(Received for publication, August 10, 1994; and in revised form, January 5, 1995)
From the
We have previously described a C-terminally truncated variant of
the chemokine neutrophil-activating peptide 2 (NAP-2) that exhibited
higher neutrophil-stimulating capacity than the full-size polypeptide.
To investigate the impact of the NAP-2 C terminus on biological
activity and receptor binding, we have now purified the novel molecule
to homogeneity. Furthermore, we have cloned, expressed in Escherichia coli, and purified full-size recombinant NAP-2
(rNAP-2-(1-70)) and a series of C-terminally deleted variants
(rNAP-2-(1-69) to rNAP-2-(1-64)). Biochemical and
immunochemical analyses revealed that the natural NAP-2 variant was
structurally identical to the rNAP-2-(1-66) isoform. As compared
with their respective native and recombinant full-size counterparts,
both molecules exhibited 3-4-fold enhanced potency in the
induction of neutrophil degranulation as well as 3-fold enhanced
binding affinity for specific receptors on these cells. All other
variants were considerably less active. The natural occurrence of a
NAP-2 variant truncated by exactly four residues at the C terminus
suggests that limited and defined proteolysis at this site plays a role
in the regulation of the biological function of the chemokine.
The chemokine neutrophil-activating peptide 2 (NAP-2) ()is a 70-amino acid residue polypeptide (1) that is
formed from platelet-derived precursors by proteolytic processing.
These precursors are homologous molecules differing in the lengths of
their N termini and are collectively termed
-thromboglobulin
antigen (
TG Ag). At least two of the polypeptides, platelet basic
protein (94 residues) and connective tissue-activating peptide III
(CTAP-III) (85 residues), were directly shown to become converted into
NAP-2 by N-terminal truncation through monocyte and granulocyte
proteases(2, 3) . Mature NAP-2 stimulates various
effector functions of polymorphonuclear neutrophil granulocytes (PMN)
including directed chemotactic migration(4, 5) ,
exocytosis of lysosomal enzymes (5) and secondary granule
contents(6) , and up-regulation of adhesion
receptors(7) . Together with structurally and functionally
related chemokines such as interleukin-8 (IL-8) and melanoma
growth-stimulating activity (MGSA), NAP-2 has been assigned to a
subfamily now termed ``
-chemokines.'' These mediators
are selective activators of granulocyte (but not monocyte) functions
and were found to act on PMN through specific binding to common
receptors, the IL-8 receptors type A and B. Only high affinity binding
was detected for IL-8, while separate high and low affinity binding
sites exist for NAP-2 and
MGSA(4, 8, 9, 10) .
The
-chemokines contain four cysteine residues at highly conserved
positions, which enclose the core region of the molecules. The first
two cysteines are separated by a single amino acid, forming a motif
(CXC) that distinguishes the
-chemokine from the
-chemokine subfamily, where these cysteines are in directly
adjacent position (CC) (reviewed in (11, 12, 13) ). Although structural analyses
of chemokines have shown that disulfide bridge formation is essential
for the maintenance of their typical molecular conformation (14, 15) as well as for biological
activity(16, 17) , additional structural features
important for receptor binding and biological function have been found.
Concerning the
-chemokines, a common sequence motif of three
successive amino acids (ELR) preceding the CXC grouping was
recognized to form an indispensable prerequisite for the induction of a
transient Ca
influx and biological responses in
PMN(18, 19) . This was directly shown for IL-8 and
MGSA, where substitution of the ELR motif for alanines resulted in a
drastic decrease in PMN-stimulating activity(20, 21) ,
while insertion of the motif conferred enhanced activity to the poorly
active chemokine homologue platelet factor 4 (PF-4)(22) .
Although now it appears clear that defined structural determinants
within the
-chemokine N terminus are required for receptor binding
and functional activation of PMN, recent investigations indicate that
further, although not yet precisely defined regions are also important.
Studies on IL-8 suggest that binding to the type A IL-8 receptor also
depends on the presence of certain residues within the core region,
whereas binding to the type B IL-8 receptor requires structures
provided by the C terminus(21) . However, it is not yet clear
whether the same principles apply to other
-chemokines. In this
context, our recent finding of a variant NAP-2 molecule (herein termed
NAP-2-(X)) that was truncated at the C terminus and exhibited enhanced
PMN-stimulating potency suggests the involvement of C-terminal
structures in the regulation of NAP-2 function(23) . However,
in the latter study, purification of NAP-2-(X) from culture
supernatants of stimulated peripheral blood mononuclear cells (PBMC)
was difficult, and we were not able to completely separate the molecule
from contaminating platelet basic protein nor could we precisely
determine the extent of C-terminal truncation. In the present work, we
have therefore expressed and characterized recombinant NAP-2 and a
series of C-terminally deleted variants. This approach enabled us to
directly analyze the impact of C-terminal truncation on NAP-2 function
and receptor binding while excluding the potential influence of other
kinds of post-translational modification. We have furthermore now
succeeded in purifying NAP-2-(X) to homogeneity. Comparison of this
molecule with the recombinant polypeptides revealed that strictly
defined C-terminal truncation participates in the regulation of NAP-2
function.
