Human brain-derived acidic and basic fibroblast growth factors: Amino terminal sequences and specific mitogenic activities

BIOCHEMICAL

Vol. 135, No. 2, 1986

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 541-548

March 13, 1986

HUMAN BRAIN-DERIVED ACIDIC AND BASIC FIBROBLAST GROWTH FACTORS: AMINO TERMINAL SEQUENCES AND SPECIFIC MITOGENIC ACTIVITIES Guillermo

Gimenez-Gallego,“+ Greg Con*,: and Kenneth A. Thomas

zictor

B. Hatcher,'

’

1

Department of Biochemistry and Molecular Biology, Merck Institute for Therapeutic Research, Merck Sharp and Dohme Research Laboratories, Rahway, NJ 07065 2 Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461 Received

January

9, 1986

Extended amino terminal sequence determinations, made on both acidic and basic Pibroblast growth factors from human brain, showed extensive homology with each other and with their respective bovine counterparts. Both human growth factors in the presence of heparin have equivalent specific mitogenic activities on human umbilical vein endothelial cells in culture whereas in the absence of heparin, the acidic mitogen is less than 1% as active as the basic growth factor. @ 1986 Academic Press, Inc. Two types

of bovine

FGFs, one

acidic

purified,

characterized

by complete

and shown

to have

55% sequence

potent

mitogens

dothelial

cells

spectrum

of

that

might

they

developed turally

for

about for

a variety

and induce target

cells

report

dothelial

here

decrease

acid

cells

sequence (3).

useful

have

been

determinations

(2,4,5

activity

(1,2)

FGFs both

are

vascular

en-

including

--in vivo

angiogenic

basic,

Bovine

in culture growth

agents

other

).

of the

The broad FGFs suggest

proteins.

If

human FGFs are

in man then

they

must be both

to be struc-

characterized. the

in culture

in the mitogenic

extended

homologies

equivalent cells

the

use as therapeutic

of human FGF including approximately

and

the

identity

vessel

be therapeutically

and functionally We

amino

of

blood

and

with

mitogenic in activity

amino

the

terminal each

other

activity presence

sequences

for of

and

bovine

heparin

leave from the Consejo Superior Centro de Investigaciones Bioldgicas, Madrid, * To whom correspondence should be addressed. FGF, f ibroblast grouth factor Abbreviations:

541

and

their

vein

en-

a substantial

of heparin.

de Investigaciones Spain. ; aFGF, acidic

p1 forms

FGFs,

human umbilical

of aFGF in the absence

+On sabbatical

of both

Cientificas,

FGF; bFGF, basic

FGF.

00&291X/86 $1.50 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol. 135, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MATERIALS

AND METHODS

Purification of FGFs - Both aFGF and bFGF were purified from human brains as described (peaks I and II, Fig. 3, ref. 6) followed by C4 HPLC reversed-phase chromatography to remove salts and minor contaminating proteins (7). The resulting peaks were analyzed for purity by electrophoresis in SDS polyacrylamide gels followed by silver staining (7). Amino acid composition and sequence analysis - Samples of the proteins (140 pmoles each) were dried from the HPLC elution solvent, hydrolyzed, derivatized with phenylisothiocyanate and analyzed (7). The amino acid derivatives were chromatographed at 44 ‘C on a C Zorbax (DuPont) column using a Varian 5500 liquid chromatograph to pump’a8 gradient from 0.53 M NaOAc, pH 6.5, 7.5% CH CN to 0.22 M NaOAc, pH 6.5, 46% CH3CN and quantitated using a Nelson Analyti a al 4400 recording integrator. Pure protein samples were reduced and carboxymethylated with iodo[214 Clacetic acid and both aFGF (400 pmoles) and bFGF (300 pmoles) were sequenced with a 91.3% and 94.4$ repetitive yield, respectively, on an Applied Biosystems 470A gas phase microsequencer (1,s). Mitogenic Assays - Pure human FGFs were assayed using 4 x 10' human umbilical vein endothelial cells (passage 6) per assay well in a 72 hr cell proliferation assay in the presence or absence of 50 ug/ml of heparin (6). RESULTS Protein eluted

as one major

of which gel band

Purification

had apparent

electrophoresis. (Fig.

