THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 260, No. 1s. Issue of August 25, pp. 10339-10345,1985 Printed in (1. S. A.

Structure and Function of Human C5a Anaphylatoxin SELECTIVE MODIFICATION OF TYROSINE 23 ALTERS BIOLOGICAL ACTIVITY BUT NOT ANTIGENICITY*

(Received for publication, February 19, 1985)

Richard J. Johnson and Dennis E. ChenowethS From the Department of Pathology, Veterans Administration Medical Center, Sun Diego and the University of California, San Diego, California 92161

Reaction of either human C5a or its d e ~ - A r g ~ ~ deriv- ative (de~-Arg~~-C5a) with tetranitromethane under nondenaturing conditions results in selective nitration of only 1 of the 2 tyrosine residues found in these glycopolypeptides. This reactive tyrosyl residue was identified as that found in position 23 of the sequence. Nitrotyro~yl~~-C5a and -de~-Arg~~-C5a were sepa- rated from their respective unmodified precursors by cation-exchange fast protein liquid chromatography. These purified derivatives served as reagents for the subsequent preparation of both aminotyro~yl~~-C5a and -de~-Arg~~-C5a as well as photoreactive analogs of C5a. Radioimmunoassays performed with C5a deriv- atives serving as competing ligands and a murine anti- human C5a monoclonal antibody employed as first an- tibody demonstrated that selective modification of tyrosinez3 did not produce a detectible alteration in the antigenic properties of C5a. By contrast, either nitro- or aminotyro~yl~~-C5a was approximately %fold less active than native C5a in both bioassays and competi- tive ligand-receptor binding assays. Additionally, pho- toreactive derivatives prepared by coupling either N-succinimidyl-6-(4’-azido-2’-nitrophenylamino)- hexanoate or p-nitrophenyl-2-diazo-3,3,3-trifluoro- propionate to aminotyro~yl~~-C5a at pH 5.0 failed to interact with the neutrophil C5a receptor. These ob- servations suggest that the tyrosylZ3 residue of C5a may participate importantly in the binding interac- tions of this chemotactic factor and its granulocyte receptor.

Human C5a anaphylatoxin is a 74-residue glycopolypeptide that is cleaved from the amino terminus of the a-chain of the fifth component of complement (C5) during complement ac- tivation (1). In serum, C5a is readily converted to its des-Arg derivative (de~-Arg~~-C5a’) by serum carboxypeptidase N (EC

* This work was supported in part by National Institutes of Health Grant AI-18731 and a Merit Review award from the Veterans Admin- istration. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore he hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$Recipient of a Clinical Investigator Award from the Veterans Administration.

The abbreviations used are: d e ~ - A r g ~ ~ - C 5 a , derivative of C5a, formerly written as C5m.. Arg.74; TMN, tetranitromethane; NO2-Tyr- C5a, nitrotyrosyl-C5a; NHZ-Tyr-C5a, aminotyrosyl-C5a; SANPAH, N-succinimidyl-6-(4’-azido-2’-nitrophenylamino)hexanoate; NO2- Tyr, 3-nitrotyrosine; TPCK, N-tosyl-L-phenylalanine chloromethyl ketone; ANPAH, 6-(4’-azido-2’-nitrophenylamino)hexanoate; FPLC, fast protein liquid chromatography; RIA, radioimmunoassay; SDS- PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography.

3.4.17.3) (2). Human C5a is a potent spasmogen that promotes smooth muscle contraction, but d e ~ - A r g ~ ~ - C 5 a is essentially devoid of spasmogenic activity (3). By contrast, both C5a and de~-Arg~~-CSa can serve as inflammatory mediators, i.e. each stimulates neutrophil responses such as chemotactic migra- tion, superoxide production, and lysosomal degranulation. However, de~-Arg’~-C5a is approximately 20- to 50-fold less active than C5a in this regard (4,5).

Cellular responses result when either C5a or de~-Arg~~-C5a binds to specific receptors that have been demonstrated on human peripheral blood neutrophils and monocytes (6, 7) as well as murine macrophages (8). Binding interactions of C5a with these receptors appear to be governed by two functionally separate regions of the C5a molecule (4). According to our current hypothesis, structural determinants located within the amino-terminal 69 residues of C5a impart ligand specific- ity. Binding interactions between this portion of the molecule and the “recognition domain” of the C5a receptor by them- selves are not sufficient to trigger cellular responses. Instead, additional binding interactions that take place between the carboxyl-terminal portion of the C5a molecule and the recep- tor “activation domain” seem to be required to achieve this result (4).

One of the primary objectives of our ongoing investigations has been to more clearly identify regions of the C5a molecule that may contribute importantly to the regulation of ligand- receptor interactions and the biological activity of this inflam- matory mediator. In the present investigations we have ac- complished this by selectively modifying a single tyrosyl res- idue in both human C5a and de~-Arg~~-C5a and by comparing the properties of these derivatives to those of the native glycopolypeptides. Furthermore, extension of these studies to include similar modifications of human C3a anaphylatoxin, which shares considerable sequence homology with C5a (9), provided evidence that human C5a displays not only unique biological properties but unique chemical properties as well.

