DNA AND CELL BIOLOGY Volume 9, Number 5, 1990 Mary Ann Liebert, Inc., Publishers Pp. 303-309

Molecular

Cloning of a Novel Bone-Forming Compound: Osteoinductive Factor

LINDA MADISEN,* MIKE NEUBAUER,* GREG PLOWMAN,* DAVID ROSEN.t PATRICIA SEGARINI,t JAMES DASCH,t ANDREA THOMPSON,! J. ZIMAN.t HANNE BENTZ.t and A.F. PURCHIO*

ABSTRACT cDNA clones

encoding osteoinductive factor (OIF) have been isolated from a bovine osteoblast library. Sequence analysis of these clones indicated that the 105-amino-acid OIF is synthesized as a larger 299-aminoacid precursor, the carboxyl terminus of which is cleaved to yield the mature protein. Northern blot analysis of bovine osteoblast mRNA revealed two OIF-specific transcripts of 1.9 and 2.4 kb. The polymerase chain reaction was used to obtain clones coding for human OIF from the osteosarcoma cell line, MG-63. The human OIF cDNA encodes a precursor of 298 amino acids that exhibits 94% identity to the bovine protein. Northern blot analysis of various cell lines and tissues indicated that expression of OIF transcripts is limited and may be restricted to cells of bone lineage. INTRODUCTION

weight of 22-28 kD, which is reduced to 12 kD after deglycosylation; the sugar residues appear necessary for aclaboratories have reported the extraction of tivity. bone and cartilage-inducing molecules from pulverized In this study we report the use of degenerate oligonubone. Urist et al (1984) isolated a 18.5-kD protein from cleotides and the polymerase chain reaction (PCR) to isobovine bone (bovine bone morphogenic protein) capable late cDNA clones encoding human and bovine OIF. The of inducing mesenchymal cells to differentiate into bone OIF protein sequence is highly conserved between human and cartilage. Osteogenin, a 28- to 43-kD glycoprotein iso- and bovine species and its expression appears to be tightly lated from dehydrated diaphyseal bovine matrix powder, regulated. lar

Several

induces new bone formation when mixed with demineralized bone matrix extracted with guanidine hydrochloride (Sampath et al, 1987; Paralkar et al, 1989). Wozney et al (1988) have isolated and expressed cDNAs encoding three new bone morphogenic proteins (BMP), each of which can induce the formation of cartilage in vivo. Cartilage-inducing factor A (found to be identical to TGF-01; Seyedin et al, 1986) and cartilage-inducing factor B (now termed TGF-02; Seyedin et al, 1987) were isolated from bovine demineralized bone and shown to cause fetal rat muscle mesenchymal cells to produce cartilage-specific macromolecules. Recently, a novel protein termed osteoinductive factor (OIF) was isolated from bovine bone which, in conjunction with TGF-/31 or TGF-/32, induces bone formation at ectopic sites (Bentz et al, 1989). The protein has a molecu-

*Oncogen, Seattle,

WA 98121.

tCollagen Corp., Palo Alto, CA 94303. 303

MATERIALS AND METHODS Cell lines and tissue culture Fetal bovine tissue (5-6 months) was obtained from a local slaughterhouse (Armour Dixon, CA). Bone was obtained from the iliac crest (cancellous bone) and was minced into pieces of approximately 1 mm3. Bone chips were rinsed three times with minimal essential medium (MEM) and 500 U/ml penicillin and 500 fig/m\ streptomycin ((5 x

Pen/strep) to remove marrow. Bone chips were digested with a mixture of 0.2% collagenase (CLS II, Worthington) and 0.1% trypsin in PBS for 30 min at 37CC. Digested bone particles were collected, washed, and placed in culture in medium (BGJb) containing 2 mMglutthen

MADISEN ET AL.

