Identification of a novel platelet-derived neutrophil-chemotactic polypeptide with structural homology to platelet-factor 4

Identification of a novel platelet-derived neutrophil-chemotactic polypeptide with structural homology to platelet-factor 4

Vol. 172, October No. 30, 2, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1990 IDENTIFICATION OF A NOVEL POLYPEPTIDE ...

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Vol.

172,

October

No. 30,

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages

1990

IDENTIFICATION

OF A NOVEL

POLYPEPTIDE

WITH

Jens-M. Niek-L.M.

PLATELET-DERIVED

NEUTROPHIL-CHEMOTACTIC

STRUCTURAL

HOMOLOGY

Schrdder'*,

Michael

Persoon2'

TO PLATELET-FACTOR

' Royal

and Enno Christophers'

Received

Republic

Gist-Brocades

September

10,

4

Sticherling',

' Department of Dermatology, University Schittenhelmstr. 7, D-2300 Kiel Federal

898-904

of

Kiel

of Germany

NV, Delft,

The Netherlands

1990

A novel protein, NAP-4, could be isolated from human platelet lysates. NAP-4 preparations induced chemotaxis of human neutrophils with an ED,, near 400 rig/ml. Purification by anti NAP-l/IL-8 affinity chromatography and reversed phase HPLC revealed a single peak showing a single line upon SDS-PAGE corresponding to a M, of 8000. NHZ-terminal sequence analysis indicated an unique sequence showing strong homology to human platelet factor 4 and weak homology to tumor necrosis factor e as well. The most interesting finding is the absence of the first two cysteins, known to be strongly conserved in members of the family of platelet-factor 4-like host defense cytokines. -' 1990Academic Press.Ire

Recent

reports

on are

able

termed

NAP-l

increasing

or

neutrophil

Interleukin

8 (l-6).

of mitogenic

show closed

as well

homology

B-thromboglobulin

production

mononuclear

form a potent

family

des which like

have shown that to

cells

upon stimulati-

activating

protein

NAP-l/IL-8 as chemotactic

to platelet-derived

as well

as platelet

NAP-l/IL-8-related proteins T-lymphocyte-preparations, like monocytes, and fibroblasts (for review see [7]) raised also

related

neutrophil

In the present acid

sequencing

are

able

Copyright Ail rights

produce

and/or

an

polypepti4. The

by different endothelial the

question

release

cells cells whether

NAP-l/IL-S-

attractants.

study

we describe

of a novel

* To whom correspondence 0006-291x/90

to

to

polypeptides-

factor

of

platelets

belongs

now

the

neutrophil should

$1.50

0 1990 by Academic Press. Inc. of reproduction in any form reserved.

898

isolation attractant

be addressed.

and partial detectable

amino in

Vol.

172,

No.

supernatats

BIOCHEMICAL

2, 1990

of

by one monoclonal

lysed

human

antibody MATERIALS

AND

platelets, against

BIOPHYSICAL

which

RESEARCH

will

COMMUNICATIONS

be recognized

NAP-l/IL-8.

