c5a fragment of bovine complement

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Eur. J . Biochem. 155 ,77- 86 (1986)0 EBS 1986

C5a fragment of bovine complementPurification, bioassays, amino-acid sequence and other structural studies

Renato GENNA ROand Domenico ROMEO

’ stituto di Fisiologia Veterinaria e Biochimica, Un iversita di M ilano

(Received October 7,198 5) - EJB 85 1108

Tatjana SIMO NIC’, Arm ando NEG RI’, Cnstina MOTTOLA

’,Camillo SECCHI’, Severino RONCHI’

Dipartimen to di B iochimica, Biofisica e Chimica delle Macrom olecole, Universita di Trieste

1. C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of thepurification was followed by measuring the chemotactic activity of the complement fragments. 2. The twopolypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/responseeffects. 3. After preparing a rabbit antiserum to bovine CSa/des-Arg-CSa, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 pg/ml. 4. The complete primarystructure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic

peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule.5. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts withan antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. 6 . Finally, thesecondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the aminoacid sequence. A comparison of the content and distribution of a-helical and/or hydropathic regions, suggeststhat the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologousC3a molecule.

Chemotaxis of inflammatory cells may be induced byproducts of humoral and cellular immune or immune-relatedreactions, and by products elaborated by bacteria or by theinflammatory cells themselves [l]. Among these pro-

inflammatory agents the complement fragment C5a is one ofthe most potent, also eliciting generation of cytotoxic oxygenradicals and secretion of granule content from bothneutrophils and macrophages [2 - 101. Furthermore, thispolypeptide exhibits classical anaphylatoxic properties, i.e. itcauses smooth muscle contraction, histamine release frommast cells and enhanced vascular permeability [I, 2, 11- 31.

A clear picture of the relation between structure and func-tion of this chemotactic and spasmogenic molecule, whichconsists of 74 amino acid residues, is not yet available [8]. Infact, (a) there is paucity of sequence-homology information,because only the primary structures of human and porcineC5a have been elucidated [14- 161, and (b) scarcity of suitablequantities of C5a has hampered the determination of its sec-

ondary and tertiary structure by physico-chemical techniques.Starting from several liters of bovine serum, we have purifiedmilligram amounts of C5a and des-Arg-CSa, whose effecton neutrophils have then been compared. The amino acid

Correspondence to D . Romeo, Dipartimento di Biochimica, Bio-fisica e Chimica delle Macromolecole, Universita di Trieste, ViaA. Valerio 32,I-34 127 Trieste, Italy.

Abbreviations. EAhx, 8-aminohexanoicacid; BI2BP, itamin-Blz-binding protein; ELISA, enzyme-linked immunosorbent assay;HPLC, high-performance liquid chromatography; SDS, sodiumdodecyl sulfate; PAGE polyacrylamide gel electrophoresis; TLC,thin-layer chromatograph y.

Enzymes. Trypsin (E C 3.4.21 .4); chymotrypsin (EC 3.4.21.1);carboxypeptidaseY (EC 3.4.16.1); carboxypeptidaseN (EC 3.4.17.3).

sequence of the purified chemotaxin, in conjunction withspectral and hydropathy analyses, have also been utilizedto predict its secondary structure and to draw a model ofconformation of C5a, based on the reported crystal structure

of the homologous C3a molecule [17].

MATERIALS AND METHODS

Materials

Small-pore diameter tubing (90 mm diameter, 3500 M ,cut-off) was purchased from A. H. Thomas Co., USA.Sephadex G-100 and protein-A - epharose CL4B wereobtained from Pharmacia, CM-cellulose CM52 fromWhatman and Bio-Gel P-10 from Bio-Rad. Trypsin (treatedwith tosylphenylalanylchloromethane) and a-chymotrypsinwere products of Worthington Biochemical Co., and yeastcarboxypeptidase Y of Boehringer. Cyan~[~’Co]cobalamin

was purchased from Amersham, and all the reagents used forsequence analyses from Pierce. Rabbit antiserum to porcineC5a was a generous gift of B. Damerau.

Functional behaviour of neutrophils

Neutrophil-rich granulocyte preparations were obtainedfrom EDTA-anticoagulated bovine blood as reported [18].Neutrophils were also isolated from human blood accordingto standard techniques [6, 81. Chemotaxis was assayed by anunder agarose method [S] and the number of migrated cellswas evaluated as in [19]. Secretion of vitamin-BI2-bindingprotein from neutrophils was determined as describedelsewhere [20].



