mouse MGP, are circled.

cDNA SEQUENCE OF MOUSE MATRIX gla PROTEIN

ACKNOWLEDGMENTS Dr. Yoko Otawara-Hamamoto is gratefully acknowledged for her comments and for providing the human and rat MGP oligonucleotide probe, as is also Prof. Tatsuo Suda for providing la,25-dihydroxyvitamin D3.Dr. H. Kodama provided the mouse osteoblastic cell line. Thanks also to Dr. Lowell Adams for his excellent editorial assistance. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.

REFERENCES 1. Price PA, Urist MR, Otawara Y 1983 Matrix gla protein, a new y-carboxyglutamic acid-containing protein which is associated with the organic matrix of bone. Biochem Biophys Res Commun 117:765-771. 2. Fraser JD, Price PA 1988 Lung, heart and kidney express high levels of mRNA for the vitamin K-dependent matrix Gla protein. J Biol Chem 263:11033-11036. 3. Price PA, Fraser JD, Metz-Virca G 1987 Molecular cloning of matrix Gla protein: Implications for substrate recognition by the vitamin K-dependent y-carboxylase. Proc Natl Acad Sci USA 84:8335-8339. 4. Soriano-Garcia M, Park CH, Tulinsky A, Ravichandran KG, Skrzypczak-Jankun E 1989 Structure of Ca” prothrombin fragment 1 including the conformation of the Gla domain. Biochemistry 28:6805-6810. 5 . Lian JB, Gundberg CM 1988 Osteocalcin, biochemical consideration and clinical applications. Clin Orthop 226:267291. 6. Gendreau MA, Krishnaswamy S, Mann KG 1989 The interaction of bone gla protein (osteocalcin) with phospholipid vesicles. J Biol Chem 264:6972-6978. 7. Ware J, Diuguid DL, Liebman HA, Rabiet MJ, Kasper CK, Furie BC. Furie B, Stafford DW 1989 Factor IX San Dimas. J Biol Chem 264:1140-11406. 8. Ryan J, Wolitzky B, Heimer E, Lambrose T, Felix A, Tam JP, Huang LH, Nawroth P, Wilner G, Kisiel W, Nelsestuen GL, Stern DM 1989 Structural determinants of the factor IX molecule mediating interaction with the endothelial cell binding site are distinct from those involved in phospholipid binding. J Biol Chem 264:20283-20287. 9. Schwalbe R, Ryan J, Stern DM, Kisiel W, Dahlback B, Nelsestuen GL 1989 Protein structural requirements and properties of membrane binding by y-carboxyglutamic acidcontaining plasma proteins and peptides. J Biol Chem 264: 20288-20296.

1017 10. Maniatis T, Fritsch EF, Sambrook J 1982 In: Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 202-203. 11. Sudo H, Kodama H, Amagi Y, Yamamoto S, Kasai S 1983 In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol %:191-198. 12. Kiefer MC, Bauer DM, Young D, Herrnsen KM. Masiarz FR, Barr PJ 1988 The cDNA and derived amino acid sequences for human and bovine matrix gla protein. Nucleic Acids Res 165213. 13. Maniatis T, Fritsch EF, Sambrook J 1982 In: Molecular Cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 197-198. 14. Sanger F, Nicklen S, Coulson AR 1977 cDNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74~5463-5467. 15. Price PA, Williamson MK 1985 Primary structure of bovine matrix gla protein, a new vitamin K-dependent bone protein. J Biol Chem 260:14971-14975. 16. Fraser JD, Otawara Y, Price PA 1988 1,25-Dihydroxyvitamin D, stimulates the synthesis of matrix y-carboxyglutamic acid protein by osteosarcoma cells. J Biol