©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Limited and Defined Truncation at the C Terminus Enhances Receptor Binding and Degranulation Activity of the Neutrophil-activating Peptide 2 (NAP-2)
COMPARISON OF NATIVE AND RECOMBINANT NAP-2 VARIANTS (*)

(Received for publication, August 10, 1994; and in revised form, January 5, 1995)

Jan E. Ehlert Frank Petersen Michael H. G. Kubbutat (§) Johannes Gerdes Hans-Dieter Flad Ernst Brandt (¶)

From the Department of Immunology and Cell Biology, Forschungsinstitut Borstel, Parkallee 22, D-23845 Borstel, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

The chemokine neutrophil-activating peptide 2 (NAP-2) (^1)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 beta-thromboglobulin antigen (betaTG 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 ``alpha-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 alpha-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 alpha-chemokine from the beta-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 alpha-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 alpha-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 alpha-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.


EXPERIMENTAL PROCEDURES

Bacterial Strains and Recombinant DNA Methods

Competent Escherichia coli cells (MAX efficiency DH5alphaF`IQ) were obtained from GIBCO BRL (Eggenstein, Germany). Bacteria were grown in LB medium using ampicillin as the selective marker. If not otherwise stated, recombinant DNA methods were performed according to Sambrook et al.(24) . Both strands of insert-containing plasmid DNA were sequenced with an automated laser fluorescent DNA sequencer (Pharmacia, Uppsala, Sweden) using the chain termination method (25) with T7 DNA polymerase (Pharmacia) and fluorescein isothiocyanate-labeled primers according to the supplier's manual. Sequencing primers PAX1-FITC (5`-CCTGGTCTTGCTGGCCAACAT-3`) and PAX2-FITC (5`-CCCGGCGGCAACCGAGCGTTCT-3`) were derived from the pAX5 (Medac, Hamburg, Germany) plasmid sequence. Sequences were evaluated on Microgenie software (Release 7.1; Beckmann Instruments, Munich, Germany). Oligonucleotides were synthesized with an automated DNA synthesizer (model DNA SM, Beckmann Instruments) using standard cyanoethylphosphoamidite chemistry.

Insert Construction

Poly(A) RNA was purified from enriched platelet preparations (obtained from Dr. B. Katzmann (Institute of Immunology and Transfusion Medicine, Medical University of Lübeck, Lübeck, Germany)) by oligo(dT) Dynabeads (Dynal, Oslo, Norway) according to the manufacturer's manual. The cDNA was prepared by reverse transcription with oligo(dT) using the Superscript kit (GIBCO BRL). Inserts were generated by polymerase chain reaction employing NAP-2 cDNA as template (corresponding to CTAP-III mRNA (positions 241-450), EMBL accession number M54995(26) ). Polymerase chain reaction conditions were chosen as described previously(27) . The following oligonucleotide termed FOR served as the 5`-primer: 5`-ATATAGATCTTGATCGAGGGTAGGGCTGAACTCCGCTGCATGTGTATAAAG-3`. The sequence written in boldface is consistent with the first 27 bases coding for NAP-2. To permit generation of a definite N terminus by site-specific proteolysis, a sequence (written in italics) coding for the endoproteinase factor Xa recognition site Ile-Glu-Gly-Arg was introduced at the 5`-end of the NAP-2 cDNA sequence. A BglII restriction site (underlined) was added to allow cloning into the pAX5 vector. To create different C-terminally deleted variants of NAP-2, disparate 3`-primers (BACK70 to BACK64) (Table 1) were used. The 17 bases at the primers' 3`-ends matched the NAP-2 cDNA sequence, followed by a nonsense codon and a SalI restriction site for cloning into pAX5. The 3`-end of BACK70 was complementary to the last 17 bases at the 3`-end of the NAP-2 sequence, whereas the complementary regions of 3`-primers BACK69 to BACK64 were successively shifted upstream by one triplet to cause stepwise deletion of C-terminal amino acids at the protein level.



Plasmid Construction

Inserts were ligated into vector pAX5, leading to expression plasmids pAXNAP70 to pAXNAP64 (see Fig. 1), coding for a tripartite fusion protein consisting of beta-galactosidase, a collagen fragment, and the respective NAP-2 variant with an N-terminal factor Xa recognition site. Expression plasmids were used for transformation of competent E. coli cells. Transformed clones were screened for inserts by polymerase chain reaction (28) using primer PAX1-FITC in combination with BACK70 to BACK64, respectively. Plasmids of transformants used for protein expression were verified by DNA sequencing.