EXPERIMENTAL PROCEDURES

Preparation of Anaphylatorins-Human C3a and C5a were isolated from zymosan-activated human serum containing 1 mM DL-2-mer- captomethyl-3-g~1anidinoethylthiopropanoic acid (Calhiochem-Behr- ing) as carboxypeptidase N inhibitor (10). Human de~-Arg’~-C5a was similarly prepared from serum after yeast activation in the absence of the carboxypeptidase inhibitor.

Preparation and Purification of Anaphylatoxin Deriuatiues-Nitra- tion reactions were performed in a 0.5-ml cuvette a t room temperature in 50 mM Tris, pH 8.0. Either C3a, C5a, or de~-Arg’~-C5a were employed a t a final concentration of 1 or 2 x 10“ M and tetranitro- methane (TNM) was added as an ethanolic solution to give a 10-fold molar excess as described by Riordan and Vallee (11). The progress of the reaction was followed by monitoring the absorbance a t 428 nm

10339

10340 Structure and Function of C5a for a period of 2 h. The reaction was then terminated by gel filtration of the reaction mixture on a 3-ml column of Bio-Gel P-6 equilibrated with 50 mM Tris, pH 8.0. Additionally, the derivatives N02-Tyr-C5a and NOz-Tyr-des-Arg7'--C5a were separated from unmodified C5a and des-Arg7'-C5a by ion-exchange FPLC using a Pharmacia Mono S column. Elution parameters are given in the legend to Fig. 2.

The aminotyrosyl derivatives of C5a and des-Arg""C5a (NHz-Tyr- C5a and NHZ-Tyr-des-Arg7'-C5a) were prepared from the correspond- ing nitrotyrosyl derivatives by reduction with a 10-fold molar excess of sodium dithionite (12). The reaction, which was carried out in 50 mM Tris at pH 8.0, was judged to be complete by the disappearance of the nitrophenolic moiety (absorbance of 428 nm) of the nitrated peptides. The aminotyrosyl analogs were separated from excess so- dium dithionite by gel filtration on a 3-ml column of Bio-Gel P-6 equilibrated in 50 mM sodium acetate, pH 5.0. The recovered product was lyophylized.

Photoaffinity analogs of C5a and des-Arg7'-C5a were made by couplingeitherN-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hex- anoate (SANPAH) or p-nitropbenyl-2-diazo-3,3,3-trifluoropropio- nate to the NHz-Tyr-C5a and NH2-Tyr-des-Arg74-C5a derivatives. For these studies, either NHz-Tyr-Csa or NH2-Tyr-des-Arg7'-C5a (12-15 nmol) was dissolved in 30 p1 of 0.15 M sodium acetate, pH 5.0. All subsequent manipulations were performed under dim light or in the dark. Either SANPAH or N-succinimidyl-6-(4'-azido-2'-nitro- pheny1amino)bexanoate was dissolved in dimethylformamide and 10 p1 (200 nmol) were added to the peptide solution. The reaction was allowed to proceed for 3 h and then the derivatized peptide was separated from the unreacted photoaffinity reagent by precipitation and washing with ice-cold acetone. The products were dried in vacuo, redissolved in HzO, and stored at -20 "C.

Photolysis of the photoreactive aminotyrosyl-C5a and -des-Arg"- C5a derivatives was accomplished by irradiation under a Blak-Ray UV lamp (350 nm) for 5-15 min. Samples were kept on ice during the photolysis.

Radioiodinated peptides were usually prepared by a solid-phase lactoperoxidase-glucose oxidase method (Bio-Rad Enzymobrads, Bio- Rad Technical Bulletin 1071). However, the ANPAH-NH-TyrZ3-C5a derivative was radioiodinated by the method of Bolton and Hunter (13) with reagent obtained from New England Nuclear. This was done to insure that iodination of Tyrz3 would not alter the properties of this particular derivative. Additionally, Bolton-Hunter labeled preparations of C5a are known to retain functional activity (6). The '251-labeled peptides were separated from free radioiodine by either gel filtration on Sephadex G-10 equilibrated with 30% acetic acid or by affinity chromatography (7).

Analytical Methods-Quantitation of the anaphylatoxins, their de- rivatives, and other peptides was achieved by amino acid analysis after acid hydrolysis (14). Samples were hydrolyzed in 5.7 N HCl with a drop of 0.1% phenol for 20-24 h at 110 "C and analysis was performed on a Duram Model 500 amino acid analyzer. Calibration was achieved with a mixture of normal amino acids (Pierce) and 3- N02-tyrosine (Sigma) which eluted between tyrosine and phenylala- nine, as standards.