78

Purijica ion of CSaldes- A rg- C5 a

Four batches of bovine serum for a total volume of 17 1(40 1 blood) were activated for 45 min at 37°C by boiledbaker’s yeast (20 g/l) in the presence of 1 M EAhx, titratedto pH 3.8 and dialyzed in small-pore tubing against 0.1 Mammonium formate at pH 5.0 (buffer A) with 0.5 mM EDTA,as described by Fernandez and Hugli [21]. CM-cellulose wasstirred into the dialyzed serum (45 ml of settled slurry/lserum), and, after standing overnight in the cold, the mixture

was filtered and extensively washed with buffer A.Proteins with chemotactic activity were recovered from

CM-cellulose by batch-wise elution with 0.5 M ammoniumformate at pH. 5.0. The eluate was brought to 45% saturationwhith solid ammonium sulfate, the precipitate was separatedby centrifugation, and further ammonium sulfate to 80%saturation was added to the supernatant. After centrifugation,the pellet was suspended in a small volume of buffer A, andthen lyophilized. The lyophilized material derived from thefour batches of serum was dissolved in 120 ml of buffer A,and dialyzed against the same buffer. The dialysate was thenloaded on a column (125 x 4.5 cm) of Sephadex G-100 whichwas eluted with buffer A at a flow rate of 1.6-2.5 ml cm-2h- I, monitoring the eluate by absorbance measurements at

280 nm. The fractions eluting from about 1250 ml to about2000 ml, which contained chemotactic material, were pooledand lyophilized. After dissolution in 16 ml buffer A, the mate-rial was loaded on a column of Bio-Gel P-10 and then elutedwith the same buffer. Fractions reacting with rabbit antiserumto hog C5a (see Results) were pooled and lyophilized. Afterdissolution in 17 ml of 50 mM ammonium formate, pH 5.0,this material was applied to a column (5.8 x 1.6 cm) of CM-cellulose, which was first washed overnight with the samebuffer and then eluted with a linear gradient formed with200ml 50mM buffer and 200ml 500mM ammoniumformate, pH 5.0, at a flow rate of 12 ml cm-2 h-’.

Electrophoresis

Microzone electrophoresis (30 min at 300 V) was perform-ed in a cold room with cellulose acetate strips (Titan 111, Hel-ena Labs, USA) soaked in 0.16M Tris/lOmM EDTA,brought to pH 8.6 with boric acid.

Polyacrylamide gel electrophoresis (PAGE) was carriedout on a 15% acrylamide gel slab containing 0.1 M phosphate,pH 7.2, 0.1% SDS [22] and 2.5 M urea, using cyanogenbromide cleavage peptides of myoglobin (LKB) as molecularmass standards.

Antiserum to C 5a, immunodiffuusion and ELISA

A rabbit antiserum to bovine C5a was obtained after re-peated immunizations of two rabbits with purified CSa/des-Arg-C5a (100 pg) mixed with complete Freund’s adjuvant.The immonoglobulins were precipitated from the antiserumwith 40% sodium sulfate and further purified by affinitychromatography on protein-A- Sepharose CL-4B. Doubleimmunodiffusion was carried out in 1% agarose in 0.9%NaCI.

For setting up an enzyme-linked immunosorbent assay(ELISA) for C5a, various concentrations of antigen (0.75-7500 nglml), used for coating the microtiter wells, weretitrated with increasing amounts (0.1- 0 pglwell) of immu-noglobulins purified from the anti-C5a rabbit antiserum [23].The antigen-antibody complexes formed were then reacted

with goat anti-(rabbit IgG) conjugated with alkalinephosphatase [23], whose activity was then measured withp-nitrophenyl phosphate at pH 9.75. The results of this titra-tion suggested the use of 75- 25 ng/ml of antigen to coat themicrotiter wells in a competitive ELISA, which was developedwith 3- 0 pg antibody per well.

Amino acid analyses

Dried aliquots of purified C5a or des-Arg-CSa, containing100pg protein, were hydrolyzed for 24 h, 48 h and 72 h at110°C in evacuated sealed tubes with 0.5 ml of 6 M HCl.Amino acids were analyzed with a C. Erba automatic aminoacid analyzer. The content of half-cystine was quantified ascysteic acid [24]. Tryptophan was determined after hydrolysiswith 4 M methanesulfonic acid in the presence of 2%tryptamine [25].

Carboxym ethylation and enzymatic digestion

Suitable amounts of freeze-dried C5a were redissolved in1 M Tris/HCl pH 8.3, containing 6 M guanidine hydrochlo-ride and 10 mM EDTA and reduced under nitrogen at room

temperature for 2 h, by addition of a 10-fold molar excess ofdithiothreitol. Alkylation of thiol groups was performed byincubation for 60 rnin in the dark with recrystallizediodoacetic acid using a 1.1-fold molar excess over the total- H concentration. Reagents were removed by exhaustivedialysis against water.