304 were obtained from the outgrowth of expiants after 2 weeks in culture. Cells grown for RNA isolation were used within the first two passages. Approximately 70% of the cells stained positively for alkaline phosphatase. The MG-63 osteosarcoma cell line was obtained from Dr. Greg Mundy (University of Texas, San Antonio) and grown in Dulbecco modified Eagle's medium (DMEM) plus 10% fetal calf serum (FCS) (Hyclone); 781 T cells were obtained from Dr. Eugenie Kleinerman (University of Texas, MD Anderson Cancer Center) and propagated in DMEM containing 10% FBS, nonessential amino acids (GIBCO), 0.3 mg/ml glutamine, and sodium pyruvate.

aminé, 10% FBS, and 1 x Pen/strep. Cells

cDNA

library construction and screening

Double-stranded cDNA was synthesized from polyadenylated RNA isolated from bovine osteoblasts as described (Sambrook et al, 1989). cDNAs larger than 500 bp were cloned into XgtlO as described (Webb et al, 1987). The complete amino acid sequence of mature OIF has been determined (Bentz et al, 1990). The library was first screened with the "P-labeled 128-fold degenerate oligonucleotide probe

[5'-GTATCATTAGCCTTACAAAA-3'] G

G

G C T

T

G

G

complementary to DNA encoding Phe-Cys-Lys-Ala-AsnAsp-Thr (amino acids 61-67 of mature OIF) as described (Madisen et al, 1988). Seventy positive clones were identified, 20 of which were selected for secondary screening with the 128-fold degenerate probe [5'-GCCTTACAAAAAGTATCATC-3'] T

G

G

T G C

G

G

complementary to DNA coding for Asp-Asp-Thr-Phe-CysLys-Ala (amino acids 58-64 of mature OIF), and subsequent screening with the 64-fold degenerate probe [5 '-GCAAAATACAACAAAAT-3 '] G C T

G

T

T

G

the sequence Ala-Lys-Tyr-Asn-Lys-Ile (amino acids 1-6 of mature OIF). From the remaining 18 positives, several overlapping clones, spanning approximately 2.14 kb, were characterized. A clone containing a 1.9-kb insert, pOIF-54, was sequenced on both strands by the dideoxy chain-termination method (Sanger et al, 1977). An overlapping clone, pOIF-32, which extended the 3'-untranslated region out to 2.14 kb, was also sequenced. Several other clones were then sequenced so that the entire OIF coding region was confirmed by at least two clones.

encoding

Human OIF clones were obtained by following a cDNA cloning strategy based on PCR techniques (Saiki et al, 1985). Single-stranded cDNA was prepared from MG-63 polyadenylated RNA and then used as a template for amplification. Primers for the PCR consisted of an oligonucleotide identical to DNA encoding the 6 amino-terminal amino acids of bovine OIF and a 36-mer (5'-ACTTTCA-

TTTATATGTTGTACCAATAGAGGTTAAAA-3 ') complementary to DNA immediately following the stop codon of human OIF (D. Rosen, unpublished observation).

Using a DNA thermal cycler (Perkin Elmer-Cetus) and Tag DNA polymerase (Stratagene), 33 cycles of amplification were performed following a step program (94°C, 1 min; 45°C, 2 min; 72°C, 3 min). The PCR reaction products were electrophoresed on an agarose gel and a band of the predicted size was stained with ethidium bromide, isolated, and subcloned for sequence analysis. The human OIF coding region was confirmed by sequence analysis of five independent clones. A partial cDNA clone was isolated from an MG-63 cDNA library confirming the 5' coding sequence of human OIF. Northern blot

analyses

extracted from various tissues using the method and polyadenylated RNA was isolated as described (Sambrook et al, 1989). RNA was fractionated on a 1% agarose-formaldehyde gel (Lehrach et al, 1977), transferred to a nylon membrane (Hybond N; Amersham), and hybridized to 32P-labeled probe (Madisen Total RNA

was

detergent lysis

etal, 1988).