Peptide maps of both nitrotyrosyl-des-Arg7'-C5a (NOz-Tyr-des- Arg7'-C5a) and nitrotyrosyl-C3a (NOz-Tyr-C3a) were obtained after digestion with 5% (w/w) TPCK-treated trypsin (Worthington) in 50 mM Tris, pH 8, at 40 "C for 5 h. The resulting peptides were resolved by direct application of the digestion mixture to Sephadex G-50 gel filtration columns which had been equilibrated with 30% acetic acid. Further peptide purification was accomplished by HPLC on a Cm p- Bondapac reverse-phase column (Waters Associates) employing a solvent system described by Mahoney and Hermodson (15).

Partial Carboxypeptidase A (Worthington) digestion of NOz-Tyr- des-Arg7'-C5a was achieved by incubating 10 nmol of NOz-Tyr-des- Arg7'-C5a with 25 pg of carboxypeptidase A in 100 p1 of 100 mM NH4HC03, pH 8.1. After incubation for 6 h at 37 "C, the sample was frozen and lyophylized to terminate enzymatic digestion. Amino acid analysis, without acid hydrolysis, was then employed to quantitate the liberated amino acids.

Polyacrylamide gel electrophoresis (PAGE) was performed on 10% gels containing sodium dodecyl sulfate (SDS) that were made and run according to Laemmli (16). In these studies, samples of photo- lyzed antigen-antibody complexes were precipitated with an equal volume of saturated ammonium sulfate and the precipitate was dis- solved by boiling in 100 p1 of sample buffer (0.15 M Tris, pH 6.8, 5% SDS, 25% glycerol, and 12.5% 2-mercaptoethanol). After electropho- resis, the gels were stained with Coomassie Brilliant Blue R, de-

stained, and dried on an LKB slab gel drier. Autoradiography was performed using Kodak X-Omat AR film.

Radioimmunoassays-Radioimmunoassay (RIA) procedures were performed as previously described (17). In these simultaneous addi- tion competitive binding assays, 100 fmol of lZ5I-C5a, varying amounts of competing ligand and a quantity of appropriately titered murine anti-human C5a monoclonal antibody (TC-9) were incubated for 15 min at room temperature. Separation of antibody-bound and free "'I- C5a was then achieved by precipitation of antigen-antibody com- plexes after addition of an equal volume of saturated ammonium sulfate (18). Precipitates were harvested by centrifugation at 11,000 X g for 2 min (Beckman Microfuge) and bound isotope determined by counting in a Packard Auto-Gamma scintillation spectrophotom- eter.

Biological Assays-For degranulation assays, human neutrophils were isolated from the heparin anticoagulated venous blood of healthy donors by Ficoll-Hypaque density centrifugation (19). These cells were suspended at a final density of 2.5 X 106/ml in Hank's balanced salt solution containing 5 pg/ml of cytochalasin B and incubated for 5 min at 37 "C. After this, varying amounts of soluble stimuli, such as C5a or NO2-Tyr-C5a, were added and the incubation conducted for an additional 10 min at 37 "C. Cells were then removed by centrifugation and supernatants were analyzed for released myelo- peroxidase (20), p-glucuronidase (211, or lactoferrin (22) by estab- lished protocols.

Ligand-Receptor Binding Assays-Both direct and competitive li- gand-receptor binding assays were performed by techniques that have previously been described in detail (6). The direct assay employed varying amounts (0.1 to 10 nM, final concentration) of "'I-C5a or Bolton-Hunter labeled ANPAH-NH-Tyr-C5a incubated with 2.5 X 10' human neutrophils for 15 min at room temperature. Simultaneous addition competitive binding assays were conducted by incubating varying amounts of unlabeled C5a or C5a derivative as competing ligand with 1 nM lZ5I-C5a and 3 X lo5 human neutrophils for 15 min at room temperature. In each case the amount of cell-bound "'1- ligand was determined as previously described (6).

RESULTS

Preparation and Characterization of Nitrotyrosyl-Anaphy- latoxins-Selective chemical modification of both human C5a and de~-Arg~~-C5a was utilized as a means to evaluate the possible contributions of these molecules' tyrosyl residues to their antigenicity and biological activity. Initially, nitrotyrosyl derivatives of each glycopolypeptide were prepared and char- acterized in the following manner.

Quantitative spectrophotometric studies indicated that in- cubation of 0.1 mM human des-Arg7*-C5a with a 10-fold molar excess of TNM under nondenaturing conditions resulted in modification of only one of the two tyrosines in this glyco- polypeptide (data not shown). These results were confirmed by amino acid analysis which demonstrated a 50% reduction in the tyrosine content of the molecule (Table I). By contrast, reaction of TNM with de~-Arg~~-C5a in 6 M guanidine HCl resulted in a loss of 80% of the tyrosine content of the polypeptide (data not shown).

