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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