RESULTS cDNA and deduced amino acid sequence bovine OIF

of

DNA sequence analysis of OIF-specific cDNA clones spanning 2.14 kb revealed a single open reading frame encoding a deduced 299-amino-acid protein (Fig. 1). The carboxy-terminal 105 amino acids were identical to the mature OIF sequence (Bentz et al, 1989, 1990). The fourth ATG is predicted to encode the initiating methionine because it is followed by a stretch of 18 hydrophobic and uncharged amino acids (overlined in Fig. 1) characteristic of a signal peptide (see hydropathy plot, Fig. 2). This initiator codon also has a purine located 3 nucleotides upstream, consistent with the census sequence for translational start

sites

(Kozak, 1986). Four potential N-glycosylation sites are located at positions 79, 215, 246, and 259 although the site at position 79 is not favorable due to the presence of a proline residue at position 82 (Bause, 1983). Mature OIF is released from its precursor following proteolytic cleavage after the Asn residue at position 194.

Sequence of human OIF precursor A human osteosarcoma cell

line, MG-63,

was

found to

produce low levels of OIF (D. Rosen, unpublished observation). Polyadenylated RNA was extracted from these

305

OSTEOINDUCTIVE FACTOR cDNA

GCTAATGCAAGCCATGACCA

-397

TCTATTGAGGAAAACCACAAAAAACTTCAAAACAGCTACAACGGTATTCTAAGAAGACTTCAATTGCGTACCTTTGTTCAAGGTACCTAATTATTAAAA AACTGAAATTATCAACATTGCTTGGATTTTTGATAGAAAAAGAACTAAAACCATTTTCTGAAACTATTTTTCAGAATATCACTGATACATAAAAACTTT TAGCATCTAATTAAAGATCACAAAGGGTTAAATACTGTTCGCTGGCCCCTGCTGCGTATACCCTGCCAAAAAGTCCTCTAAGCTTTTAAATATTGCTTC GATGGTCTGCATTTTTATTTCCAGGGAAAAAGAGAGTATTGTCCCACAGTCAGCAGGCCACTAGCTTATTAGTTTTCAGTCACCTTAATTTCTATTAAA

-298

-416

20 _10 Thr Leu Gin Ser Thr Leu Leu Leu Phe Leu Phe Val Pro Leu Ile Lys Pro Ala Pro Pro Ser Gin Gin CCA GCA CCA CCA TCT CAG CAG ATG AAG ACT CTG CAA TCT ACA CTT CTC CTG TTC CTG TTT GTG CCT CTG ATA AAG Met

Lys

_

_

35

-199 -100 -1

75

45

Tyr Glu Asp Lys Asp Ser Arg Ile Ile Tyr Asp Tyr Gly Thr Asp Asn Leu Glu Glu Thr Phe Phe Ser Gin Asp GAT TCA CGC ATT ATC TAT GAT TAT GGA ACA GAT AAT CTT GAA GAA ACC TTT TTT AGC CAA GAT TAT GAG GAT AAA

150

70 60 Leu Asp Gly Lys Ser Thr Lys Glu Lys Glu Thr Met Ile Ile Val Pro Asp Glu Lys Ser Phe Gin Leu Gin GAG AAA CCC GAT ATA GTA AGT TTT CAA TTA CAA TAC CTG GAT GGA AAA AGT ACT AAG GAA AAA GAA ACT ATG ATA

225

Tyr

#

85

95

Lys Asp Glu Asn Ile Thr Pro Leu Pro Pro Lys Lys Glu Asn Asp Glu Met Pro Thr Cys Leu Leu Cys Val Cys AAA GAT GAG AAT ATA ACG CCA TTA CCC CCC AAG AAA GAA AAT GAT GAA ATG CCC ACA TGC CTG CTA TGT GTT TGT 110

300

120

Leu Ser Gly Ser Val Tyr Cys Glu Glu Val Asp Ile Asp Ala Val Pro Pro Leu Pro Lys Glu Ser Ala Tyr Leu TTA AGT GGC TCT GTA TAC TGT GAA GAA GTT GAC ATT GAT GCT GTA CCA CCT TTG CCA AAG GAA TCA GCC TAT CTT