?A)

-

Activity

and one minor

peak from

masses of 16.7 The larger

and had

an amino

A

peak

Figure

from

(6)

from

second

peak

acid

heparin-Sepharose

the reversed-phase

kDa as determined

column,

by SDS polyacrylamide

electrophoresed

composition

both

that

as a single was very

similar

B

8

-

I

1)1

-

92.5

-

31.0

-

21.5

-

14.4

1. SDS polyacrylamide electrophoretic the C.. reversed-bhase HPLC burification

66.2 45.0

gels. Samples (200 ng each) of both human (A) aFGF and (B)

bFCF were Rest dena’tured and’ reduced with SDS/2-mercaptoethanol, electrophoresed and silver stained as described (7). The protein mass standards are phosphorylase B (92.5 kDa), bovine serum albumin (66.2 kDa), ovalbumin (45 kDa), carbonic anhydrase (31 kDa), soybean trypsin inhibitor (21.5 kDa) and lysozyme (14.4 kDa).

542

to

Vol.

135,

No. 2, 1986

BIOCHEMICAL

Table

AND

BIOPHYSICAL

I. Amino Acid Compositions

RESEARCH

of human FCFs

aFGF Humana Observed Integer

Amino Acid Asx

14.8

GlX Ser GlY His Aw

2.5 4.4 5.6 7.0 4.1 a.0 7.4 5.2 1.4

Thr Ala Pro W Val Met Ile Leu Phe LYS CYS Tv

bFGF Bov ineb Sequence

0.8 5.0 2.3 9.6 5.3

4

(8) (7) (5)

(l-2) (5)

(18) (5) (11)

5.6 6.6 2.0 3.9 2.6 7.7 4.7 ND ND

a The human aFGF and bFGF were

scaled to the assuming 1 Trp and 3 (aFGF) or 4 (bFGF) The bovine aFGF composition was calculated g The bovine bFGF composition was calculated ND. not determined.

brain-derived

nificantly II

different

material

peak

aFGF

(6)

that

(Table

composition

eluted

from

apparent

masses of 16.6

and 17.6

of

material

silver

staining

at this

Acid

to that

load.

Sequence

endothelial

cell

both

mitogenic

by reversed-phase

amino

sequence

determination

terminal stitutions

residues are

identical

to bovine

Leu

Pro

for

and

bFGF (Table

I).

than

543

for

His

peak broader with

composition The protein contaminant

very

pure.

of the first

peak

of

from heparin-Sepharose

with

brain-derived Tyr

a 0.5%

HPLC was confirmed 21,

a sig-

as a doublet acid

eluted

(Fig.

had

as a single

human FGFs are

activity

purified

peak

The amino

less

bovine

Heparin-Sepharose

- The identity

and subsequently terminal

smaller

gels

1B).

can detect

Determinations

7 7 2 4

(8)

column

of bovine

Therefore,

(6) (7) (2) (4)

(15)

data).

kDa (Fig.

9 10

(13)

SDS polyacrylamide

used here

200 ng protein

--Amino vascular

similar

procedure

the

the reversed-phase in

is

whereas

(unpublished

electrophoresed

this

I)

(10) (9)

masses of the equivalent Cys residues. from ref. 1. from ref. 2.

{GFs

bovine

(10) .(5!

