a form of human basic fibroblast growth factor with an extended amino terminus

Vol. 144, No. 2, 1987

April 29, 1987

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 543-550

A FORM OF HUMAN BASICFIBROBLAST GROWTH FACTOR WITH ANEXTENDEDAMINO

TERMINUS

Andreas Sommer* ] ,

David Moscatelli 2

Michael T. Brewer ] Robert C. Thompson ]

, Marco P r e s t a + ' 2 , a n d D a n i e l B. R i ~ k i n 2

]Synergen, Inc., 1885 33rd Street, Boulder, Colorado 80301

2 Department of Cell Biology and Kaplan Center,

New York University Medical Center, 550 First Avenue,

New York, New York 10016

Received February 24, 1987

The amino acid sequence of a human placental bFGF was determined by a combination of protein and cDNA sequencing. The placental bFGF consists of 157 amino acid residues with a calculated molecular weight of 17,464 and is highly homologous to bovine pituitary bFGF. The human protein contains an amino terminal extension when compared to the sequence established for bovine bFGF (Esch et al., 1985) and to the sequence of the predicted translation product based on human bFGF cDNA clones (Abraham et al., 1986). ©1987AcademicP ..... Inc.

We have isolated from term human placenta a protein which induces

the production of the proteases plasminogen activator and collagenase,

is a potent chemotactic factor, and is mitogenic for bovine capillary

endothelial cells (i). These three activities have been postulated to

be part of the angiogenic response (2). Indeed, the protein has been

shown to be angiogenic on the chick chorioallantoic membrane (i). We

*To whom correspondence should be addressed.

+ Present Address: Institute of General Pathology, Faculty of Medicine,

University of Brescia, Italy.

Abbreviations: ~bFGF, basic fibroblast growth factor; aFGF, acidic

fibroblast growth factor; TFA, trifluoroacetic acid; PTH, phenylthiohydantoin; MES, (2[N-Morpholino]ethanesulfonic Acid).

543

0006-291X/87 $1.50 Copyright © 1987 by Academic Press, Inc.

All rights of reproduction in any form reserved.

Vol. 144, No. 2, 1987 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

have also characterized a protein from the human hepatoma cell line SK-

HEP-I which has similar biological activities and chemical

characteristics, and which cross-reacts with antibodies prepared against

the placental protein (3).

These two proteins have many physical and biological properties in

common with bFGF. Like bFGF (4,5), they bind strongly to heparin-

Sepharose, have isoelectric points greater than pH 9.0, have molecular

weights of approximately 18,000, and are mitogenic for a variety of

mesenchymal cells. Bovine pituitary bFGF has also been shown to be

angiogenic (6).

The amino acid sequence of bovine pituitary bFGF has been

determined (6), and a cDNA clone encoding bovine bFGF has been isolated

(7). Human bFGFs have been isolated from brain and placenta and from

hepatoma cells in culture (8-11). A partial sequence of human brain

bFGF shows it to have the same N-terminal sequence as that originally

reported for bovine pituitary bFGF (8,9). Partial cDNA clones encoding

human bFGF have also been isolated byAbraham et al. (12).

We report here the amino acid sequence of anangiogenesis factor

from placenta which appears to be a human bFGF. The protein contains

157 amino acids and has an amino terminal extension relative to all

other bFGF's for which primary structural information has been reported.

METHODS

Isolation of the Placental Protein The protein was isolated from human placenta bythe procedure

described previously (I).

Proteolytic Digestion of the Placental Protein Digestions with Lys-C, submaxillaris protease, and V-8 protease

were performed as reported (13) except that the digestion was performed in the presence of 0.5 M NaCI.