The freeze-dried carboxymethylated protein (150 nmol)was resuspended in 0.2 ml of 50 mM phosphate buffer,pH 8.0, and digested with trypsin (1y0,w/w) for 15 ha t 37°C.An identical amount of freeze-dried material, resuspendedin 0.5% ammonium bicarbonate, pH 8.0, was digested witha-chymotrypsin (2% w/w) for 4 h at 37°C. Both reactionswere stopped by adding a drop of 50% trifluoroacetic acid.

Purijication and amino acid analysis of tryptic andchymo rypt c pep ides

Separation of peptides was achieved by reverse-phaseHPLC, using either a Waters Associates pBondapak CI8column (3.9 mm x 30 cm) or a Brownlee Labs Aquapore RP

300 column (10 pm, 7 mm x 25 cm). Peptides were elutedwith a linear gradient of 0-30% acetonitrile in 0.1%trifluoroacetic acid over 64 min at a flow rate of 2.0 ml/min,using a Jasco HPLC instrument equipped with an Uvidec-100 V spectrophotometer set at 220 nm. Eluted material cor-responding to the absorbance peaks was submitted to aminoacid analysis as described above.

N - and C-terminal analyses

The N-terminal residue of the intact C5a was determinedby the method of Gray [26] and its dansyl derivative wasidentified by TLC [27]. The C-terminal residue was identifiedaccording to Hayashi [28]. Carboxypeptidase Y digestion ofintact C5a or des-Arg-C5a was performed in 0.1 M pyridineacetate, pH 5.5, at 37°C for 5- 0 min, at an enzyme substrateratio of 1 100 (w/w).

C-terminal analyses of C5a peptides were performed byincubating for 15- 20 rnin and increasing the carboxy-peptidase concentration to 2% (w/w). The residues released bycarboxypeptidases Y were identified by aminoacid analysis.



79

Sequencing methodologies

Manual N-terminal sequence analyses of tryptic andchymotryptic peptides were performed by Edman degrada-tion as described by Tarr [29]. Phenylthiohydantoins wereidentified by HPLC on a Spherisorb S5 ODSI column asdescribed by Tarr [30]. About half of the N-terminal aminoacid sequence was also controlled by an automated Beckman890 M sequencer.

A computer search through published protein sequences

was conducted with the assistance of M. A. Cimbala (Depart-ment of Biochemistry, University of Massachusetts MedicalCenter, USA), by utilizing the Intelligenetics library.

Other analytical techniques

Protein was routinely determined by the method of Lowryet al. [31], with bovine serum albumin as reference protein.Concentrations of purified C5a samples were also checked byamino acid analysis, assuming integral values for Asp, Glu,Gly, Ala and Phe. The results of these analyses yielded acorrelation factor of 0.75 with respect to the Lowry’s method.

;t”I

.i

2

%

Y

aB

”

I A II I I I 1 I I

10 ao 10 70

Cracti.n We

Fig. 1 . Bio-Gel P-I0 hromatography of chemotactic material fr om aprevious gel filtration through Sephadex G -10 0. The Bio-Gel P-10

column (125 x 2.6 cm) was eluted with 0.1 M ammonium formate,

pH 5.0, at a flow rate of0 .8-1.1 ml - cm-’ .h-’. Fractions 26-52

and 65- 3 exhibitcd optimal chemotactic activity at about 10 pg and0.1 pg protein/ml. respectively

RESULTS

Purification of C 5 a

The initial purification steps of CSa/des-Arg-CSa weredesigned to recover all the chemotactic material present inyeast-activated bovine serum (1 300 g protein). They consistedin a batch-wise CM-cellulose adsorption step, followed byammonium sulfate (45- 0% saturated) precipitation, andgel permeation through Sephadex G-100, yielding 40 g, 10 gand 0.33 g protein, respectively.As shown in Fig. 1, the activeprotein pool derived from Sephadex G-100 was further re-solved into four peaks by gel permeation through Bio-Gel P-10.Fractions 26- 2 (pool A) and 65- 3 (pool B, 30mg protein)

exhibited chemotactic activity, with the latter fractions beingseveral-fold more active than the former ones. In a double-immunodiffusion experiment, pool B (1 pg protein) but notpool A (up to 25 pg protein) reacted with a rabbit antiserumto porcine C5a. Finally, pool B showed a single band afterSDS/urea/PAGE with an apparent M , of 8000.

When subjected to CM-cellulose chromatography, pool Bwas resolved into four components (Fig. 2) . Two minor ones,around peaks I and 11,were chemotactically inactive, whereasfractions pertaining to peaks 111and IV, totalling 25 mg pro-tein, were very active. After microzone electrophoresis,fractions 143of peak I11and 164 of peak IV migrated as singlebands, with the former migrating to a more anodal positionthan the latter (Fig. 2). Furthermore, fraction 141 had the

same amino acid composition as pooled fractions 164- 166,but lacked one amino acid, Arg (Table 1). We thus concluded

1 I V I

I ? :-*

.. - ... . .. . . .. . .. . . .. .. .