Identification of the single tyrosyl residue that was modified under nondenaturing conditions was facilitated by the fact that the 2 tyrosyl residues of des-Arg7'-C5a are found in different tryptic peptides that can be readily separated from each other by gel filtration. The results of such an experiment are illustrated in Fig. IA. For these studies NOz-Tyr-des- Arg7'-C5a, containing a trace of 'z51-des-Arg74-C5a was di- gested with TPCK-trypsin and the peptides were resolved on a column of Sephadex G-50. The tyrosyl residue in position 23 is contained within the large tryptic fragment that is labeled fraction I, while the tyrosyl residue in position 13 elutes as part of the Tyr-Lys dipeptide located at the end of the profile (fraction V). As demonstrated by the absorbance profile at 428 nm, only a single nitrotyrosyl moiety, that coelutes with the large tryptic fragment, could be identified. Confirmation that tyrosyl residue 23 had been selectively

Structure and Function of C5a 10341

TABLE I I A Amino acid compositions of des-Arg''-CSa and nitrated d e ~ - A r g ~ ~ - 2.0

- C5a derivatives

Native Nitrated derivatives Amino acid

des-Arg-C5aa des-Arg-C& T.Ib T-V

6.04 (6) 6.12 (6) 3.77 (4) ASP Thr' 3.16 (3) 2.97 (3) 2.04 (2) Sef 3.84 (4) 3.80 (4) 1.16 (1) Glu 8.92 (9) 9.04 (9) 4.95 (5) Pro GlY 3.48 (3) 3.18 (3) 1.44 (1) Ala 7.63 (8) 7.61 (8) 2.83 (3) Val 4.26 (4) 4.46 (5) 2.51 (3) FRACTION NUMBER

I- - 0.040 V z

0.5 - NDd ND ND

v) m

CYS ND '

Met 0.69 (1) Ile 5.22 (5) Leu 4.18 (4) TY r 1.94 (2) Phe 1.13 (1) LYS 7.67 (8) His 2.01 (2) '4% 3.99 (4)

Per cent yield NOZ-TyP

ND 0.54 (1) 4.68 (5) 3.99 (4) 0.88 (1) 1.18 (1) 8.06 (8) 2.27 (2) 4.00 (4) 1.24 (1)

ND

0.92 (1) 1.10 (1)

0.96 (1) 1.07 (1) 1.75 (2) 1.00 (1)

1.93 (2) 0.58 (1)

39 29

(9). Numbers in parentheses are based on published sequence data

* Peptide T-I is comprised of three separate tryptic peptides that are disulfide-linked. These peptides are Lys-20 through Arg-37, Cys- 47 through Lys-49, and Ala-50 through Arg-62 (see Ref. 9).

Values are corrected for loss during hydrolysis, 5% for Thr and 10% for Ser.

ND, not determined. e Determined by comparison with 3-nitrotyrosine standard.

modified by TNM was obtained by collecting fractions I and V, further purifying fraction V by HPLC and submitting these purified peptides to amino acid analysis. As shown in Table I, all of the tyrosine at position 23 had been converted to the nitro derivative while the tyrosine at position 13 remained unmodified. Similar results were obtained when human C5a, rather than de~-Arg~~-C5a was nitrated with TNM under similar conditions (data not shown).

The unique reactivity of the tyrosyl residue in position 23 of C5a was confirmed by additional experiments performed with human C3a. This molecule and C5a are quite homolo- gous, i.e. they share 29 amino acids including the tyrosine at position 13 (although the corresponding tyrosyl residue in C3a is numbered 15 due to an amino-terminal extention) (9). By contrast with the results obtained when either C5a or des- Arg74-C5a were nitrated, reaction of C3a with TNM resulted in partial modification of both t y r ~ s i n e ' ~ and the second tyrosine of C3a found in position 59. As shown in Fig. 1B, after trypsin treatment and gel filtration, the tyrosine at position 59 eluted as part of a large fragment (fraction I), while the tyrosine at position 15 eluted with the Tyr-Pro-Lys tripeptide near the end of the elution profile (fraction IV). These observations were confirmed by subjecting both frac- tions I and IV to HPLC purification and then amino acid analysis. As shown in Table 11, both t~ros ine '~ (peptide NOz- C3a-T-IV-E) and tyrosine59 (peptide N02-C3a-T-I-C) of C3a had been significantly modified after reaction with TNM under nondenaturing conditions.

Both N02-Tyr23-C5a and NOz-Tyr23-des-Arg74-C5a were separated from their respective unmodified precursors by ion- exchange FPLC. As shown in Fig. 2, after reaction of des- Arg74-C5a with TNM, N0z-Tyr23-des-Arg74-C5a (retention time, 46.5 min) could be completely resolved from native des- Arg74-C5a (retention time, 52.4 min) by chromatography of the reaction mixture on a Pharmacia Mono S column eluted