Isolation of Peptides. Peptides from each digestion mixture were separated by reverse-phase high performance liquid chromatography (Beckman system) using a Synchrom RP-8 column and a linear gradient of acetonitrile made 0.1% (v/v) in TFA. Fractions that contained a mixture of peptides were dried in a Speed-Vac (Savant), resuspended in i00 ul of 50 mM Tris-HCl, pH 8.5, 8 M urea and repurifiedby reverse-phase

544


chromatography using an Altex C-3 column and a linear gradient of acetonitrile made 0.1% (v/v) in TFA (0-60% acetonitrile in 120 min).

Audomated Edman Degradation and PTHAmino Acid Identification Purified, uncleaved placental protein or purified peptides (100-500

pmols) were sequenced as described previously (13) using an Applied Biosystems 470A gas-phase protein sequencer.

cDNA Synthesis and Molecular Cloning SK-HEP-I cells were grown in Eagles minimum essential medium

supplemented with 10% fetal calf serum and non-essential amino acids. RNA was isolated from these cells using the NP-40 lysis procedure described elsewhere (14) and used to establish a lambda gtl0 library (see Results section). Two probes (see Results section) used for screening of the cDNA library were synthesized on an Applied Biosystems DNA synthesizer, gel purified, and 5' end labeled with [ 32p]ATP (Amersham) to a specific activity of 4-6xi06 cpm/pmol using T4 polynucleotide kinase (Pharmacia).

RESULTS

Protein Sequence of Human Placental Angiogenesis Factor

Because of the availability of limited amounts of the placental

protein, peptide isolation protocols were designed to minimize potential

losses due to multiple HPLC desalting steps. Thus, all proteolytic

digestions were performed (see Methods) in the buffer from the final

purification step (i), 20 mM MES buffer, 500 mM NaCl. Reduction and

carboxymethylation were carried out after proteolytic digestion of the

protein. A single reverse-phase HPLC was then used to desalt

simultaneously and separate the peptides.

The primary structure of the placental protein is shown in Fig. 1.

The N-terminal amino acid sequence was established byautomated Edman

degradation of purified, uncleaved placental protein. Table 1 shows the

yield of PTH amino acids obtained when 400 pmol of intact placental

protein was applied to the gas-phase protein sequencer. The data

indicate that the PTH amino acid yield is 20-40% of that normally

expected. Two additional experiments gave the same result. Thus, a

substantial portion of the placental protein may be N-terminally

blocked. Protein sequence analysis of endoproteinase Lys-C generated

peptide LC-II (see Fig. i) gave the same sequence as the N-terminus of

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Fig. i.

G - T - M - A - A - G - S - I - T - T - L - P - A - L - P - E - D - G - G - S - I' ,o I zo

hPAF; LC-II

G- A - F - P - P - G - H - F - K - D - P - K - R- L - Y - C - K - N - G - G - 3O I 40

I I SM 5 - 3 IV8 - 14 I

LC-8 F - F - L - R - I - H - P - D - G - R - V - D - G - V - R - E - K - S - D - P -

SM 5--~3 5o t 2 V 8 - 1 0 60

LC-8 I

H - I - K - L - Q - L - Q - A - E - E - R - G - V - V - S - I - K - G - V - C - 8O

2V8 - I 0 I ZO t S M - 5 - 1

I LC-7 It LC-9

A - N - R - Y - L - A - M- K - E - D - G- R- L - L - A - S - K - C - V - T - 90 IO0

S M - 5 - 1 I I SM_I 4 I ~ L C - 1 6

LC -9 I 2V8-7

D - E - C - F - F - F - E - R - L - E - S - N - N - Y - N - T - Y - R - S - R - I10 120

LC-16 I 2V8-71

K - Y - T - S - W - Y - V - A - L - K - R - T - G - Q - Y - K - L - G - S - K - I" S M - 3 - 3 "1 13o I S M - 6 14o

I " L C - 9 - S I

T - G - P - G - Q - K - A - I - L - F - L - P - M - S - A - K - S 150 157

S M - 6 I I I LC-IO

Primary protein structure of human placental angiogenesis factor. Protein sequence identified by automated Edman degradation of the intact protein or of selected peptides derived by proteolytic digestion of purified placental protein are shown (LC, endoproteinase Lys-C peptides; SM, submaxillaris protease peptides; V8, staphylococcal protease V8 peptides).