Figure 1:
Recombinant expression of NAP-2 and
variants in E. coli. Shown is a schematic diagram of the
construction of pAXNAP expression vectors. Inserts were generated by
polymerase chain reaction with the 5`-primer FOR and the 3`-primers
BACK70 to BACK64 (Table 1) using platelet-derived NAP-2 cDNA as
template. The 3`-ends of the primers were complementary to the template (openboxes). The primers' 5`-ends were used to
introduce the coding sequence for the factor Xa recognition site (FXa-rs) and the restriction site for BglII (Bgl2) at the 5`-ends as well as a site for SalI (Sal1) and a stop codon (Stop) at the 3`-ends of the
variants' sequences. Inserts were cloned into the multiple
cloning site (MCS) using restriction enzymes BglII
and SalI. The lacZ gene and the t
terminator are shown as blackboxes. The
Amp
antibiotic resistance marker and the collagen fragment (CS) gene are shown as grayboxes. The lacZ gene is preceded by its specific promoter
(P
). Inset, Western blot analyses using mAb C-24
of fusion protein from enriched inclusion bodies (lane1) and of the peptide released upon factor Xa proteolysis (lane2). Lane3 shows silver
staining of the immunopurified peptide upon
SDS-PAGE.
Figure 2:
Comparison of antibody reactivities of
native and recombinant NAP-2 variants. Shown are Western blot analyses
of native NAP-2 (lanesN), NAP-2-(X) (laneX), and recombinant rNAP-2-(1-70) to
rNAP-2-(1-64) (lanes70 to 64,
respectively) separated by SDS-PAGE (20 ng/lane). Blots were
immunochemically stained with the polyclonal antisera R-
TG (upperpanel), R
-70 (centerpanel), and R
-NAPII/55-70 (lowerpanel).
Figure 3:
Comparison of net charges of native and
recombinant NAP-2 variants. Native NAP-2 (lanes N), NAP-2-(X) (laneX), and recombinant rNAP-2-(1-70) to
rNAP-2-(1-64) (lanes70 to 64,
respectively) were separated by IEF (0.2 µg/lane) and immunoblotted
using polyclonal antiserum R-
TG as the detecting reagent. pH
values in the gel were determined by means of a
microelectrode.
Figure 4: Relative potencies of native and recombinant NAP-2 variants for receptor binding and degranulation. A, relative potencies of native NAP-2 (columnN), NAP-2-(X) (columnX), and recombinant rNAP-2-(1-70) to rNAP-2-(1-64) (columns70 to 64, respectively) to induce degranulation in neutrophils (potency of native NAP-2 = 100%); B, relative potencies of the same variants to compete with 10 nM radiolabeled native NAP-2 for receptor binding (potency of native unlabeled NAP-2 = 100%). Data are given as means ± S.D. of three to seven experiments.
Figure 5:
Receptor binding and biological activities
of NAP-2-(X) and rNAP-2-(1-66) in comparison with the full-size
molecules. A, induction of lysosomal elastase release in
cytochalasin B-pretreated PMN by increasing concentrations of NAP-2 and
truncated variants; B, displacement of 10 nMI-NAP-2 binding to PMN by the same polypeptides (dashedline indicates 50% competition). In A and B, one representative experiment (out of five) is
shown.
Corresponding results were obtained in receptor
competition assays. As shown in Fig. 4B, the ability of
recombinant NAP-2 variants to compete for binding with a fixed
concentration of 10 nMI-NAP-2 increased with
C-terminal deletions of up to four amino acids and sharply declined
upon further truncation. The very prominent increase in binding potency
obtained with rNAP-2-(1-66) was practically identical to that of
NAP-2-(X), with both polypeptides exhibiting
280% of the potency
observed with the full-size chemokine. Improved binding of NAP-2-(X) to
PMN could not be ascribed to a selective increase in affinity for one
of the two binding sites known for NAP-2 (determined to K
0.4 and 20 nM, as described
previously(10) ). In competition assays performed with 1 nM
I-NAP-2, a concentration selectively addressing the
high affinity binding site, the binding potency of rNAP-2-(1-66)
over the full-size molecules was
180%. When using a concentration
of 20 nM
I-NAP-2 involving both binding sites,
the potency of rNAP-2-(1-66) increased to
300% (data not
shown), indicating that enhanced binding seen with C-terminal
truncation is a phenomenon mediated by both sites.