12 12 10 15 3 11 4

(2)

10.0 9.4

7 7 4 1 6 19 7 12 3 1

Sequence

(13) (14) (11) (15)

3.8

(6)

85:: 4.9 0.6 NDd ND

Integer

12.8

(7) (4)

E4ov ineC

Humana

Observed

(15) (16) (10) (13) (4)

5.8 9.5

COMMUNICATIONS

30

to be aFGF by of the

aFGF. at

positions

The

32 amino only 5 and

sub21,

Vol. 135, No. 2, 1986

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

1 HaFGF BaFGF BbFGF HbFGF

PHE-ASN-LEU-PRO-PRO-GLYPHE-RSN-LEU-PROmGLYPRO-PRO-GLYPRO-PRO-GLY-

PRO-ALA-LEU-PRO-GLU-RSP-GLY-GLY-SER-GLY-RLR-PHE PRO-RLR-LEU-PRO-GLU-RSP-GLY-GLY-SER-GLY-RLR-PHE I

10 10

HaFGF BaFGF EbFGF HbFGF

20

-ASN-TYR-LYS-LYS-PRO-LYS-LEU-LEU-TYR-CYS-SER-RSN-GLY-GLY -RSN-TYR-LYS-LYS-PRO-LYS-LEU-LEU-LEU-TYR-CYS-SER-RSN-GLY-GLY

20

30 30

-PHE-LEU-RRG-ILE-LEU-PRO-RSP-GLY-THR-VRL-RSP-PHE-LEU-RRG-ILE-LEU-PRO-RSP-GLY-THR-VRL-RSP-

HaFGF BaFGF BbFGF HbFGF

40

--Figure 2. Homology of human and bovine FCFs. Human (H) and bovine (B) aFCF and bFGF are aligned to give the optimal number of identical amino acid residues as shown enclosed in boxes. The numbers above and below the alignment correspond to the aFGF and bFGF sequences, respectively. The XXX designation refers to an unidentified residue.

respectively.

The

microheterogeneous

forms

The Asn residue in the

form

protein

(Fig. human

show extensive amino Relative activity

was

vein

brain-derived

stored at

-20’

or

amounting

HPLC dilute

-7O’C

II

(6)

following

to

of

Asp

endothelial

reversed-phase

identified

amino bFGF.

HPLC

terminal

41

Human aFGF and bFGF

to 68% identity

- A lo-fold

under

but

II

cells

in

the

had shown

its

after

trifluoroacetic argon

(8).

specific

compared in the

Previous

results).

stable

higher

(bFGF)

endothelial

(unpublished

focusing in the

the

two

Phe residue.

deamidated

peak

to bovine

peak

aFGF, however,

isoelectric

terminal

of

Phe residue.

sequenced

Activities

vascular

confirmed

an amino

terminal

identical

homology

mixture

was quantitatively

40 of

heparin-Sepharose

human umbilical (6)

All

equal

commonly

regions.

Mitogenic

of

was also

bFGF are

terminal

an

in heparfn-Sepharose

2).

sequence

was without

the amino

activity

of

and

was missing

that

chromatography

aligned

with

position

mitogenic

residues

sample

in the second

The principal cell

protein

activity

to peak I (aFGF) presence

experience to be labile

reversed-phase acid/acetonitrile

Therefore, 544

mitogenic

to compare

of

on

heparin

with

bovine

following

chromatography elution

solvent

relative

specific

if

Vol. 135, No. 2, 1988

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

PROTEIN(pg/ml)

Figure 3. Relative specific mitogenlc activities of human brain-derived FGFs. Both (A) aFGF and (B) bFGF were assayed in the absence (0) or presence (0) of 50 ug/ml of porcine heparin as described (6). The arrows denote the half-maximal stimulation of mitOgeneSis.

activities ing

of the

elution

from

chromatography sample

was

stable

two mitogens

its

assayed

acid

(Fig.

equivalent

mediately

active

(Fig.

umbilical

vein

eluted

3A,

however,

activity) >lO

rig/ml

since

The bFGF

it

appeared

half-maximal pg/ml

130

cells.

heparin-Sepharose

from

the HPLC column

activity) the

have

presence

When assayed

(unpublished less

aFGF was about activity)

reversed-

column,

bFGF was slightly

almost

and bFGF from

half-maximal

the

half-maximal

the

activity)

in

to be

in the acidic

from

activities

endothelial

whereas

to lose

chromatography

aFGF eluted

mitogenic

from

of heparin,

half-maximal

38,

vein

elution to that

absence

p&ml

specific

Both pg/ml

(Fig.

umbilical

activity

the

230

human after

identical In

column

reversed-phase

and has been reported

(9).