375

145 135 Asn Lys Ile Lys Lys Leu Thr Ala Lys Asp Phe Ala Asp Ile Pro Asn Leu Arg Arg Leu Asp TAT GCA CGA TTC AAC AAA ATT AAA AAG CTG ACC GCC AAA GAT TTT GCA GAC ATA CCT AAC TTA AGA CGA CTT GAT

450

170 160 Phe Thr Gly Asn Leu Ile Glu Asp Ile Glu Asp Gly Thr Phe Ser Lys Leu Ser Leu Leu Glu Glu Leu Thr Leu TTT ACG GGA AAT TTG ATT GAA GAC ATA GAA GAC GGT ACT TTT TCA AAA CTT TCT CTG TTA GAA GAA CTT ACA CTA

525

Tyr Ala Arg Phe

185

r195

-

Ala Glu Asn Gin Leu Leu Lys Leu Pro Val Leu Pro Pro Lys Leu Thr Leu Phe Asn Ala Lys Tyr Asn Lys Ile GCT GAA AAT CAA CTA CTG AAG CTT CCA GTT CTC CCT CCC AAG CTT ACT TTA TTT AAT GCA AAA TAT AAC AAA ATC

210

220

Lys Ser Arg Gly Ile Lys Ala Asn Thr Phe Lys Lys Leu His Asn Leu Ser Phe Leu Tyr Leu Asp His Asn Ala

AAG AGT AGA GGA ATC AAA GCA AAT ACA TTC AAA AAA CTG CAT AAC CTC TCC TTC CTC TAC TTG GAT CAC AAT GCT

235

600

675

245 -#-

Leu Glu Ser Val Pro Leu Asn Leu Pro Glu Ser Leu Arg Val Ile His Leu Gin Phe Asn Asn Ile Thr Ser Ile TTG GAA TCT GTG CCT CTT AAT TTA CCA GAA AGT CTG CGT GTA ATT CAT CTT CAG TTT AAC AAC ATA ACT TCA ATT

750

270 -*- 260 Thr Asp Asp Thr Phe Cys Lys Ala Asn Asp Thr Ser Tyr Ile Arg Asp Arg Ile Glu Glu Ile Arg Leu Glu Gly ACA GAT GAT ACA TTC TGC AAG GCT AAT GAC ACC AGT TAT ATT CGG GAC CGC ATT GAA GAG ATA CGC TTG GAG GGC

825

295 285 Asn Pro Val Ile Leu Gly Lys His Pro Asn Ser Phe Ile Cys Leu Lys Arg Leu Pro Ile Gly Ser Tyr Ile AAC CCC GTC ATC CTG GGA AAA CAT CCC AAC AGT TTT ATC TGC TTA AAA AGA TTG CCT ATA GGG TCA TAC ATT TAA