AND METHODS

Human platelets were separated from venous Platelet isolation: blood as a byproduct of the isolation of mononuclear cells and granulocytes as recently described (1). Briefly the platelet containing plasma was centrifuged at 4000 x g for 30 min. The platelet containing sediments were suspended in PBS containing 10 mM EDTA. Further purification of platelets was performed by two step centrifugation by the use of a 5 min. centrifugation at 200 x g, whereby the sediment was discarded and platelets were spun down by subsequent centrifugation at 4000 x g. Platelet sediments were pooled and frozen below -70' C until further use. Neutrophil isolation: Human polymorphonuclear leukocytes (PMN) from healthy human volunteers were isolated as described (1). Bioassavs: Chemotaxis by human neutrophils was estimated using a well established indirect cell-counting method as recently described in detail (1). Enzyme release by human neutrophils pretreated with Cytochalasin B was performed as recently described in detail (1). Myeloperoxidase was used as marker enzyme. Purification of platelet-derived neutrophil attractinq orotein Platelet sediments were suspended in 2 M NaCl and acidified to pH 3.0 by the use of formic acid. After threefold freeze/thawing insoluble proteins were spun down (4000 x g, 30 min.) and supernatants were applied to a G-75 gel column (2.6 x 65 cm), which previously was equilibrated using 0.1 M ammonium formate, pH 5.0. Fractions containing PMN-chemotactic activity (usually the area corresponding to proteins with M, between 5 kDa and 40 kDa) were pooled and concentrated over an Amicon YM-5 membrane. The pool containing platelet-derived neutrophil attracting proteins was diafiltered against PBS, pH 7.4, and thereafter passed through an anti-NAP-l/IL-8 column (0.4 x 2 cm), which was prepared by coupling our anti NAP-l/IL-8 monoclonal antibody 14 E 4 (8) to Affi-Gel 10 (Bio Rad). The affinity column was washed with 3 ml PBS followed by 5 ml 2 M NaCl adjusted to pH 7.4, The eluate was diafiltered against 0.1 % (V/V) trifluoroacetic acid (TFA) and applied to a narrow pore reversed phase (RP-18) HPLC column (Nucleosil, 5 ,llrn octadecyl silica column, 250 mm x 4.6 mm, Bischoff, Leonberg, FRG) previously equilibrated with 0.1 % (V/V) TFA containing 10 % (V/V) acetonitrile. Proteins were eluted from the column by the use of a gradient of increasing concentrations of acetonitrile. Polvacrvlamide se1 electrophoresis: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for polypeptides was performed as described (9) in the presence of 8 M urea. Proteins were visualized by silver staining. Amino acid sequence analysis: The intact protein was analysed using an Applied Biosystems gas phase sequencer model no. 470 A with on line HPLC analysis of the phenylthiohydantoin derivatives of amino acids, model no. 120 A. 899

Vol.

172,

No.

2, 1990

BIOCHEMICAL

AND

RESULTS When lysates fractions

neutrophil corresponding

40000

(data

not

neutrophil

shown).

chemotactic

reproducibly 14E4 monoclonal The

eluted

with

Both,

the

in

In

order

HPLC profile

bind

major

assay

at

(Fig.

additional

the

of

phase

to

peak

activity

PMN-chemotactic (8)

the

we

with

the

and data

not

column

M glycine

could buffer,

pH

glycine-buffer-eluate, Fig.

Only

1 shows fractions

were

active

30

40

in the

the

RP-18-

containing chemotaxis

1).

NL” 0.:28

!0

Fiq.

20 10 RETENTION-TIME

hIIN

1

Reversed phase (RP-18)-HPLC

of the 2 M NaCl-eluate obtained from PMN-chemotactic activity the anti-NAP-l/IL-8 affinity column. (0) of 10 pl aliquots of each HPLC-fraction was measured in the Boyden chamber system. Elution times of NAP-l and NAP-2 are indicated by arrows. 900

be

material.

as the HPLC.

2 M NaCl-eluate.

215 mm absorbing

this

and the

prepared

described 0.1

5000

experiments

the

PMN-chemotactic as well

bind

column

with

in

characterize

to

bound

Stripping

between

In initial

some

exclusion

is detectable

further

we tried

we recently

by reversed of

to

column.

activity

2 M NaCl-eluate

separated

the

masses

affinity

of

2 M NaCl.

resulted

were

molecular

to

antibody majority

COMMUNICATIONS

by size

to

to an anti-NAP-l/IL-8

shown). 3.0,

able

separated activity

affinity

were

activity

were chemotactic

activity

to an anti-NAP-l/IL-8

RESEARCH

AND DISCUSSION

of human platelets

chromatography,

BIOPHYSICAL

Vol.

BIOCHEMICAL

172, No. 2, 1990

AND BIOPHYSICAL

2

1

3

RESEARCH COMMUNICATIONS

5

4

6

Ficr. 2 SDS-PAGE of NAP-4. NAP-4 as well as some other structurally related polypeptides were analyzed by SDS-PAGE using the tricine/SDS-system. Lane 1 and lane 5 contained myoglobin (17.4 (8.1 kDa) as kDa), cytochrome c (12.4 kDa) as well as ubiquitine standards. Whereas in lane 2 70 ng platelet factor 4 were applied lane 3 was loaded with 100 ng NAP-4. Lane 4 was loaded with NAP-3

50

ng

authentic

NAP-l/IL-S.