3A, 110

after

follow-

from heparin-Sepharose.

reversed-phase

solutions

the heparin-Sepharose

for

elution

immediately

or

HPLC chromatography

following

HPLC column

heparin

to

either

column

heparin-Sepharose

activity

virtually

after

prior

from

trifluoroacetic phase

heparin-Sepharose

done directly

as eluted

90% of

the

, aFGF was assayed

1

on this

active percent culture

of im-

aFGF has data). (Fig. or less

3B, as

of human

cells. DISCUSSION

The amino very

similar

FGFs and recently binding

growth

acid to

compositions

those reported factors

of both

previously for (10).

acidic

determined

the synonymous

and for

basic

human

FGFs are

the corresponding

bovine

human brain-derived

Human aFGF shows extensive 545

sequence

heparinhomology

Vol. 135, No. 2, 1986 with

bovine

BIOCHEMICAL

brain-derived

throughout

the amino

The second

biguously

peak of

terminal

proteins

have

the

terminal

terminal

between identified

same

amino

end then

unrelated

it,

reported

and for

extension

full

human

tity

observed

identity (12)

between

compares

terminal

factor-a

too,

or,

amino

unam-

human

FGF by amino

acid

Only

amino

the

the

in

implies

amino

that

either

of the human bFGF sample one form

has a blocked

form sequence

of

identity

with

1 j-residue

amino

terminal

amino

bFGF and

of 40 of the

complete

bovine

not

an

first

bFGF thus

41 both

sequence

previously

terminal

g-residue

(11) regions,

following in

of sequence

FGFs in this

the amino

aFGF,

identity

nearly

aFGF and

the (68

region.

to exhibit bovine

a single

similarity

sample,

if

terminal

from

has now been

is a microheterogeneous The

eluted

p1 forms

percent)

Therefore,

bFGF

(3 1.

we would

growth

This

factor

and

human between

expect

the

sequence

amount

growth

of

observed

the same 55% overall

human insulin-like

human epidermal

two

of

iden-

sequence

factors

I and II

transforming

growth

(13).

Previous pared

reports

to aFGF for

of a higher

specific

human umbilical

vein

at least

partially

attributable

mitogen

following

heparin-Sepharose

mechanism

and the

(14,151.

Both

the

identical

(61,

considering

sequence

to 62% between

and 44% between

941

activity

identification.

on bFGF but missing

sequences

of

electrophoresis

aligned

bovine

form

by SDS gel

FGFs show the same degree the equivalent

basic

which,

the human protein

present

mitogenic

bFGF and this

extending

In the commonly

are

to be acidic

sequence

of human bFGF exhibits

confirming

proteins

bovine

contaminant.

residues

thought

was detected

composition

both

cell

to be the

and amino

two

32 residues.

originally

sequence

The

endothelial

established

composition

acid

aFGF.

terminal

heparin-Sepharose,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

activities

the

diminished precautions

of the acidic

to

mitogenic endothelial

selective

cells

of

described and basic 546

aFGF in

here

FGFs and the presence

the

as yet,

absence

and elsewhere

bFGF com-

appear of

by an, the

of (2,6)

inactivation

chromatography

activity

activity

of

acidic

undefined of

(8,9)

to be

heparin

to protect heparin

in

Vol. 135, No. 2, 1986 the

mitogenic

activity

aFCF

of

dothelial

(161,

reflect

different

compared

donor

differences

in

cells

from

donors

a variety

certain

of other

observed

(2).

is

data).