900

CCACTATCAATGCAGCATAGCTAAAGTACACACATACTTATAATCTGTCTCAACAATGTCTAAAGGAGCATAAATATTTAATATTAATTTTGCATCTGA

999

CTATTGAAGGAACTTATGCTTTAAGCAAGAATGTTAAAAAAAAAGTCTTATATATAACAACAAGTAAAAAGTAAGATTGAATCTCTAAGTTCAAAACAG

1098

AAATGTGAAAATATTTGAACAGAATTACAAAATCCCTGTAGTTCGTAATAGAGTAACACTTAAAAGGTACGTTTTTATATAAATACCCAAAATTAAGAA

1197

GTGTTACAAAGTTAAAAGATAAGTCCAAGAAACTTTCAACTGTCTTTCCTGGCTTCCACTGGATCCCTAAAGCTTTTAAGGCATATGTTCCAAAGACTT

1296

TGAAAAGCTGAATATTTCTAAGGATCTCTCACTTTTCTTTCTTTTATGATTTATACATTATTCATTATGAAGTAGGAACTCTGTTTTCTTTCTTTTAAG

1395

GCAGCTACTATATTTTTACTTAGCCTGAGAAATAGGGTATAGTCTTATACCTAAGCAAAACTTTCCAAATAAAGCATAATTTTACTCTCTTGACAAAGA

1494

GCTAATATAGTAATGTTCAATTATTCTGTTTTGTGGTTACACAATTAAAAGCTTTAGTGAAGTAGTACGAAAATTTAGTTTAACTCATAAATAATCAAC

1593

ATTCTAAGTCCACCACAAAACATTTTAGGAAGCTAGGCACATCAAAATCAGTATGCTTATTAAAAAAACTGAAAGTCCACTCTGTTTCTCAGAATATAG

1692

CCATAAAGTTCTCCACTGTAGGTATGTCAT

1722

Nucleotide sequence of bovine OIF cDNA and deduced amino acid sequence. Several cDNA clones (pOIF-54, pOIF-18, and pOIF-32) spanning 2.14 kb were sequenced using the dideoxy chain-termination method (Sanger et al, 1977). The mature OIF sequence is boxed and the putative signal peptide is overlined. Potential glycosylation sites are indicated by asterisks.

FIG. 1.

306

MADISEN ET AL.

HYDROPATHY

50

100

150

200

250

300

AMINO ACID FIG. 2. Hydropathy plot of bovine OIF cDNA sequence. The 299-amino-acid bovine OIF precursor was analyzed using the algorithm of Kyte and Doolittle (Kyte and Doolittle, 1982) for domains having a hydrophilic or hydrophobic nature. A leu-rich stretch of hydrophobic and uncharged amino acids, characteristic of a signal peptide, follows the initiating methionine. A second hydrophobic region is located around amino acid 100.

cells and used as a template for isolating cDNA clones encoding human OIF using PCR techniques. By minimizing the length and number of PCR cycles and sequencing several independent clones, we avoided possible errors due to the use of Tag polymerase. A comparison of the deduced amino acid sequences of human and bovine OIF is shown in Fig. 3. The two proteins share 94% amino acid homology in the pro-region (including a stretch of 94 identical residues) and 96% homology within the mature region. The human precursor is 298 amino acids, one shorter than its bovine counterpart. Two potential TV-glycosylation sites are found in the human OIF precursor corresponding to those at positions 215 and 259 in the bovine protein (Fig. 3A). The human and bovine squences share 92% homology throughout the coding region (Fig. 3B).

Northern blot

analyses

Hybridization of 32P-labeled bovine pOIF-54 to polyade-

nylated RNA isolated from bovine osteoblasts revealed two major bands of 1.9 and 2.4 kb (Fig. 4A). Similar analysis of mRNA from the human osteosarcoma cell lines

781T (Fig. 4B, lane 1) and MG-63 (Fig. 4B, lane 2) revealed two predominant mRNA species of 3.6 and 1.9 kb, as well as a less abundant 2.4-kb band. The amount of OIF-specific mRNA in 781 T cells is higher than in MG-63 cells (see legend to Fig. 4B) mRNA from other human osteosarcoma cell lines including HOS, K-HOS, MNNG-OS (obtained from ATCC), UT-OS, and SA-OS (obtained from Dr. G. Mundy, University of Texas, San Antonio) were screened for OIF-specific transcripts and were all negative. Similarly, total RNA from various human tissues including brain, thymus, placenta, kidney, and colon was also negative for OIF transcripts (Fig. 4D).

DISCUSSION Bovine and human cDNA clones have been isolated for novel growth factor, OIF. Sequence analysis of these clones predicts that OIF is synthesized as a larger precursor that has been highly conserved between these two species. The remarkable degree of homology within the pro-region a

OSTEOINDUCTIVE FACTOR cDNA

307

Bovi ne Human

MKTLQSTLLLFLFVPLIKPAPPSQQDSRIIYDYGTDNLEETFFSQDYEDKYLDGKSTKEK

Bovine Human

ETMIIVPDEKSFQLQKDENITPLPPKKENDEMPTCLLCVCLSGSVYCEEVDIDAVPPLPK ..-V.I.N...L.A.