Due to an improved purification protocol (described under ``Experimental Procedures''), it was finally possible to isolate sufficient amounts of native NAP-2-(X) for mass spectroscopic analysis and determination of the C-terminal sequence. The molecular weight measured by matrix-assisted laser desorption/ionization mass spectroscopy was 7227 and differed by <0.1% from the theoretical value(7222) for a NAP-2 molecule truncated by four residues at the C terminus. In a further approach to directly identify the C-terminal sequence of NAP-2-(X), digestion of the protein with endoproteinase Lys-C yielded a peptide fragment with the sequence KLAGD. This pentapeptide unambiguously corresponds to positions 62-66 in NAP-2. Thus, both analyses directly identify NAP-2-(X) as NAP-2-(1-66).
As shown in Fig. 5, the functional properties of NAP-2-(X) and its recombinant homologue rNAP-2-(1-66) were also in perfect agreement. Identical elastase release rates were obtained with both polypeptides over a wide range of concentrations, and this was paralleled by exact alignment of the ligand binding curves obtained with either molecule in receptor competition experiments with radiolabeled full-size NAP-2. It may thus be concluded that enhanced functional activity and receptor binding in NAP-2-(X) are due to defined proteolytic truncation and not to other kinds of post-translational modification.
This study was brought about by our recent discovery of a molecular variant of the chemokine NAP-2 (herein termed NAP-2-(X)) that was truncated at the C terminus and exhibited enhanced biological activity(23) . These findings indicated that the C terminus could be important for the function of the chemokine. However, direct proof was still lacking because it could not be excluded that post-translational modification other than proteolytic cleavage was responsible for increased biological activity in NAP-2-(X). A further incertitude was inferred by the circumstance that the final preparation of NAP-2-(X) was still contaminated by platelet basic protein and that the extent of truncation could not be exactly determined. Thus, in the present study, we first cloned and expressed NAP-2 and a series of C-terminally truncated variants in E. coli. We then examined the capacities of the molecule to induce degranulation in PMN and to bind to specific receptors on these cells. Moreover, the successful purification to homogeneity of NAP-2-(X) from PBMC-derived culture supernatants allowed for a direct comparison of this naturally occurring molecule with the recombinant chemokines, in both structural and functional respects.
Although the biologically active
-chemokines IL-8(35) , MGSA(36) , and PF-4 (37, 38) have been successfully expressed in
prokaryotes, no such reports exist for NAP-2. A possible reason for
this could be problems in establishing stable rNAP-2-producing E.
coli clones, as was reported by others(39) . We
encountered similar problems with E. coli strain JM109, (
)but successful expression of NAP-2 as part of a fusion
protein was achieved in strain DH5
F`IQ. The chemokine could then
easily be released by digestion with endoproteinase factor Xa.
Comparison of purified recombinant NAP-2 (rNAP-2-(1-70)) with
native NAP-2 revealed identity of the molecules in all biochemical and
biological parameters analyzed (Fig. 2, 3, and 5). Apparently,
the chemokine's structure responsible for degranulation activity
as well as receptor binding on PMN is exclusively determined by its
primary amino acid sequence and not by potential post-translational
modifications. These results are in accordance with those obtained for
synthetic NAP-2 by Clark-Lewis et al.(39) . In fact,
the only modification observed in
TG Ag consisted of the
nonenzymatic addition of glucose to variable lysine side
chains(40) . Although the NAP-2 sequence contains a potential
phosphorylation site, such modification has never been detected.
Functional analyses performed with recombinant C-terminally deleted NAP-2 variants demonstrated a biphasic impact of truncation on chemokine activity. Whereas stepwise truncation by up to four residues successively enhanced degranulation activity and receptor binding, further shortening reduced these activities even to below the level of the full-size molecule (Fig. 4). These results demonstrate that the absence or presence of even a single amino acid residue may considerably affect the chemokine's function. Dependence of functional activity on the length of the C terminus has also been demonstrated for IL-8, where a synthetic analogue truncated by three residues exhibited slightly enhanced activity, while truncation by six residues abolished this effect(18) . However, the influence of single residue deletions was not analyzed.