30 OD DO 120 150 180

Fract ion N O

Fig. 2. CM-cellulose chromatography o pool B of Fig. 1. The lyophilized proteins of pool B were dissolved in 17 ml of 50 mM ammonium

formate, pH 5.0, and applied to a column (5.8 x 1.6 cm) of CM-cellulose. After washing overnight with the same buffer, the column was eluted

with a linear gradient formed with 20 0 ml of 50 mM buffer and 200 ml of 500 m M ammonium formate, pH 5.0, at a flow ra k of 1 2 m l cm-’

h - ’. Insert: microzone electrophoresis of fractions 162 (a, peak IV), 143 (b, peak 111) and 145- 60 (c, I11- V trough)



80

Table 1. Amino acid composition of bovine C5a as compared to humanand porcine CSaFractions 141 (A) and 164-166 (B) from th e CM-cellulosechromatography step (Fig. 2) were pooled and subjected to aminoacid analysis.Values in parenthese are nearest integers

Amino acid Amount in polypeptide

LysineHistidineArginineAspartic acidThreonineSerineGlutamic acidProlineGlycineAlanineHalf-cystineValine'Methionine

Isoleucine'Leucine"TyrosinePheny alanine

Tryptophand

Total amino acidresidues

M,

A B human porcineC5a [26] C5a [26]

nmol/mol

9.0 (9)2.9 ( 3 )

5. 2 ( 5 )

6.9 (7 )1 . 1 (1 )1.9 (2)

9.9 (10)1.0 (1)3.2 (3)

10.9 (11)5.5" (6)

1.5 (2 )1.0 (1 )

4.5 (5)2.2 (2)

2.0 (2)1.7 (2)

0.7 (1)__

73-

9.1 (9)3.0 ( 3 )

6.0 (6)6.8 (7)1. 3 (1)1.9 (2)10.3 (10)

1. 2 (1)

3.3 (3)10.8 (11)5.6b (6)1 .3 (1)

1.6 (2)

4.5 (5 )2.2 (2)

2.1 (2)

1.9 (2)

0. 7 (1)

748537

8 1 1

2 1

5 5

6 73 1

4 1

9 1 1

1 1

3 38 97 65 21 2

5 54 3

2 5

1 1- -

74 748286 8621

" Determined as cysteic acid after hydrolysis in the presence ofdimethylsulfoxide.

Determined as carboxy methylcysteine.

Quantified after hydrolysis in methan sulfonic acid in the pres-' Referred to 72-h hydrolysis.

ence of tryptamine.

that peaks 111 and IV represented des-Arg-C5a and C5a, re-

spectively. This was confirmed by N- and C-terminal analysesof carboxymethylated material from peaks 111and IV , which

showed that both possessed methionine as N-terminal residue,

with glycine and arginine at the C-terminal end, respectively.

Modulation of neutrophil unctions by C 5a and des-Arg-C5 a

When the effects of C5a on neutrophil migration were

compared with those of des-Arg-CSa, it was found that thetwo peptides elicited a maximal response at the same concen-tration (Fig. 3), with an identical pattern of cell desensitiza-

tion. However, the shape of the response curve of neutrophilsto C5a and des-Arg-C5a varied in different experiments, and

the number of cells which migrated towards the chemotaxin

well was some times higher when the stimulus was provided

by C5a, some others when des-Arg-C5a was the chemotactic

agent. Bovine C5a also induced oriented migration of human

neutrophils.

Also the pattern of activation of Bl z BP secretion from

neutrophils was practically the same with C5a and its desArg

derivative, the latter being slightly more active at the lowestconcentrations (Fig. 4).

300-

C5a

I1 I I I '

1 10

Chemotaxin concn (pg/ml)

.001 .0 1 .1

Fig. 3. Chemotaxis under agarose of bovine neutrophils responding to

CSa or des-Arg-CSa. Th e figure shows results of three individual

experiments

30

20-

10-

O--O C5a

Id' C5adesAr~

ai 10 100

Secretagogue concentrailon @/mi)

Fig. 4. Secretion of vitamin-B12-binding rotein ( B I 2 B P ) rom bovineneutrophils exposed to C5a or des-Arg-CSa. The ~yano[~'Co]co-balamin binding capacity of supernatants derived from neutrophilsincubated (30 min, 37°C) with th e purified peptides was determinedas reported elsewhere [20]. Data are means of five experiments (theSE M was lower than 25% of the means)

Immun ochemical detection of C5a

The rabbit antiserum to CSa/des-Arg-CSa gave a single

precipitation line with pool B (Fig. l), but did not react with

the chemotactic material of pool A. This specifity was ex-ploited for setting up an ELISA aimed at an immunochemical

detection of C5a. As shown in Fig. 5, the competitive

immunoassay had a sensitivity which was greater than that of

the chemotactic assay, allowing measurements of C5a in a

concentration range of about 5- 000 ng/ml.