SDS-PAGE-analysis

of

this

M,

near

corresponding

to

a

We analysed

31

residues

platelet-derived termed 111)

In

lane

5

90

ng

of

authentic

a

single

(MGSA/gro) was loaded.

(Fig.

8000 (Fig. of

neutrophil

NAP-4

(the

3).

Sequence

guous sequence:

the

aminoterminal protein

- NAP-3 have

analysis

line

2).

activating

NAP-l

terms

revealed

fraction

revealed

end -

of

been used

the

this

tentatively [5,6,10,

following

unambi-

Glu-Ala-Glu-Leu-Gln-Asp-Leu-Gln-Val-Lys-Thr-Val-

Lys-Gln-Val-Ser-Pro-Val-His-Ile-Thr-Ser-Leu-Glu-Val-Asp-Lys-AlaGly-Arg.

Data

search

using

the

Micro

Genie

Protein

Data

Bank

(Beckmann Instruments Inc.) revealed that NAP-4 is a unique protein. However, as shown in Fig. 3, sequence of 31 aminoterminal amino acids platelet-factor PMN-chemotactic on product of

PF-4

of NAP-4 shows strong (71 %) homology with human 4, but only weak homology to known protein-like factors platelet

like NAP-l/IL-8, basic protein)

NAP-2 (10) (a truncatias well as NAP-3 (11)

:

NAP-4: TNF-a:

.. .

NAP-l: NAP-2: NAP-3: Fiq. 3 Sequence alignment of NAP-4 with other closely tides. Sequences of PF-4 (platelet factor 4) necrosis factor cy (14), NAP-l (2,3,4,7), NAP-2 The single letter code for amino acids is

indicated

by

(-).

cytokines

are

boxed

Residues conserved with

a solid

line.

901

related

polypep-

(17),

TNFu (tumor

(10) used.

NAP-3 Gaps

in NAP-4 and the

(11).

are other

Vol.

172,

No.

Fiq.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

4

PMN-activating properties of NAP-4 preparations. PMN-chemotactic activity (-O-) as well as degranulating activity (myeloperoxidase release) (..A..) of NAP-4-preparations were determined using established assays. Chemotactic activity is expressed as chemotactic index, degranulation is whereas expressed as percentage of release by a total control (prepared by lysis of PMN with 0.1 % hexadecyltrimethylammonium bromide). The mean of three in duplicate performed experiments is shown. For comparism the dose response curve of NAP-l/IL-8 related PMN-

chemotaxis (which

is

(.a@..) is included.

identical

[MGSA] or the Interestingly

there

sequences

found

21

Although

(14).

sequence

NAP-4

absence

of

When aligned

is

melanoma growth

overall

towards TNFe.

Cys-residues with

other

NAP-4 is lacking

as well

as localisation

stipulatory

oncogene

factor

at position

gro

of

homology

possible Another

of

part at

this

of

residue shared

relationship finding is the

10 and 12 of NAP-4.

Cys-X-Cys

these

low,

evulutionary interesting

known to position

the motif

with

Q starting is

activity

[20,21]).

NAP-4

members of the family

4-like proteins, which are residues at identical relative that

the

necrosis

the

and

of

some homology

in tumor

points

between

with

gene product

of platelet

contain (7), it (Fig.

Cys-residues

factor

four cysteinbecomes evident 3). is

The presence a conserved

structural species

element of the supergene family independent from the (7). Therefore it is interesting to find NAP-4 as the which lacks the major C-Xmember of PF-4-like proteins,

first C-structural

element.

isolated by anti NAP-l/IL-8-affinityNAP-4-preparations chromatography followed by RP-18-HPLC constantly were found 902

to

Vol.

172,

be

No.

BIOCHEMICAL

2, 1990

active

in

an

degranulation

AND

established

assay

4).

however,

when

compared

of

The dose

to evoke halfmaximal which is more rig/ml,

to

be near

that

400

found

for

(number

of migrating

8 (Fig.

4).

Although

it

contained

traces

the

only

cannot

however

is

not

is III

present not

be

(10)

NAP-4-

appears

to

Although against

binding NAP-l/IL-8

8

serum

in

sufficient amino

in

our

be

of

This of

for

NAP-l/IL-

contaminant, potency

connective

anti-NAP-l/IL-8

our

found than

efficiency

high

platelets

will

was higher

PMN-chemotactic

by

tissue (16)

and

of

protein activa-

and therefore

(16).