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(0) terminator are shown as blackboxes. The Amp^r 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.



Expression and Purification of Recombinant NAP-2 Variants

After fermentation to a culture density of A = 1.0, transformants were induced to express fusion protein with isopropyl-beta-D-thiogalactopyranoside at a final concentration of 1 mM. Upon 5 h of incubation at 37 °C, cells were harvested, and the pellet was resuspended in lysis buffer (50 mM Tris-HCl, pH 7.4, 10 mM beta-mercaptoethanol, 10 mM EDTA, 10 mM MgCl(2), 10 µg/ml DNase I (Boehringer, Mannheim, Germany)) and sonicated. Expressed fusion protein stored in inclusion bodies was purified according to Schoner et al.(29) and subsequently refolded following a protocol optimized for the refolding of beta-galactosidase(30) . Upon digestion with factor Xa(31) , the solution was acidified with trifluoroacetic acid to pH 2.1 and afterwards was reneutralized to pH 7.2 by sodium hydroxide. Precipitated protein was pelleted, and the supernatant was applied to an immunoaffinity column coated with betaTG Ag-specific monoclonal antibody C-24 (see below)(3) . Immunopurified peptides were stored in 0.1% trifluoroacetic acid at -20 °C.

Purification of Native Cytokines

Native NAP-2 was purified to homogeneity from culture supernatants of stimulated PBMC using sequential immunoaffinity chromatography, cation-exchange chromatography, and reversed-phase HPLC as described previously(3, 32) . NAP-2-(X) was purified to homogeneity from the same source. After its separation from NAP-2 by cation-exchange chromatography as described previously(23) , further separation from platelet basic protein and other contaminants was achieved by reversed-phase HPLC on an analytic cyanopropyl column (4.6 times 250 mm, 5 µm, wide pore; J. T. Baker Inc.). Samples acidified with trifluoroacetic acid were directly loaded and eluted at 0.5 ml/min with a gradient of 0-35% 1-propanol in 0.1% trifluoroacetic acid. NAP-2-(X) eluted at 12% 1-propanol as a distinct symmetrical protein peak. A single sequence (AELRXMXI . . . , where X stands for an unidentified residue, probably C, since samples were not reduced) was obtained upon N-terminal amino acid sequencing, and a single band was detectable by silver stain analysis of SDS-polyacrylamide gels as well as on immunoblots of SDS-polyacrylamide and IEF gels stained with betaTG Ag-specific antibodies (see below). In comparison with a control of NAP-2 run in parallel, NAP-2-(X) migrated slightly faster on SDS-PAGE and focused at a clearly higher pI (8.9 versus 9.4) on isoelectric focusing (IEF).

Mass Spectroscopy

Determination of the molecular weight of NAP-2-(X) was performed by matrix-assisted laser desorption/ionization mass spectroscopy (33) by Eurogentec (Seraing, Belgium).

N-terminal Amino Acid Sequencing

N-terminal sequence analyses of native and recombinant NAP-2 and its variants were performed by Dr. A. Petersen (Department of Clinical Medicine, Forschungsinstitut Borstel, Borstel, Germany) on a gas-phase sequencer (Model 473A, Applied Biosystems Inc., Foster City, CA).

Identification of the NAP-2-(X) C-terminal Sequence

Purified NAP-2-(X) was subjected to digestion with endoproteinase Lys-C. Upon separation of the resulting peptides by reversed-phase HPLC on a MicroRPC C(2)-C(18) column (Pharmacia), the fragment representing the C terminus of NAP-2-(X) was identified by complete sequence analysis by Edman degradation(34) . All procedures were performed as a custom service by Eurogentec.