Amino acid sequence of C5 a

Tocharacterize the complete amino acid sequence of CSa/

des-Arg-CSa, samples of about 150nmol, after reduction and



81

' 7 1

0.03 0.1 a3 1

c5a/c5adesArg ( m / r n l )

Fig. 5.Calibration curvefor a competitiveE LI SA . Wells of a microtiterplate coated with CSa/des-Arg-CSa(overnight incubation at roomtemperature with 0.27 ml of a solution in 50 m M NaHC03with 75 ngprolein/ml) were reacted with 10 pg protein of a rabbit IgG fraction,containing anti-C5aantibodies (2 h at 37"C), n th e absence or in the

prescnce of various amounts of CSa/des-Arg-C5a in the fluid phase.T he immune complexes remaining in the wells were detected with goatanti-(rabbit immunoglobulins),conjugated with alkaline phospha-tase, which then catdyzed the formationof -nitrophenol(maximum

absorbance at 405 nm) from p-nitrophenyl phosphate [23]

carboxymethylation, were submitted to tryptic or chymo-tryptic digestion. Separation of tryptic peptides was achievedby reverse-phase HPLC (Fig. 6 ) . Amino acid and sequenceanalyses of eluted fractions allowed the identification of 19tryptic peptides (Table 2), which accounted for the wholeamino acid composition of CSa/des-Arg-CSa. Some of thetryptic fragments were originated by incomplete cleavage atcontiguous bonds, involving two or more Lys residuesoccurring next to each other in the sequence. Since the

unresolved mixture CSa/des-Arg-CSa was used for this analy-sis, two C-terminal peptides (referred as T15and des-Arg-T15in Table 2 ) were obtained.

Of the reported peptides 17 were completely sequenced bymanual Edman degradation, and as shown in Table2, apartial sequence of T7-8-9 and T8-9 (eight N-terminal andfour C-terminal residues) was established. Alignment of thetryptic peptides was achieved by sequence analysis of

chymotryptic peptides, as well as by automatic Edman degra-dation of about half of the whole chemotaxin.

Fig. 7 shows the HPLC elution profile of the chymotrypticpeptides, which were characterized by amino acid composi-tion and sequence (Table 3) . Two fragments, C6 and C6-7-8,

Fig. 6. Elution pro$le of' C5a tryptic peptides, separated on Aquapore R P 300 using a linear gradient of 0-30% of aretonitrile in 0.1%

triyuorocetic acid. Flow rate 2.0 ml/min

. i - - i a

Fig. 7. Elution profile of C5a chymotryptic peptides. separated on pBondapak C18 Waters using a linear gradient of 0-30% (facetonitrile in

0.1% trifuoracetic acid. Flow rate 2.0 ml/min



Table 2. Amino-acid composition and sequence of bovine C5a tryptic peptides

Molar ratios of each amino acid, calculated from analyses performed on 24-h hydrolysates, are indicated under corresponding symbols,appearing for the first time in the sequence. The sequence of residues in brackets was not determined. Positions refer to the intact polypeptides

Tryptic Amino-acid composition and sequencepeptides

T1

T1-2

Tl-2-3-4

T2-3-4

T3-4

T5

T6

T6-7

T7-8

T7-8-9

T8-9

T9

T10

T11

T12

T13

T14

Met -Leu -Lys (1 -3)0.7 0.9 1.0

Met -Leu -Lys -Lys (1 -4)

1.0 0.9 2.0

Met -Leu -Lys -Lys -Lys-Ile -Glu-Glu-Glu-Ala-Ala-Lys1- 2)0.8 0.9 3.6 0.8 2.9 1.9

Lys -Lys -1le -Glu -Glu -Glu -Ala -Ala-Lys (4- 12)3.0 1.0 2.7 2.2

Lys -1le2.0 0.9 3.1 2.0

Tyr -Arg (13-14)0.8 1-0

Asn -Ala -Trp -Val -Lys(l5-19)1.1 1.1 - 0.8 1.0(1.2) (1.3) (0.7) (0.7) (1.0)

Asn -Ala -Trp -Val -Lys-Lys(15-20)

Lys -Cys -Cys -Tyr -Asp-Gly-Ala-His -Arg(20-28)0.6 1.4 0.5 1.0 1.1 1.0 0.9 1.0

Lys -Cys -Cys -Tyr -Asp-Gly-Ala-His -Arg- Asn (As0 Asp, Glu, Thr),Cys-Glu-Glu-Arg (20-37)0.9 3.0 1.1 4.1 1.0 1.1 1.0 2.0 3.0 1.1

Cys -Cys -Tyr -Asp -Gly-Al a-Hi s-Arg-Asn (Asp, Asp, Glu, Thr), Cys -Glu-Glu-Arg(21-37)2.9 0.8 3.8 0.9 0.9 0.9 2.0 3.1 1.0