I - B ' \

3.0 - I -

0.40 1 E

z

1.0 - 2

f 2.0 - - d

- 0.30 0

X

z n V

- 0.20 z U

- 0 . 1 0 a

20 30 40 50 60 70 80 FRACTION NUMBER

FIG. 1. Sephadex G-50 chromatography of anaphylatoxin tryptic peptides. A, resolution of nitrated des-Arg-C5a tryptic pep- tides by Sephadex G-50 chromatography. Human des-Arg7'-C5a (60 nmol) was nitrated, mixed with 6 X lo5 cpm of '251-des-Arg74-C5a as tracer and digested with TPCK-trypsin (5% w/w). The digest was resolved on a 1.5 X 23.5-cm of Sephadex G-50 (superfine) eluted with 30% acetic acid. Fractions (0.5 ml) were analyzed for radioactivity then dried in vacuo and redissolved in 0.5 ml of 1 mM NH4OH for determination of the absorbance at 428 nm. B, elution profile of nitrated C3a tryptic peptides resolved by Sephadex G-50 chromatog- raphy. Human C3a (120 nmol) was nitrated, mixed with 5 X lo5 cpm lZ5I-C3a as tracer, and digested with TPCK-trypsin (5% w/w). The digest was resolved on a 1.5 X 46-cm column of Sephadex G-50 eluted with 30% acetic acid and analyzed as described above.

with a linear salt gradient. The NOZ-Tyrz3 derivative of human C5a was separated from native C5a under similar conditions.

In addition to the 2 tyrosyl residues, another potential site for modification by TNM is the single methionyl residue of C5a. To determine if this reaction had taken place, purified N02-Tyrz3-des-Arg74-C5a (10 nmol) was subjected to carbox- ypeptidase A digestion. Analysis of the released amino acids demonstrated Gly (9.74 nmol), Leu (10.48 nmol), Gln (8.22 nmol), Met (7.82 nmol), and Asp (2.83 nmol). These findings demonstrated that the important methionyl residue in posi- tion 70 (4) had not been significantly modified during the nitration reaction. As will be described, these FPLC-purified nitrotyrosyl derivative of C5a and de~-Arg~~-C5a were also employed both as starting materials for the preparation of additional anaphylatoxin derivatives and in specific biological and immunoassays.

Preparation of Additional C5a and de~-Arg~~-C5a Deriua- tiues-The aminotyrosyl derivatives of C5a and d e ~ - A r g ~ ~ - C5a were prepared by reduction of the respective FPLC- purified nitrotyrosyl derivatives with a 10-fold molar excess of sodium dithionite (12, 23). Quantitative reduction of the NOz-tyrosyl moiety was confirmed by amino acid analysis of the resulting derivatives which demonstrated complete loss of the nitrotyrosyl residue. Photoreactive derivatives of the an-

10342 Structure and Function of Cria

TABLE 11 Amino acid compositions of nitratrd-C.7a and nitratrd-C:h tryptic

prptides ~ ~~~~ ~~ . -

Amino arid NOz-C9n NOZ-C3a-T-I-C NOz-C3n-T-IV-E __ " .~

Asp 4.71 (5Y 3.00 ( 3 ) Thr 3.3 (3) 1.03 (1) Serb 4.01 (4) 1.96 (2) G l u 6.5 (7) 4.31 (4) Pro ND' ND ND (ily 3.68 (4) 2.17 (2) Ala 3.56 (4) 1.03 (1) Val 2.85 (3) 0.89 ( 1) cys N D ND ND Met 1.75 (2) 0.49 (1) Ile 1.97 (2) 1.72 (2) Leu 6.44 (7) 3.16 (3) 'l'v r I'he

0.64 3.21 (3) 2.97 ( 3 )

Lys 6.97 (7) 2.19 (2) 1.00 ( 1 ) His 2.42 (2) A rg 11.09 (11) 3.41 (3) NO,-TyP 1.25 0.80 ( 1) 0.88 (1) Per rent yield 18 23

' Numhers are based on published sequence data (25). *Values are corrected for loss during hydrolysis, 5% for Thr and

' ND, not determined.

. - .~ ~

~~ ~ ~ ~ ~ _ _ _ _ _ _ _

IO'% for Ser.

Determined by comparison with 3-nitrotyrosine standard.

I 1 1 I I I I

0 10 20 30 40 50 60 TIME (minutes)

FIG. 2. Resolution of nitrated dea-Arg7'-C5a from native d e s - A r ~ ~ ~ " C R a h y F P I X chromatography. After reaction with 'I'NM, f i 0 nmol of peptide was applied to a I'harmacia Mono S column that had heen equilibrated with 50 mM Na,HP04, pH 7.2. After isocratic elution for 15 min at a flow rate of 1 ml/min, a linear gradient inrreasing to 84 mM Na,HPO,, pH 7.2, was applied over the next 50 min.

aphylatoxins were then synthesized by coupling the photosen- sitive reagent, N-succinimidyl-6-(4'-azido-2'-nitrophenylam- ino)hexanoate to the arylamino group of NH2-Tyr-C5a and NHa-Tvr-des-Arg'"-(5a in 0.15 M sodium acetate, pH 5.0. This produced an orange derivative whose properties will he described.