(15)

of

heparin cells in

cultures,

noteworthy

part, or lots

that

we have

to FGFs of endothelial

--in vitro

that

the lability

and the absence

relative

The physiological

of the heparin

might,

We conclude

activity

en-

endothelial

of particular it

in the

vascular

effect

vein

responsiveness

lower

active

including

The greater

the

specific

aFGF is highly

umbilical

conditions

to stabilize

of the magnitude

regard

(unpublished

to its

help

equivalent

cells,

histories

mitogenic

storage

cells

on aFGF is

of

previously

In this

might

Since

human

sources,

the

have contributed

effect

for

RESEARCH COMMUNICATIONS

observe

generality

to those

seen

might

to

cultures

activity

various

in part,

cells.

the

serum and heparin.

of aFCF under

least

of aFCF is uncertain.

aFGF mitogenic here

AND BIOPHYSICAL

required

on other

on the activity

of calf

were

(4,7,15),

reported

at

on these

heparin cells

effect

which,

activities

absence

on

assays

of

mitogenic

BIOCHEMICAL

of heparin

to bFGF observed

significance

with

of the heparin

unknown. ACKNOWLEDGEMENTS

KAT and GGG thank The

work

03049,

was

also

HL 16387

Associations

and

Drs.

funded

E. Cordes by grants

HL 34171)

and

and

E. Scolnick

from

the

U.S.

Public

both

the

New York

for

their

Health and

support. Service

American

(AG Heart

to VBH. REFERENCES

1.

2. 3. 4. 5. 6. 7.

Gimenez-Gallego, G., Rodkey, J., Bennett, C., Rios-Candelore, M., DiSalvo, J. and Thomas, K. (19851 Science 230, 1385-1388. Esch, F., Baird, A., Ling, N., Ueno, N., Hill, F., Denoroy, L., Klepper, R., Gospodarowicz, D., Bohlen, P. and Guillemin, R. (1985) Proc. Natl. Acad. Sci. USA 82, 6507-6511. Thomas, K. A. and Gimenez-Gallego, G., Trends in Biochem. Sci., in press. Thomas, K. A., Rios-Candelore, M., Gimenez-Gallego, G., DiSalvo, J., Bennett, C., Rodkey, J. and Fitzpatrick, S. (1985) Proc. Natl. Acad. Sci. USA 82, 6409-6413. Lobb, R. R., Alderman, E. M. and Fett, J. W. (1985) Biochemistry 24, 4969-4973. Conn, G. and Hatcher, V. B. (1984) Biochem. Biophys. Res. Commun. 124, 262-268. Thomas, K. A., Rios-Candelore, M. and Fitzpatrick, S. (1984) Proc. Natl. Acad. Sci. USA 81 , 357-361. 547

Vol. 135, No. 2, 1986

8. 9. 10. 11. 12. 13. 14.

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Thomas, K. A., Methods in Enzymology, Peptide Growth Factors, Academic Press, Orlando, USA, in press. Bohlen, P., Baird, A., Esch, F., Ling, N. and Cospodarowicz, D. (1984) Proc. Natl. Acad. Sci. USA 81, 5364-5368. Lobb, R. R., Strydom, D. J. and Fett, J. W. (1985) Biochem. Biophys. Res. Commun.131, 586-592. Bohlen, P., Esch, F., Baird, A., Jones, K. L. and Gospodarowicz, D. (1985) FEBSLett. 185, 177-181. Rinderknecht, E. and Humbel, R. E. (1978) FEBSLett. 89, 283-289. Derynck, R., Roberts, A. B., Winkler, M. E., Chen, E. Y. and Goeddel, D. V. (1984) Cell 38, 287-297. Thornton, S. C., Mueller, S. N. and Levine, E. M. (1983) Science 222, 623-625.

15.

Schreiber, A. B., Kenney, J., Kowalski, J., Thomas, K. A., GimenezGallego, C., Rios-Candelore, M., DiSalvo, J., Barritault, D., COUrty, J ., Courtois, Y., Moenner, M., Loret, C., Burgess, W. H., Mehlman, T., Friesel, R., Johnson, W. and Maciag, T. (1985) J. Cell Biol. 101, 1623-

16.

Schreiber, A. B., Kenney, J., Kowalski, W. J., Friesel, R., Mehlman, T. and Maciag, T. (1985) Proc. Natl. Acad. Sci. USA 82, 6138-6142.

1626.

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