Asn -Asp -Asp -Glu -Thr-Cys-Glu-Glu-Arg(29-37)2.9 3.1 1.0 0.6 1 o

-Glu -Glu -Glu-Ala-Ah-Lys (5-12)

1.2 1.0 - 0.6 2.0

Ala -Ala -Arg (38-40)2.1 1 o

Ile -Ala -1le -Gly -Pro -Glu -Cys -Ile -Lys (41 -49)

2.8 1.1 1.0 1.0 1.1 0.5 1.o

Ala -Phe -Lys (50-52)1.0 0.9 1.0

Ser -Cys -Cys -Ala -1le -Ala -Ser -Gln-Phe-Arg(53-62)1.8 1.7 2.2 1.1 1.1 1.1 1.0

Ala -Asp -Glu -His -His -Lys (63-68)1.0 1.0 1.0 1.9 1 o

des-Arg-T15

T1S

Asn -Met -Gln -Leu -Gly(69-73)1.1 0.7 1.0 0.9 1.1

Asn -Met -Gln -Leu -Gly-Arg(69-74)1.1 0.7 1.1 1.0 1.0 1.0

16and 28 residues long, respectively, were partially sequenced.

Analyses of chymotryptic peptides allowed the alignment oftryptic peptides T1 to T10 (covering residues 1-40) and T11to T15 (residues 41 -74). In addition, alignment of amino

acids 1- 2, covering tryptic peptides T1- T9, was confirmed

by automatic sequence analysis, with a repetitive yield of 92%.

The chymotryptic fragment C6-7-8 contained the overlap-

ping region of the two halves of the C5a molecule. Its se-

quence, although not completely determined, could be utilized

for the alignment of peptides T8-9, T10, T11 and T12,yielding a result fully supported by both the characterization

of the two halves of the molecule and the homology between

bovine and other C5a sequences [15].

The complete amino acid sequence of bovine C5a is re-

ported in Fig. 8, where all the experimental evidences for theproposed structure are also summarized.

Spectroscopic properties of C5a

The nature and the extent of ordered conformation ofbovine C5a was then investigated by submitting des-Arg-C5a

to circular dicroic spectroscopic analysis. The a-helix content



83

Table 3. Amino a cid composition and sequence of bovine C5a chymotryptic peptidesMolar ratios of each amino acid, calculated from analyses performed on 24-h hydrolysates, are indicated under corresponding symbols,appearing for the first time in the seq uence. The sequence of residues in brackets was n ot determined. Positions refer to the intact polypeptide

Chymotrypticpeptides

C2-3

Amino acid composition and sequence

Leu -Lys - Lys -Lys - Ile - Glu - Glu - Glu- Ala -Al a -Lys -Tyr (2- 3)1. 1 4. 0 0.9 2.7 2.3 0.8

c 3

c 4

c 5

C6

Lys -Lys -Lys -1le -Glu -Glu -Glu -Ala-A la-Lys-T yr (3-13)

3.9 0.9 3.0 2.2 0.8

Arg -Asn -Ala -Trp (14-17)1 .0 1 .1 0 .9 -

Val -Lys -Lys -Cys -Cy s -Tyr (18-23)1. 0 2.0 1.4 0.7

Asp -Gly -Ala -His -Arg -Asn -Asp -Asn (Gly Thr, Cys, Glu), Glu -Arg -M a -Ala (24- 39)3.4 1.0 3. 1 1 . 1 1.9 2.9 0.8

C6-7-8 Asp -Gly - A h -His -Arg -Asn -Asp -Asp-Glu-Thr (Cys, Glu, Glu, Arg,4.0 2.1 4.7 0.9 3.0 3.9 0.9 1. 8

Ala, Ala, A rg, Ile, Aka, Ile, Gly, Pro, Glu,Cys), Ile -Lys - A h -Phe (24- 51 )2.7 1. 2 1 . 3 1 .o

C8

c9

c 1 0

Ala -1le -Gly -Pro -Glu -Cys -1le -Lys-A la-Phe (42-51)2.1 2.0 0.8 1.0 1.2 0. 7 1 o 0.9

Lys -Ser -Cys -Cys -Ala -1le -Ala -Ser -Gln-P he(52-6 1)1.0 1.8 1.3 2.1 0.9 1. 1 0.9

Arg -Ala -Asp -Glu -H is -His -Lys -Am -Me t (62-70)0.9 1.2 1.4 0.9 1.4 1.1 0.8

c11 Gln -Leu -Gly -Arg (71-74)0.9 1 .1 1.0 0.8

of the polypeptide, as derived from this spectrum in the region

190-250 nm (Fig. 9) , was calculated [32] to be about 42%.