Electrophoresis, Immunoblotting, and Antibody Reagents

SDS-PAGE under reducing conditions, IEF on polyacrylamide gels in the presence of 8 M urea, transfer of protein bands onto polyvinylidene difluoride membranes, and immunochemical detection of betaTG Ag polypeptides were carried out as described previously(23) . A monoclonal antibody (mAb C-24) reacting with all variably truncated isoforms of betaTG Ag that are presently known was induced in mice, cloned, and purified as described previously (3) . Furthermore, the following rabbit polyclonal antisera were used: Ralpha-betaTG, raised against a purified preparation of native betaTG Ag, and Ralpha-NAPII/55-70, raised against a synthetic peptide consistent with the 16 C-terminal amino acids of NAP-2 (Ile-Asp)(23) . This antiserum reacted against different epitopes within the peptide structure, one located at the ultimate C terminus (Glu-Asp) and other(s) located more upstream. Separation of Ralpha-NAPII/55-70 by sequential affinity chromatography with immobilized peptides (NAP-2 Ile-Ala and Ile-Asp, respectively) yielded an antibody fraction (Ralpha-70) that bound to the Glu-Asp motif and indispensably required the presence of the ultimate C-terminal amino acid in NAP-2 (Asp) for binding, as described previously(23) .

Neutrophils: Preparation and Degranulation Assay

Human PMN were isolated from citrated blood of single healthy donors by gradient centrifugation on Ficoll-Hypaque as described previously (3) to a purity >95% in all events. Activities of stimuli (tested in 2-fold serial dilutions) were assessed by their ability to induce the release of lysosomal marker elastase from cytochalasin B-treated cells. Elastase activity released into the supernatant was measured as described previously(3) . Release rates for elastase are expressed as the percentage of total content in detergent-treated PMN lysates prepared in 0.1% hexadecyltrimethylammonium bromide. Relative potencies of NAP-2 variants are expressed as percentages of the potency of native full-size NAP-2 according to the following equation: % potency = ([A]/[B]) times 100. [A] and [B] represent concentrations of stimuli that elicit identical release rates.

Receptor Binding Competition Assay

The interaction of unlabeled NAP-2 and NAP-2 variants with chemokine receptors on PMN was investigated in binding competition assays using native radiolabeled NAP-2 as a tracer. NAP-2 was chemically modified by the introduction of additional tyrosine residues prior to labeling with I, exactly as recently published(10) . The specific radioactivity of I-NAP-2 was 565 Ci/mmol. Receptor binding competition assays were performed as described(10) , using a constant concentration of I-NAP-2 in the absence and presence of increasing concentrations of unlabeled NAP-2 and NAP-2 variants (up to a 100-fold molar excess). Nonspecific binding of I-NAP-2 was subtracted. Competition by full-size NAP-2 up to a 100-fold molar excess was measured in every assay, serving as a reference. Determination of relative binding potency was performed according to the equation described above. In the receptor binding competition assay, [A] and [B] represent concentrations that cause 50% competition with labeled NAP-2.


RESULTS

Cloning, Expression, and Purification of Recombinant NAP-2 and Its Variants

We have established a prokaryotic expression system in E. coli for recombinant full-size NAP-2 (rNAP-2-(1-70)) and NAP-2 variants deleted by up to six C-terminal amino acids (rNAP-2-(1-69) to rNAP-2-(1-64)). For this purpose, we used expression vectors pAXNAP70 to pAXNAP64, which were constructed as depicted in Fig. 1and as described under ``Experimental Procedures.'' The insert sequences in plasmids of transformed E. coli subsequently used for protein expression were verified by DNA sequencing. Induction of pAXNAP70-containing bacteria led to the high level expression of a protein that proved to be reactive with betaTG Ag-specific mAb C-24 upon SDS-PAGE and Western blotting (Fig. 1, inset, lane1). The apparent M(r) of 132,000 was consistent with that expected for the tripartite fusion protein. The major part of this immunoreactive protein (70%) was insoluble in lysis buffer and, according to phase-contrast microscopy, was stored in inclusion bodies. Enrichment of inclusion bodies by differential centrifugation and subsequent solubilization by 8 M urea, 1% beta-mercaptoethanol led to a fusion protein solution of 80% purity as estimated by semiquantitative analysis of Coomassie Blue-stained SDS-polyacrylamide gels. Upon refolding by dialysis, digestion of enriched fusion protein with endoproteinase factor Xa released several mAb C-24-reactive protein fragments visible on Western blots (data not shown). The smallest evolving fragment had a size of 8 kDa, consistent with the molecular mass of native NAP-2. Subsequent acidification and reneutralization of the solution led to precipitation of all immunoreactive protein, except for the 8-kDa fragment (Fig. 1, inset, lane2). After immunopurification on mAb C-24-Sepharose, the 8-kDa polypeptide was homogeneous as confirmed by silver stain analysis of SDS-polyacrylamide gels (Fig. 1, inset, lane3) and by N-terminal sequence analysis, where only the expected sequence AELRXMXI . . . , consistent with the N terminus of native NAP-2, was obtained. Expression and purification to homogeneity of the C-terminally deleted variants rNAP-2-(1-69) to rNAP-2-(1-64) were achieved by the same methods. The final products all exhibited a correct N terminus.