the intact protein. Identification of the carboxy-terminal residue as

serine is based on: (i) sequence analysis of the Lys-C peptide LC-10

(Fig. i), which terminated with the sequence ---S-A-K-S, and (ii) cDNA

sequence data (Fig. 2) (see below) which revealed a termination codon

immediately following DNA coding for the sequence ---S-A-K-S. Residues

18-21, 30-33, 117-120, and 131-135 were not unambiguously identified by

protein sequencing and were deduced from the cDNA sequence (Fig. 2) (see

below) .

SK-HEP-I cDNA Sequence

Since the human hepatoma cell line SK-HEP-I had been shown to

synthesize a protein that cross-reacts with antibodies prepared against

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Table i. Amino terminal sequence analysis

of placental protein

Cycle # PTH Amino Acid Yield (pmol)

1 G 179

2 T 19

3 M 50

4 A 58

5 A 67

6 G 65

7 S 15

8 I 49

9 T 15

I0 T 20

ii L 31

12 P 47

13 A 32

14 L 38

15 P 38

16 E 21

Analysis of phenylthiohydantoin amino acids showed

no evidence for protein contamination of the sample.

Amount of protein applied to gas-phase sequencer:

400 pmol.

the placental protein (3), these cells were used as a source of mRNA for

construction of a cDNA library. SK-HEP-I mRNA was isolated, and 5 ug of

this mRNA was used to synthesize 8 ug of cDNA. Two hundred ng of cDNA

was ligated into 2 ug of EcoRI-digested lambda gt-10 DNA, and a library

containing 1.7x106 recombinants was established.

Oligonucleotide probes were designed from the sequence of the

placental protein and were used to screen the SK-HEP-I cDNA library to

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Vol . 144 , No. 2, 1 9 8 7 B I O C H E M I C A L A N D B I O P H Y S I C A L RESEARCH C O M M U N I C A T I O N S

50 60 GGG ACC ATG GCA GCC GGG AGC ATC ACC ACG CTG CCC GCC TTG CCC GAG GAT GGC GGC AGC Gly Thr Met Ala Ala Gly Ser I le Thr Thr Leu Pro Ala Leu Pro Giu Asp Gly Gly Ser

90 120 GGC GCC TTC CCG CCC GGC CAC TTC AAG GAC CCC AAG CGG CTG TAC TGC AAA AAC GGG GGC Gly Ala Phe Pro Pro Gly His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Giy

150 180 TTC TTC CTG CGC ATC CAC CCC GAC GGC CGA GTT GAC GGG GTC CGG GAG AAG AGC GAC CCT Phe Phe Leu Arg I le His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp Pro

210 240 CAC ATC AAG CTA CAA CTT CAA GCA GAA GAG AGA GGA 6TT GTG TCT ATC AAA GGA GTG TGT His I le Lys Leu Gin Leu Gin AIo Glu Glu Arg Gly Val Val Ser I le Lys Gly Vel Cys

270 300 GCT AAC CGT TAC CTG GCT ATG AAG GAA GAT GGA AGA TTA CTG GCT TCT AAA TGT GTT ACG AIa Ash Arg Tyr Leu AIo Met Lys Glu Asp Gly Arg Leu Leu Ale Ser Lys Cys Val Thr

330 360 GAT GAG TGT TTC TTT TTT GAA CGA TTG GAA TOT AAT AAC TAC AAT ACT TAC CGC TCA AGG Asp Glu Cys Phe Phe Phe Glu Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg

390 420 AAA TAC ACC AGT TGG TAT GTG GCA CTG AAA CGA ACT GGG CAG TAT AAA CTT GGA TCC AAA Lys Tyr Thr Ser Trp Tyr Vol AIo Leu Lys Arg Thr Gly Gin Tyr Lys Leu Gly Ser Lys