DISCUSSION

Starting from large volumes of bovine serum, we have

purified to homogeneity milligram amounts of the comple-

ment fragment C5a and its desArg derivative. Treatment ofserum with EAhx, the inhibitor of serum carboxypeptidase Ngenerating des-Arg-C5a [2], was indeed expected to prevent

the removal of the C-terminal amino acid from C5a. It is

likely, however, that some activation of complement occurred

during the process of coagulation (37 "C, 2 h) and separation

of serum from the coagulum, before addition of eAhx.

The yield of the purification procedure applied appears to

be very high. In fact, the purified polypeptides account for

about 50% of the maximal quantity of C5a which can be

generated in serum from C5 [ 3 ] . Recently, a synthetic geneencoding the human C5a has been cloned and expressed inEscherichia coli [33]. However, the expression level for C5a

was only 0.007°/~of the total bacterial protein, thereby makingpurification of C5a from serum still a viable approach.

From a rabbit antiserum to bovine CSa/des-Arg-CSa we

have isolated the immunoglobulin fraction, which was then

utilized to set up a competitive ELISA for C5a. This sensitive

and specific method of quantifying C5a provides a useful

alternative to the chemotactic assay, which does not dis-

tinguish among the various serum chemotaxins.

Bovine C5a, like human and porcine C5a [14-161,contains 74 residues. Unlike the former, it does not contain acarbohydrate moiety, as indicated by failure to react with the

periodic acid/Schiff stain in polyacrylamide gels (not shown)

and by the identity ofM , values, as determined by SDSjPAGEand amino acid analysis (absence of the carbohydrate moiety

is justified by substitution of Asn at position 64 [14] with

Asp, see below).Human C5a has been reported to be more potent than

its desArg derivative in activating two typical functions ofneutrophils, chemotaxis and secretion of granule content [8].

In contrast, dose/response curves of neutrophils exposed to

bovine C5a and des-Arg-C5a are practically identical. This

may suggest that removal of the C-terminal amino acid fromC5a decreases the potency of its chemotactic and secretagogue

activity only when there are the concurrent modulatory effectsof the carbohydrate moiety of the molecule [34].

Elucidation of the primary structure of bovine C5a was

obtained by sequencing tryptic and chymotryptic peptides, as

well as about half of the intact polypeptide. A large portion

of the molecule was thus sequenced at least twice.

Bovine C5a has a sequence homology of 78% and 70%with porcine and human CSa, respectively (Fig. lo),

consistently with the observation that it cross-reacts with bothantibodies to porcine C5a and C5a receptors on humanneutrophils. In particular, 47 positions in the three molecules

are occupied by the same amino acid residue. Two base

changes per codon explain the variations of residues 17, 53and 66 of bovine C5a with respect to both human and porcine

sequences; of residues 9, 27, 28, 46 and 65, with respect to

human C5a; and of residues 42 and 56 with respect to the

porcine sequence. A distinctive feature of bovine C5a is thepresence of Trp-17, a residue which occurs in a variable por-

tion of the molecule. In fact, all the other anaphylatoxins (C3a,

C4a, C5a) from different animal sources so far characterized



84

10 20NH - k t - I.cu - Lys - Lys - Lys - Ile - Clu - Glu - Gl U - M a - Al a - Lys - Tyr - Arg - Asn - A1a - l r p - Val - I.ys - i.ys -

2

70I lr - Arg - Ala - Asp - Glu - tlis - llis - Lys - Asn - Met - Cl n - Leu - C ly - Arg-COM

- 1 c 10 9c 11-Fig. 8. Summary o proofs o bovine C5aaminoacid sequence. (+) eptides partially sequenced by manual Edman degradation; (+) eptidespartially sequenced by carboxypeptidase Y digestion;(H)eptides completely sequenced by manual Edman degradation; T trypsin,C chymotrypsin; (b b) ntact C5a, automatically sequenced

(90 20 0 210 22 0 230 240 210

w nght (nm,

Fig. 9. Far-ultraviolet circular-dicroic spectrum o bovine des-Arg-C5a (0.07mglml) dissolved in 10 mM sodium phosphate p H 6.95,

0.1 M NaCI at 25°C

[14- 16, 35 -391, contain no tryptophanyl residue. While thesubstitution at position 53, which changes a charged residue(Glu in human and Asp in porcine C5a) into a polar one (Ser),has very likely a minor effect on the general character of themolecule, the replacement of Ser (both in human and porcine

C5a) by His in position 66 increases the already considerablecharge density of the C-terminal part of bovine C5a. Actually,both N- and C-terminal moieties of the bovine polypeptideare more highly charged than the corresponding regions ofC5a from other sources. In fact, a Lys-Lys-Lys-Ile-Glu-Glu-

Glu sequence is found a t the N-terminus from the third to theninth residue, and in the region between Arg-62 and Lys-68there is only one residue which is uncharged at neutral pH.