Comparison of Native and Recombinant Full-size NAP-2

To investigate whether native NAP-2 and rNAP-2-(1-70) were structurally and functionally equivalent, the polypeptides were directly compared in a set of different assays. According to SDS-PAGE and Western blot analyses, the molecules were of the same size, migrating at identical positions corresponding to 8 kDa (Fig. 2, lanesN and 70). Furthermore, comparable reactivities with the three different antisera used for detection indicated the presence of identical epitopes in both molecules. Especially, positive reactivity with Ralpha-70, an antiserum dependent on the presence of the ultimate residue (Asp) in native NAP-2, confirmed that the recombinant polypeptide had an intact C terminus. Finally, as seen by IEF and subsequent immunoblotting, native and recombinant NAP-2 exhibited an identical pI of approx8.9 (Fig. 3, lanesN and 70), providing further evidence that these molecules are structurally equivalent. Results from functional analyses of the polypeptides paralleled these findings. Thus, rNAP-2-(1-70) and its native counterpart exhibited comparable biological activities as seen by their identical potencies to stimulate the release of lysosomal elastase from cytochalasin B-treated PMN (Fig. 4A) and by practically identical dose-response curves for a wide range of concentrations (Fig. 5A). On the other hand, degranulation in response to a high concentration (80 nM) of either polypeptide was dose-dependently inhibited in the presence of betaTG Ag-specific mAb C-24. Total inhibition occurred at a 2-fold molar excess of the antibody over native NAP-2 as well as rNAP-2-(1-70) (data not shown). These results confirmed that the PMN-stimulating activity observed with rNAP-2-(1-70) was associated with the polypeptide and was not due to potential contamination with bacterial formylated peptides. Corresponding results were obtained in receptor binding assays where rNAP-2-(1-70) exhibited the same potency as native NAP-2 in competing with radiolabeled native NAP-2 for binding to specific receptors on neutrophils. As shown in Fig. 5B, a 10 nM concentration of either unlabeled polypeptide reduced the specific binding of 10 nMI-NAP-2 by 50%.


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 Ralpha-betaTG (upperpanel), Ralpha-70 (centerpanel), and Ralpha-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 Ralpha-betaTG 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.



Impact of C-terminal Truncation on Biological Function of NAP-2

The successful preparation of a recombinant NAP-2 molecule that was functionally equivalent to native NAP-2 provided an adequate basis for further studies aimed at elucidating the potential role of the chemokine's C terminus. Comparison of the biological activities of the full-size molecules with those of C-terminally truncated variants yielded the results shown in Fig. 4A: deletion of up to three amino acids (rNAP-2-(1-69) to rNAP-2-(1-67)) led to a slight but reproducible increase in the chemokine's potency to stimulate neutrophil degranulation (to 190% of control). A more prominent increase in potency to 400% was observed with a polypeptide truncated by four residues (rNAP-2-(1-66)), while further truncated variants (rNAP-2-(1-65) and rNAP-2-(1-64)) exhibited activities even lower than those of the full-size chemokines. Interestingly, the still undefined native variant NAP-2-(X) (purified to homogeneity from PBMC culture supernatants; see ``Experimental Procedures'') was as potent as the most active variant, rNAP-2-(1-66).

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(d) approx 0.4 and 20 nM, as described previously(10) ). In competition assays performed with 1 nMI-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 approx180%. When using a concentration of 20 nMI-NAP-2 involving both binding sites, the potency of rNAP-2-(1-66) increased to approx300% (data not shown), indicating that enhanced binding seen with C-terminal truncation is a phenomenon mediated by both sites.