Moreover

it

monoclonal

Furthermore, NAP-2 as well as from the reversed phase column

be separated

preparations

NAP-l/IL-8.

NAP-4-preparations

isolated shown). times

be a

found

proteases

freshly

PMN-

to

with

monocyte

recognized

by

NAP-2

from or

NAP-4.

Contamina-

NAP-l/IL-8

therefore

one of our monoclonal future determination

antibodies of NAP-l/IL-

be unlikely. of NAP-4 to will render more

amounts acid

to

(10,15).

via

as

PMN-chemotactic

by truncation

1) and therefore of

that

known

antibody 14 E 4 (data not NAP-l elute at different (Fig. tion

to that

potent

well

appears

concentrations

similar

excluded

NAP-2

produced

peptide

is

be

of a highly

origin

will

PMN) is

COMMUNICATIONS

PMN-chemotaxis than 100 fold

At highest

PMN-chemotaxin

platelet ting

potency

NAP-l/IL-8.

as

NAP-4,

PMN-chemotaxin necessary

RESEARCH

PMN-chemotaxis-

(Fig. lower

BIOPHYSICAL

sequence

difficult, of

it

NAP-4

and

for

further

will allow determination biological

purification of of its complete

studies.

ACKNOWLEDGMENTS

This work was supported by Deutsche Forschungsgemeinschaft, grants SCHR 305/l-2 and Sti 95/2-l. We are gratefully indebted to Jutta Quitzau, Christine Gerbrecht-Gliessmann and Anke Rose for excellent technical assistance and Ilse Brandt and Hildegard Kuhlmann for editorial help. REFERENCES

1. Schroder J.- M., Mrowietz, U., Morita, E. Christophers, E. (1989) J. Immunol. 139, 3473-3483. 2. Yoshimura T., Matsushima, K., Tanaka, S., Robinson, E. A., Apella, E., Oppenheim, J. J., Leonard, E. J. (1987) Proc. Natl. Acad. Sci. USA 84, 9233-9237. 3. Walz A., Pevari, P., Aschauer, H., Baggiolini, M. (1987) Biochem. Biophys. Res. Commun. 149, 755-761.

903

Vol.

172,

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17.

No.

2, 1990

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Van Damme J., Van Beeumen, J., Openakker, G., Billiau, A. (1988) J. Exp. Med. 167, 1354-1376. Westwick J., Li, S. W., Camp, R. D. (1989) Immunology Today 10, 146-147. Gronhoj Larsen C., Anderson, A. O., Apella, E., Oppenheim, J. J., Matsushima, K. (1989) Science 243, 1464-1466. Baggiolini M., Walz, A., Kunkel, S. L. (1989) J. Clin. Invest. 84, 1045-1049. Sticherling M., Schroder, J.M., Christophers, E. (1990) J. Immunol. 143, 11628-1634. Schroder J. - M., Sticherling, M., Henneicke, H.- H., Preissner, W. C., Christophers, E. (1990) J. Immunol. 144, 2223-2332. Walz A., Baggiolini, M. (1989) Biochem. Biophys. Res. Commun. 159, 969-975. Schroder J. - M., Persoon, N. L., Christophers, E. (1990) J. Exp. Med. 171, 1091-1100. Richmond A., Balentien, E., Thomas, H. G., Flaggs, G., Barton, D. E., Spiess, J., Bardoni, R., Francke, U., Derynck, R. (1988) EMBO J. 7, 2025-2033. Anisowicz A., Bardwell, L., Sager, R. (1987) Proc. Natl. Acad. Sci. USA 84, 7188-7192. Pennica, D., Nedwing, G. E., Hayflick, J. S., Seeburg, P. H., Derynck, R., Palaldino, M. A., Kehr, W. J., Aggarwal, B. B., Goeddel, D. V. (1984) Nature 312, 724-728. Walz A., Dewald, B., von Tscharner, V., Baggiolini, M. (1989) J. Exp. Med. 170, 1745-1750. Walz A., Baggiolini, M. (1990) J. Exp. Med. 171, 449-454. Deuel, T. F., Keim, P. S., Farmer, M., Heinrikson, R. L. (1977) Proc. Natl. Acad. Sci. USA 74, 2256-2258.

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