120 119

Bovine Human

ESAYLYARFNKIKKLTAKDFADIPNLRRLDFTGNLIEDIEDGTFSKLSLLEELTLAENQL

180 179

Bovi ne Human

LKLPVLPPKLTLFNAKYNKIKSRGIKANTFKKLHNLSFLYLDHNALESVPLNLPESLRVI .A_N. .T.

.L.L.T.F..SI.NI...

60 60

*

.S. i

*

240 239

*

*

Bovi ne

*

HLQFNNITSITDDTFCKANDTSYIRDRIEEIRLEGNPVILGKHPNSFICLKRLPIGSYI

A Human

.A.IV.F *

HUMAN BOVINE

ATGAAGACTCTGCAGTCTACACTTCTCCTGTTACTGCTTGTGCCTCTGATAAAGCCAGCA

HUMAN BOVINE

CCACCAACCCAGCAGGACTCACGCATTATCTATGATTATGGAACAGATAATTTTGAAGAA

HUMAN BOVINE

TTT

TCCATATTTAGCCAAGATTATGAGGATAAATACCTGGATGGAAAAAATATTAAGGAAAAA G

HUMAN BOVINE

ACACCATTACCTCCCAAGAAAGAAAATGATGAAATGCCCACGTGTCTGCTGTGTGTTTGT

HUMAN BOVINE

TTAAGTGGCTCTGTATACTGTGAAGAAGTTGACATTGATGCTGTACCACCCTTACCAAAG

HUMAN BOVINE

GAATCAGCCTATCTTTACGCACGATTCAACAAAATTAAAAAGCTGACTGCCAAAGATTTT

HUMAN BOVINE

GCAGACATACCTAACTTAAGAAGACTCGATTTTACAGGAAATTTGATAGAAGATATAGAA

HUMAN BOVINE

GATGGTACTTTTTCAAAACTTTCTCTGTTAGAAGAACTTTCACTTGCTGAAAATCAACTA

HUMAN

CTAAAACTTCCAGTTCTTCCTCCCAAGCTCACTTTATTTAATGCAAAATACAACAAAATC

G

G

T

C

G

T

T

C

A

G

C

HUMAN BOVINE

TTGGACCATAATGCCCTGGAATCCGTGCCTCTTAATTTACCAGAAAGTCTACGTGTAATT

HUMAN BOVINE

CATCTTCAGTTCAACAACATAGCTTCAATTACAGATGACACATTCTGCAAGGCTAATGAC

HUMAN BOVINE

ACCAGTTACATCCGGGACCGCATTGAAGAGATACGCCTGGAGGGCAATCCAATCGTCCTG

HUMAN BOVINE

GGAAAGCATCCAAACAGTTTTATTTGCTTAAAAAGATTACCGATAGGGTCATACTTTTAA

A

TT

537 597

IT

T

A

477

A

AAGAGTAGGGGAATCAAAGCAAATGCATTCAAAAAACTGAATAACCTCACCTTCCTCTAC C

657

T

T

717

G

TA

777

T

T

AC

417

C

HUMAN BOVINE

T

357

G

C C

C

297

ACA

T

T

237

AAT

T

G

180

C

GAAACT---GTGATAATACCCAATGAGAAAAGTCTTCAATTACAAAAAGATGAGGCAATA G

120

C

ATT

ATGA A

60

T

HUMAN BOVINE

BOVINE

B

C

A

299 298

C

T

C

G

T

CG

837

A

Al

897

FIG. 3. A. Amino acid alignment of the bovine and human OIF precursors. Sequences are shown using the single-letter amino acid code. The top line shows the 299-amino-acid bovine precursor. The second line shows only those residues of the 298 human OIF precursor that differ from the bovine sequence. Asterisks above the bovine sequence and below the human sequence identify potential 7V-glycosylation sites. The cleavage site for release of the mature protein is indicated by the arrow. B. Nucleotide sequence alignment of cDNAs encoding the human and bovine OIF precursors. The sites at which the bovine sequence differs from the human are shown; all other residues are identical. Mature OIF is boxed.