Antigmic Propcrtim of dps-Arg7'I-CSa and Its Derivatives- RIA techniques were employed t,o determine if selective chem- ical modification produced an akeration in the antigenic properties of des-Arg7"-C5a. It was possible to address this question in a specific manner by utilizing a murine anti-

1

100 -

80 -

m o 60 - m \

40 -

20 -

0 I

-9 -8 -7 -6 -5 - LOG CONCENTRATION OF ANTIGEN IM)

FIG. 3. Inhibition of '2nI-CBa binding to murine monoclonal antibody TC-9 hy den-Arg7'-CRa and its analogs. ('ompeting ligands included des-Arg-Cm (0); SO,-'l'vr-des-Arg"-(':ia (0); A S - PAH derivative of' NH,-Tyr-des-Arg"-C:a (AI. Inwt. nutr)r;diogra- phy of SDS-PAGE analysis performed after photolysis o f the ra- dioiodinated ANPAH derivative of SH2"I'yr"-('5a pre-t)r~unti t o a n - tibody TC-9. Imnr I demonstrates total hintling. I,anv 2 clrmonstrates nonspecific binding observed in the presence of' a 1 o O O - f o l r i molar excess of native C5a.

, .

human C5a monoclonal antibody (TC-9) that cross-reacted exclusively with a C5a-derived tryptic peptide that contained the tyrosyl residue in position 23 of (sa.' The results of RIA'S performed with TC-9 as primary antibody and ""1-C:ia as radioiodinated antigen are depicted in Fig. 3 . These studies demonstrate that native des-Arg"-CFia, NOa-Tyr-des-Arg"- C5a, and the photoreactive (ANPAH) derivative of NH,- TyrZ3-des-Arg74-C5a were all essentially equivalent with re- gard to their ability to inhibit binding of ""I-Cfia to the monoclonal antibody (ID5, of 1.3 x lo-: M, 1.0 X 10" M, and 0.7 x lo-' M, respectively). These observations suggest that selective chemical modification of Tyr'" did not prorlr~ce a significant perturbation of the antigenic properties of human des-Arg""C5a.

Additionally, these findings suggested that the ANPAH- derivative of NH,-Tyr""-des-Arg"-(::ia might serve as a useful photoaffinityprobe. To explore this possihilitv, ANPAH-NH- Tyr-des-Arg"I-Cfia was first radioiodinated, then incuhated with TC-9 in the dark. After binding was complete. the mixture of antigen and antihodv was photolyzed, subjected to SDS-gel electrophoresis, and analyzed by autoradiography. As shown in the inset in Fig. 3 , radioisotope-labeleri hands were observed a t 14,000, 28,000, and 42,000 daltons. These presumably represent monomer, dimers, and trimers, respec- tively, of t h e "'II-ANPAH-NH-Tyr-des-Ar~~'-~5a derivative. These conclusions are based on the ohservat ion that the apparent molecular weight oft he C5a monomer. when defined by SDS-PAGE techniques, is approximately 16,000 (24 ) . Ad- ditionally, the hands observed at 28,000 and 42,000 daltons were produced only after photolvsis of the photoreactive des- ArgiJ-C5a analog. Oligomers of NOa-Tyr"'-('5a were not nh- served under similar conditions (data not shown). These findings not only imply that nitration of C5a or cies-ArgT5a

* R. J. Johnson and I). E. Chenoweth. manuscript in preparation. - ~-

Structure and Function of C5a 10343

does not result in dimerization of the molecule but they establish the photoreactivity of the ANPAH-NH-TyrZ3-des- Arg-C5a analog as well. Furthermore, a uniquely labeled band noted a t 64,000 daltons most likely represents the murine IgG heavy ~hain-"~I-ANPAH-NH-Tyr-des-Arg~~-C5a covalent adduct (lane I ) , since this material is not produced in the presence of excess unlabeled d e ~ - A r g ~ ~ - C 5 a (lane 2) . Addition- ally, of the 8 other monoclonal antibodies tested in this fashion, 5 formed a similar covalent adduct while 3 did not. These findings clearly demonstrate that a functionally active C5a photoaffinity probe had been prepared by the techniques described.

Biological Activity and Receptor Binding Properties of C5a Deriuatives-As shown in Fig. 4, both native C5a and des- Arg74-C5a inhibit binding of lZ5I-C5a to specific receptors found on neutrophils, although they differ in apparent affinity for these receptors, with d e ~ - A r g ~ ~ - C 5 a being about 100 times less effective than C5a. Both nitrated C5a and de~-Arg~~-C5a, as well as aminotyrosyl d e ~ - A r g ~ ~ C5a, all differ from their respective parent molecules when evaluated in these compet- itive binding assays. In each case, the analogs containing modified tyrosyl residues are approximately %fold less active than their respective native precursors. Furthermore, the subtle differences between C5a and NOZ-Tyrz3-C5a that were noted in these receptor binding assays were also manifest when the biological activities of C5a and N02-TyrZ3-C5a were compared. As shown in Table 111, N0z-Ty?3-C5a was approx- imately 3-fold less active than native C5a when this derivative was employed to promote selective release of lysosomal en- zymes from cytochalasin B-treated human neutrophils.