Apart from demonstrating
the impact of C-terminal truncation on the recombinant molecules, we
also found evidence that this principle may be important under
physiological conditions. This was indicated by the occurrence of the
natural isoform NAP-2-(X) purified from PBMC-derived culture
supernatants, which we have shown here to be truncated to the same
extent as the recombinant variant rNAP-2-(1-66). Both molecules
exhibited the same enhanced capacity to stimulate PMN degranulation and
to bind to receptors on these cells, indicating that their improved
function was due to the precise truncation behind Asp and
not to other post-translational modifications. Our present finding that
NAP-2-(X) was truncated by four residues is not consistent with results
from our previous work(23) , where we assumed that maximally
three residues were missing. This difference is probably due to the
circumstance that the epitope specificity of R
-NAPII/55-70
in that study was determined by means of short synthetic peptides,
which were not long enough to include all the epitopes required for
binding.
There are several possibilities how this truncation could
influence receptor binding and function. Structural analyses of the
related polypeptides IL-8(14) , bovine PF-4(41) , and
MGSA (15) have revealed that the C-terminal stretches of these
molecules are all arranged into amphiphilic -helices that interact
with stretches of underlying
-sheets. Homologies in primary and
secondary structure allow these conformational features to be
superimposed on NAP-2. Previous studies on IL-8 showing that a molecule
lacking the complete C-terminal
-helix was still active while a
corresponding C-terminal synthetic peptide was not suggested that this
region stabilizes the chemokine's tertiary structure and does not
directly interact with the receptor (18) . Although our own
studies (
)showing that a synthetic peptide homologous to the
C terminus of NAP-2 (residues 48-70) was likewise inactive are
consistent with the above model, our results obtained with the stepwise
truncated polypeptides favor an alternative hypothesis. The biphasic
course of binding affinities peaking with the removal of exactly four
residues indicates that there may exist an optimal size of the
molecule's C terminus with best fit into the receptors. As
indicated by a concomitant increase in biological activity, this
mechanism may lead to improved interaction of functionally important
sites (e.g. the ELR motif(18) ) with their
counterstructures within the receptors. Our observation that binding of
NAP-2-(X) and rNAP-2-(1-66) was enhanced to both the high and low
affinity receptors to a similar extent is in agreement with our
previous findings that simultaneous interaction of NAP-2 with both
receptors is required to induce an optimal degranulation response in
PMN(10) .
The occurrence of precise truncation in NAP-2-(X)
raises questions regarding the mechanism involved in the generation of
such an optimally tailored molecule. The fact that -helical
structures are generally highly resistant to proteolytic attack would
preclude cleavage at the NAP-2 C terminus. In this respect, it is
interesting to note that the ultimate amino acids in IL-8 (one
residue)(42) , bovine PF-4 (three residues)(41) , and
MGSA (four residues) (15) have been found to form fraying ends
with high mobility instead of participating in the rigid
-helical
structure. Although no such structural analyses have been performed on
NAP-2, its sequence homology with bovine PF-4 at the C terminus (NAP-2,
. . . GDESAD; and PF-4, . . . GDES) implies that a corresponding
fraying end comprising five residues may follow the
-helix in the
former chemokine. Such a disordered structure would facilitate the
access of proteolytic enzymes, as has previously been reported for
MGSA(43) . Nevertheless, the conditions under which NAP-2-(X)
is formed are not yet clear. Since we discovered NAP-2-(X) in
stimulated PBMC culture supernatants(23) , but not upon
coincubation with purified PMN(3) , its formation probably
depends on proteolytic enzymes associated with leukocytes other than
neutrophils. Apart from various carboxypeptidases, a likely candidate
would be an endoproteinase termed granzyme B, which is released by
activated cytotoxic T-cells. The unique specificity of this enzyme,
which is one of the few proteases known to preferentially attack
peptide bonds C-terminal to aspartic acid residues(44) , would
most easily explain the generation of NAP-2-(X) in stimulated PBMC
cultures. Experiments to verify this are underway.
At present, we
can only speculate on the potential physiological role of NAP-2-(X).
Given that this truncated variant is simultaneously formed with NAP-2,
it could participate in the very first line of defense to injury as a
more potent activator of PMN. However, according to our data, it cannot
be excluded that NAP-2-(X) formation is dependent on the presence of
activated leukocytes and would thus be generated at an advanced stage
of inflammation, where other much more potent chemokines such as IL-8
are formed. In such a situation, the prevailing function of NAP-2-(X)
could be to down-regulate the PMN response. As we have previously found
for the full-size chemokine, very low (i.e. nonstimulatory)
concentrations of NAP-2 can desensitize PMN to subsequent challenge
with other chemokines, while IL-8 is inactive in this
respect(10) . Preliminary experiments performed with NAP-2-(X)
indicate that this variant exhibits a desensitizing capacity 4-fold
higher than that of NAP-2. Thus, the truncated molecule
could participate in the termination of an inflammatory response.