450 ACA GGA CCT GGG CAG AAA GCT ATA CTT TTT CTT CCA ATG TCT GCT AAG AGC TGA Thr Gly Pro Gly Gin Lys Ale I l e Leu Phe Leu Pro Met Ser AIo Lys Ser End

Fig. 2. Nucleotide sequence of a cDNA clone derived from human hepatoma mRNA (SK-HEP-I cell line). The predicted translation product of the open reading frame is also shown and is completely in agreement with the primary structure of the human placental angiogenesis factor as established by protein sequencing (see Fig. i).

find a clone for the placental protein. A 192-fold degenerate 17-mer

corresponding to the amino acid sequence I-K-G-V-C-A (residues 76-81)

and a 256-fold degenerate 20-mer corresponding to the amino acid

sequence Y-C-K-N-G-G-F (residues 35-41) were synthesized and used to

screen approximately 1.2x106 plaques. Five plaques were found to

hybridize to both probes, and one of these was selected for DNA

sequencing as described in Materials and Methods. A partial sequence of

this cDNA is shown in Fig. 2. The sequence contains an open reading

frame for an amino acid sequence which is completely in agreement with

the primary structure of the placental protein.

DISCUSSION

We describe here the primary structure of a placental angiogenesis

factor as determined by a combination of protein and cDNA

548


sequencing. Inspection of the primary structure (Fig. i) reveals the

presence of 157 amino acid residues with a calculated molecular weight

of 17,464.

' From a comparison of the primary structure of the protein with that

of bovine pituitary bFGF (6), it is evident (a) that the placental

protein is a member of the bFGF family, (b) there exists an extension at

the amino terminus of the placental protein that is not present in the

bovine bFGF isolated by Esch et al. (6) and that has not been predicted

from the sequence of human basic FGF cDNA clones (12), and (c) there are

two amino acid substitutions (residues 123 and 139) relative to bovine

bFGF (6).

The N-terminal amino acid sequence of human brain FGF (8) has been

reported as P-A-L-P..., which is identical to that of bovine pituitary

bFGF (6). In support of the longer sequence reported here, we note that

Uneno et al. (15) suggested the presence of a nine-amino-acid extension

in bovine brain bFGF based on amino acid analysis and that a tryptic

peptide isolated from a human hepatoma-derived growth factor was found

to contain the sequence (T)-L-P-A-L-P...(II). The hepatoma cell line

used to isolate the protein from which this tryptic peptide was derived

is the same cell line we used to establish the cDNA sequence. Except

for the amino terminal extension, the amino acid sequence of the

placental protein agrees with the predicted translation product deduced

from a series of partial cDNA clones of human bFGF (12). It remains to

be determined whether the apparent amino terminal heterogeneity in

various bFGFs is an isolation artifact or indicates the existence of

different proteins produced by a mechanism generating amino terminal

diversity.

The isolation of an extended form of human bFGF from placenta

indicates that a diversity of bFGFs differing at their amino termini may

exist. Whether this diversity is generated at the level of

transcription or occurs post-translationally remains to be determined.

549


ACKNOWLEDGEMENTS

The authors would like to thank Ms. Julie A. Wilson and Mr. R.

Manejias for their excellent technical assistance. This work was

supported in part by grants from the NIH and the American Cancer Society

to DBR. D.M. was supported by an investigatorship from the New York

Heart Association. M.P. was supported by a fellowship from the Juvenile

Diabetes Foundation International.

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4. Bohlen, P., Baird, A., Esch, F., Ling, N., and Gospodarowicz, D. (1984). Proc. Natl. Acad. Sci. USA 8_!1 , 5364-5368.

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12. Abraham, J.A., Whang, J.L., Tumolo, A., Mergia, A., Friedman, J., Gospodarowicz, D., and Fiddes, J.C. (1986). EMBO J. 5, 2523-

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a form of human basic fibroblast growth factor with an extended amino terminus

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