Structure of NAP-2-(X): Comparison with Recombinant C-terminally Deleted NAP-2 Variants and Direct Analyses

The similarities of natural NAP-2-(X) and recombinant rNAP-2-(1-66) in biological activity and receptor binding led us to examine whether these molecules were structurally identical. This was first done by comparing the biochemical and immunochemical characteristics of NAP-2-(X) with those of variants rNAP-2-(1-69) to rNAP-2-(1-64) and the full-size chemokines. In immunoblots of SDS-polyacrylamide gels developed with Ralpha-betaTG antiserum, NAP-2-(X) and the recombinant variants all migrated slightly faster than the full-size polypeptides (Fig. 2). The failure of antiserum Ralpha-70 (specific for the ultimate betaTG C terminus) to detect the recombinant variants as well as NAP-2-(X) reconfirmed that all these molecules were truncated at the C terminus. More relevant information could be deduced from the reactivity pattern of polypeptides with an antiserum (Ralpha-NAPII/55-70) that reacted to an additional epitope located more upstream within the NAP-2 C-terminal sequence. As seen in Fig. 2, this antiserum detected not only full-size NAP-2, but also part of the recombinant variants, namely those deleted by up to four residues (rNAP-2-(1-69) to rNAP-2-(1-66)). By contrast, immunoreactivity with further truncated variants was almost completely abolished, indicating that the epitope for antibody recognition had become destroyed in these molecules. Interestingly, reactivity with NAP-2-(X) was fully preserved, suggesting that this polypeptide is truncated by at least one but maximally four residues. A more precise estimate on the number of residues missing in NAP-2-(X) was possible after further comparative analyses by IEF and immunoblotting. As depicted in Fig. 3, all truncated variants focused at more basic pI values than the full-size molecules. A first shift in net charge was observed with variants rNAP-2-(1-69) to rNAP-2-(1-67), probably due to the elimination of one negatively charged amino acid, the ultimate Asp (C-terminal sequence in NAP-2, . . . Lys-Lys-Leu-Ala-Gly-Asp-Glu-Ser-Ala-Asp (negatively charged residues are depicted in boldface)). A further increase in positive net charge resulted upon deletion of Glu with rNAP-2-(1-66), while no additional changes were detectable with variants rNAP-2-(1-65) and rNAP-2-(1-64). The band representing NAP-2-(X) focused at a pI value identical to that of variants rNAP-2-(1-66) to rNAP-2-(1-64). According to these results, NAP-2-(X) should represent a molecule lacking at least four residues. Since truncation of the molecule by maximally four residues was obvious from its reactivity patterns with the different betaTG Ag antisera (as shown above in Fig. 2), NAP-2-(X) should represent a NAP-2 isoform truncated by exactly four C-terminal amino acids.

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.


DISCUSSION

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 alpha-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, (^2)but successful expression of NAP-2 as part of a fusion protein was achieved in strain DH5alphaF`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 betaTG 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 Ralpha-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 alpha-helices that interact with stretches of underlying beta-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 alpha-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 (^3)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 alpha-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 alpha-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 alpha-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.^3 Thus, the truncated molecule could participate in the termination of an inflammatory response.


FOOTNOTES

*
This work was supported in part by Sonderforschungsbereich 367 Projekt C4. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Ludwig Inst. for Cancer Research, St. Mary's Hospital Medical School, Norfolk Place, London W2 1PG, United Kingdom.

To whom correspondence should be addressed. Tel.: 49-4537-10444; Fax: 49-4537-10404.

(^1)
The abbreviations used are: NAP-2, neutrophil-activating peptide 2; rNAP, recombinant NAP; betaTG Ag, beta-thromboglobulin antigen; CTAP-III, connective tissue-activating peptide III; PMN, polymorphonuclear neutrophil granulocyte(s); IL-8, interleukin-8; MGSA, melanoma growth-stimulating activity; PF-4, platelet factor 4; PBMC, peripheral blood mononuclear cell(s); HPLC, high pressure liquid chromatography; IEF, isoelectric focusing; PAGE, polyacrylamide gel electrophoresis; mAb, monoclonal antibody.

(^2)
J. E. Ehlert, unpublished data.

(^3)
E. Brandt, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. A. Petersen for performing sequence analyses of native and recombinant NAP-2 and variants, C. Wohlenberg for performing sequence analyses of plasmid DNA, and Dr. B. Katzmann for providing platelet preparations. We especially thank C. Pongratz and G. Hucß for perfect technical assistance.


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