These observations demonstrated that selective modifica- tion of tyrosylZ3 of the C5a might result in a modest pertur- bation of this molecule's activity. However, the fact that both the NO2-TyrZ3 and NHz-TyrZ3 derivatives of C5a and des- Arg74-C5a still retained the capacity to interact with the neutrophil C5a receptor suggested that it might be possible to synthesize a photoreactive C5a analog that could be used to probe the C5a receptor. Therefore, an ANPAH-NH-Tyr-C5a derivative of human C5a was prepared and radioiodinated with Bolton-Hunter reagent at pH 7.0. This lZ5I-labeled deriv- ative was then evaluated for its ability to bind directly to the

LOG CONCENTRATION OF LIGAND (M) FIG. 4. Inhibition of lZ5I-C5a binding to human neutrophils

by C5a, de~-Arg'~-C5a, and their derivatives. Simultaneous addition assays were performed with 1 nM lZ5I-C5a, 3 X lo5 human neutrophils, and varying concentrations of each competing ligand. Competing ligands included: C5a (A), NOZ-Tyrz3-C5a (W), des-Arg7'- C5a (O), N0z-Tyr23-des-Arg74-C5a (O), NHZ-Tyrz3-des-Arg7'-C5a (A).

TABLE I11 Ligand-induced degranulation of human neutrophils

Cytochalasin B-treated human neutrophils were incubated with either native or NOZ-Tyr-C5a at final concentrations ranging from 0.1 to 50 nM and the quantity of enzyme specifically released into the supernatant determined as described under "Experimental Proce- dures." The concentration of either glycopolypeptide that promoted half-maximal release (EDW) of each neutrophil granule marker was determined by linear regression analysis of the dose-response profiles obtained from duplicate determinations in two separate experiments.

Constituent released C5a N02-Tyr-C5a

EDm, nM

Myeloperoxidase 1.0 1.8 @-Glucuronidase 1.0 2.9 Lactoferin 1.0 3.5

RADIOLIGAND CONCENTRATION (nM) FIG. 5. Binding of radioiodinated native C5a and ANPAH-

NH-TyrZs-C5a to human neutrophils. Varying quantities of either lZ5I-C5a (0) or the radioiodinated photoreactive derivative (0) were incubated with 3 X lo5 human neutrophils for 15 min at 22 "C and the amount of bound ligand determined by standard methods (6).

neutrophil receptor. As shown in Fig. 5 the binding of lZ5I- labeled native C5a to neutrophils is saturable and half-maxi- mal binding is observed at a concentration of approximately 1 nM. By contrast, the photoreactive C5a analog displays only a nonspecific binding isotherm (Fig. 5) and fails to specifically label any neutrophil membrane component even though this derivative can be covalently cross-linked to the various mono- clonal antibodies after photolysis. Given these results, we reasoned that the p-azido(2-nitropheny1amino)hexanoyl group might be too bulky to permit ligand-receptor interac- tion. Therefore, we also prepared a C5a derivative that con- tained a smaller photoactivatable group. This was accom- plished by substituting the p-nitrophenylester of 2-diazo- 3,3,3-trifluoropropionate for SANPAH in the coupling reac- tion with NHZ-Tyr-C5a. However, even after preparation, radioiodination, and direct evaluation of this derivative, we were unable to demonstrate specific binding of this probe to intact human neutrophils.

DISCUSSION

Both human C5a anaphylatoxin and its somewhat less active d e ~ - A r g ~ ~ analog are thought to function as important

10344 Structure and Function of C5a

mediators of the inflammatory response. These complement- derived chemotactic factors initiate granulocyte responses after they bind to specific cellular receptors (6-8). The current investigations demonstrate that selective modification of the tyrosylZ3 residue of C5a can produce a reduction in both the receptor binding properties and the biologic activity of this glycopolypeptide without resulting in a significant perturba- tion of this molecule's antigenic properties. These observa- tions suggest that this specific tyrosyl residue may contribute importantly to productive ligand-receptor interactions.

Selective nitration of tyrosinez3 in C5a was achieved by reaction with TNM under nondenaturing conditions. Detailed analysis of tryptic peptides obtained from NOZ-Tyr-de~-Arg74- C5a clearly demonstrated that the nitration reaction was limited to this site and did not take place on the tyrosyl residue in position 13. This observation is somewhat surpris- ing when one considers the facts that: (a) the analogous tyrosyl residue of human C3a was readily modified under similar conditions; and (b ) the tyrosyl residue in position 13 or C5a is in a considerably more basic environment (Lys"- Tyr'3-Lys'4-His'5) than that immediately surrounding TyrZ3 (CyszZ-Tyr23-Asp24-Gly25) and would be expected to react pref- erentially (11). Thus, while the tyrosyl residue in position 23 of the C5a sequence may be selectively modified by TNM, TyrI3 seems to exhibit a rather unique lack of reactivity with this reagent. These findings suggest that certain steric factors may limit the reactivity of TyrI3.