A comparison of the sequence of bovine C5a with thatof other anaphylatoxins indicated a homology of 42% withbovine C4a [38] and of 32% with human C3a [35] . On thecontrary, a computer search of homologies with other knownprotein sequences provided minor scores.

The position of the potential intrachain disulfide bondsbetween the six Cys residues of bovine C5a has not beendetermined in the present study. However, considering thehigh degree of homology between porcine [15,16] and bovineC5a, it is likely that disulfide bridges are established betweencysteines 21 -47,22-54 and 34-55.

Information on the secondary structure of bovine C5a hasbeen achieved by both circular dicroic spectroscopic analysisand predictions of ordered regions. Roughly half of the

polypeptide appears to assume an a-helical structure. In fact,this spectrum provided a value of 42%, which is in goodagreement with the reported data obtained by the same proce-dure on porcine C3a and C5a, and on human C3a [40,41].

Furthermore, by applying the rules of Chou and Fasman[42], we calculated that a-helical regions are present in 57%of the whole molecule. These regions are located from Lys-5to Ala-11, from Asn-29 to Ile-41 and from Pro-45 to His-66.No B-regions were predicted.

The percentage of the a-helical structure in bovine C5a isvirtually identical to that of another anaphylatoxin, humanC3a, whose conformation has been determined by X-raycrystallography [17]. The distribution of the a-helical regionsappears to be different in the N-terminal moiety of the two



8 5

10 20

I#-Lw -K-I-E-E-E-A-A-K-Yf -W -ov ine C5 a

P o r c i n e C5a

Human C5a

Bov ine C5 a

P o r c i n e C 5 a

Human C5a

B o v i n e C5 a

P o r c i n e C5a

Human C5a

L - Q - K - K - I - E - E

M - L - Q - K - K - I - E - E - E - A - A - K - Y - K

30 40

C - C - Y - 0 - G - A H R - N - D - D - E - T - C - E - E - R - A - A - R

7n-Bov ine C5a F V 1 D W H - K - N - M - Q - L - G - R l

R - A E E - S - H - K - N W Q - L - G - R

Lvu- A N - I4 - H - K

P o r c i n e C5 a

Human C5a

Fig. 10. Comparison of primary structures ojb ov ine , porcine and human C5a. Homologous sequences are boxed with solid lines. Data forporcine and hum an C5 a are taken from [14- 161

10 20 3UIbO! 11 lC i ;.I \I I, E K li I li li I A A K Y K N A W V K K C C Y I) I; A II R N I1 II I: I I: 1; H -

111 5U hU 70

1 l 1 u 11 . 1 1 i 1 i . i I K I I S , G I: A C K K V I’ I. I1 I: C N Y I I li I. K K (1 I1 A H A S I1 I, I; 1. A H

Fig. 1 1 . Comparison between primary structures o bovine C5a andhuman C3a.Sequences have been aligned to maximum homology as proposedby Smith et al. [38]. Coils represent a-helical regions determined from secondary structure predictions (C5a) and from crystallographic studies

(C34

1

10 30 50 70

residue nurnbei

Fig. 12 . Hydropathy profile o bovine C5a (-) and human C3a(----) calculated using the parameters of Kyt e and D oolitt le [44 ]

with span set at nine residues

polypeptides (Fig. 11). In particular, in bovine C5a two re-

gions are predicted between residues 5- 1, and 29 and 41,whereas in human C3a an a-helical portion extends fromTyr-15 to Met-27. However, the two polypeptides share a

common secondary structure in the C-terminal half of themolecule. In fact, both Pro-45 -His-66 of bovine C5a and the

corresponding Gly-46- er-71 of human C3a are a-helicalregions. It is interesting that this ordered structure isconsidered to be essential for the biological activity of

anaphylatoxins [43].Another common feature between the two anaphylatoxins

is brought to light by a hydropathy analysis, according toKyte and Doolittle [44]. As shown in Fig. 12, both moleculesdisplay a highly hydrophylic character with only one regionof predominant hydrophobicity. It is worthwhile pointing outthat crystallographic studies on human C3a showed that thisregion (residues 45 - 0) is indeed the less accessible part ofthe molecule to solvent.

All the above considerations, concerning disulfide bond

arrangements, a-helical content, and extent and portions of



hydropathic regions, suggest that the C3a conformation, asdeduced from X-ray crystallography,could provide a suitablemodel of conform ation also for the bovine C5a fragment. Asimilar con clus ion has recently been reached by Greer [45], yusing computerized, comparative modeling method s.

We thank G . Benussi and B. Gazzin for help with the manuscriptand the photography. This work was supported by grants from theItalian M inistry of Education and f rom the Italian National R esearchCouncil (Progetto Finalizzato ‘Controllo delle malattie da infezione’,

grant 83.00688.52).

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c5a fragment of bovine complement

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