MADISEN ET AL.

308

D

FIG. 4. Northern blot analyses of OIF specific transcripts. RNA was fractionated on an agarose-formaldehyde gel, transferred to Hybond N filters, and hybridized to 32P-labeled OIF probe. RNA concentrations were determined from absorbance at 260 nm. A. A total of 1 fig of bovine osteoblast polyadenylated RNA probed with 32P-labeled pOIF-54. Numbers on the left indicate the position of migration of molecular weight standards in kilobase pairs. B. A total of 5 fig of 781T polyadenylated RNA (lane 1) or 10 pg of MG-63 polyadenylated RNA (lane 2) was probed with 32P-labeled human OIF cDNA. C. A 20-/¿g amount of total RNA from 781 T cells was probed with 32P-labeled human OIF cDNA (exposure time, 40 hr). D. A 25-fig amount of total RNA from various human tissues was probed with 32P-labeled human OIF cDNA. Lane 1, adrenal gland; lane 2, brain; lane 3, breast; lane 4, colon; lane 5, duodenum; lane 6, epidermus; lane 7, ovary; lane 8, palcenta; lane 9, testis; lane 10, thymus; lane 11, liver; lane 12, kidney (exposure time, 120 hr).

proteins implies that this region may serve a specific function as has been suggested for the conserved pro-regions of other growth factors (Derynck, 1986). A search of the DNA (GenBank, release 61) and protein (Protein Identification Resource, release 21) data bases reveals no strong homology to known sequences. However, a 19-amino-acid stretch, containing 4 of the precursor's 6 cysteine residues (beginning at amino acid 95), shares 52% homology with a cysteine-rich segment from the N-propeptide region of human and chicken type III collagen (Yamada et al, 1984; Toman et al, 1988). Consistent with the observation that OIF is heavily glycosylated (Bentz et al, 1989), bovine OIF contains three potential A^glycosylation sites within the mature region. A potential site in the pro-region has unfavorable flanking residues (Bause, 1983) and is probably not used. Two of these sites are conserved in the predicted human OIF sequence (Asn-215 and Asn-259), suggesting that glycosylation of these residues might be important for OIF bioactivity. However, Bentz et al reported the lack of glycosylation at one of these two conserved sites, Asn-215 (Bentz et al, 1989). Proteolytic cleavage of the human and bovine OIF-precursor occurs following the asparagine residue within the sequence NH2_... Thr Leu Phe Asn I Ala Lys Tyr... COOH. This recognition site for processing appears, thus far, to be unique in that it is not recognized by any known protease (Bairoch, 1989). Perhaps such specificity is necesof the two

activity only to those sites of new bone formation, and expression of this processing enzyme could be restricted to osteoblasts. Attempts to isolate this protease and characterize it biochemically are in progress. In addition, both human and bovine OIF precursors contain a second hydrophobic region following Cys-107 that conforms to the (-3, -1) rule of Von Heijne (1986) for potential secretory signal sequences. Cleavage at this site could sary to restrict OIF

result in the formation of an alternative OIF precursor. Northern blot analysis showed that bovine osteoblasts contain two major OIF-specific transcripts of 1.9 and 2.4 kb. Two human osteosarcoma cell lines (MG-63 and 781T) have major messages of 1.9 and 3.6 kb along with a minor species of 2.4 kb. The lack of OIF-specific mRNA in the various cell lines and tissues examined to date suggests that expression of this gene is tightly regulated and may be restricted to cells involved in bone formation and remodeling. Identification of factors that control the expression of OIF will increase our knowledge of the various mechanisms involved in bone growth.

ACKNOWLEDGMENTS We thank Dr. Greg Mundy and Dr. Eugenie Kleinerman for various osteosarcoma cell lines used in this study. We also thank Cynthia Hagen for excellent assistance in preparation of this manuscript.

OSTEOINDUCTIVE FACTOR cDNA

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