Nitrotyrosylz3-C5a and -de~-Arg~~-C5a were readily reduced to their respective aminotyro~yl'~ analogs under mild condi- tions with sodium dithionite (12). These reduced derivatives were prepared for two reasons. First, after reduction of the nitro group, the pK, of the tyrosyl hydroxyl group returns toward a more normal value, i.e. approximately 10 (11, 12). Thus, evaluation of aminotyrosyl analogs permits delineation of the contributions of charge effects that might have been produced by the original nitration reaction. Second, the pK, of the arylamino group is near 4.8 (12). Therefore, we were able to selectively introduce two different photoreactive probes at this specific site by performing the appropriate substitutions at pH 5.0.

Before the properties of these various analogs could be compared in a meaningful way, we felt that it was essential to separate both N0z-Tyr23-C5a and -de~-Arg~~-C5a from their respective unmodified precursors. To accomplish this goal, we devised a cation-exchange FPLC purification protocol (Fig. 2). By employing this technique to purify the nitrotyrosyl derivatives we insured that defined polypeptides could be utilized in subsequent modification studies and that the native glycopolypeptide themselves would not be present in subse- quent bio- and immunoassays.

Furthermore, FPLC purification of NOz-TyP3-C5a and - des-Arg7'-C5a also facilitated chemical analysis of these de- rivatives. For example, we felt that it was important to dem- onstrate that the single methiony17' residue of the C5a se- quence had not been oxidized during reaction with TNM (11). This was done by subjecting the FPLC-purified nitro-des- Arg74-C5a to carboxypeptidase digestion and demonstrating that methionine could be quantitatively released.

Radioimmunoassays performed with murine anti-human C5a monoclonal antibodies were employed to define the cross- reactivity of the des-Arg-C5a derivatives produced by selective modification of Tyr23. The data obtained with one monoclonal antibody, TC-9, are shown in Fig. 3. This particular antibody was selected for these studies because prior investigations' had demonstrated that TC-9 specifically cross-reacted with a C5a-derived tryptic peptide that contained TyrZ3. Therefore,

we reasoned that this antibody could provide a rather specific means of detecting minor antigenic perturbations that might result from modification of TyrZ3. Interestingly, when RIA evaluations were performed with this antibody, we found that nitration of the tyrosylZ3 side chain, reduction of this group by sodium dithionite, and/or coupling a p-azido(2-nitrophen- y1amino)hexanoyl group to the arylamino group did not sig- nificantly alter the antigenic character of de~-Arg~~-Csa.

We consider these observations to be important for several reasons. First, these RIA procedures demonstrate that tyrosinez3 of C5a may be modified without producing signifi- cant changes in the immunodominant portion of the molecule. Second, the fact that reduction with sodium dithionite did not alter the antigenicity of NOz-Tyr'3-des-Arg74-C5a implies that this mild reductive procedure did not significantly effect intrachain disulfide bonds (25). Third, as shown in the inset in Fig. 3, studies performed with this monoclonal antibody clearly demonstrated that a functionally active photoaffinity probe was actually produced after reaction of NHz-TyrZ3-C5a with SANPAH.

In marked contrast with the result obtained in immunoas- says, cellular studies suggest that selective modification of C5a's TyrZ3 residue might result in a dimunition of this molecule's ability to bind to the granulocyte receptor. Both competitive binding (Fig. 4) and bioassays (Table 111) sug- gested that nitrotyrosyl and aminotyrosyl C5a and d e ~ - A r g ~ ~ - C5a are approximately %fold less active than their native precursors. Initially, we doubted that these slight differences were significant and felt encouraged to prepare photoreactive analogs of NHZ-Tyrz3-C5a that could be employed to specifi- cally label the granulocyte C5a receptor. However, when either the ANPAH or the somewhat smaller 2-diazo-3,3,3-trifluoro- propionate derivatives of aminotyrosyl C5a were prepared and evaluated, we found that neither of these derivatives retained the capacity to bind specifically to the C5a receptor even though their ability to bind to and become cross-linked with anti-C5a antibodies after photolysis could be readily demon- strated. Taken together, these findings suggest that nitration of TyrZ3 results in a modest perturbation of the biological activity of C5a, while insertion of bulkier groups at this site results in a significant abrogation of this molecule's ability to bind to the granulocyte C5a receptor even though the general antigenic topography of the glycopolypeptide is not signifi- cantly altered.

In summary, we would like to propose that our current data suggest the following hypothesis. In the C5a molecule, the tyrosyl13 residue is not modified by TNM while TyrZ3 is. The implication of this observation may be 2-fold. Since TyrI3 is now known to be located in an a-helical portion of the C5a molecule (26), we would like to suggest that the hydrophobic face of this a-helical region may be in opposition to other hydrophobic domains found on the surface of the molecule. By contrast, the side chain of T Y ~ ' ~ is readily modified. This implies that this residue is accessible and could participate in ligand-receptor interactions. The facts that nitrotyrosyl and aminotyrosyl derivatives are less active than their precursors and that insertion of bulky photoreactive probes at this spe- cific locale results in the abolition of ligand-receptor interac- tions supports this contention. For these reasons, we would propose that structural features found in the region of TyrZ3 may contribute importantly to the biological activity and receptor binding properties of human C5a anaphylatoxin.

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