characterization of tryptic fragments of human complement factor c3

Molecular Immunology, Vol. 22, No. 8, pp. 833-841, 1985 Printed in Great Britain

0161-5890/85 $3.00 + 0.00 0 1985 Pergamon Press Ltd

CHARACTERIZATION OF TRYPTIC FRAGMENTS OF HUMAN COMPLEMENT FACTOR C3

G&TA EGGERTSEN,* ULF HELLMAN,* AKE LUNDWALL,*? JBRGEN FOLKERSEN~ and JOHN W&UIST*

*Department of Medical and Physiological Chemistry, Box 575, BMC, 751 23 Uppsala, Sweden; and fInstitute of Medical Microbiology, Odense University, DK-5000 Odense C, Denmark

(First received 20 August 1984; accepted in revised form 18 December 1984)

Abstract-C3c and C3d fragments were prepared in pure form from trypsin-digested human C3, and the individual chains of tryptic C3c were isolated by gel filtration on Sepharose 4B in 6 M guanidinium hydrochloride. No low mol. wt (A4,) fragments were identified. The polypeptide chains were characterized with regard to M,, amino acid composition and N-terminal amino acid sequence. Tryptic C3c consisted of one fragment from the /?-chain (M, 64,000) and two from the a’-chain (M, 40,000 and 23,000). The B-chain fragment was derived from the C-terminal part of the chain, and the 23,000-M, component constituted the amino terminal end of the a-chain. The 40,000-M, fragment emanated from the C-terminal end of the a-chain. Tryptic C3d displayed microheterogeneity on polyacrylamide gel electrophoresis in sodium dodecyl sulfate, but possessed a homogeneous N-terminal, identical to that described by Tack et al. (1980) (Proc. narn. Acad. Sci. U.S.A. 77, 57645768). By utilization of antisera against subunits of C3 and C3c in immunoblotting a degradation scheme for C3 by trypsin was proposed and the positions of the fragments in the intact molecule indicated.

INTRODUCTION

Complement factor C3 is one of the key components in the complement cascade, taking part in both the classical and the alternative pathway. C3 is composed of two disulfide-linked polypeptide chains with mol.

wts (M,) of 125,000 (a-chain) and 75,000 (b-chain) (Bokisch et al., 1975; Nilsson ez al., 1975). On activation* an N-terminal peptide consisting of 77 amino acids (C3a) is cleaved off from the a-chain. In its activated state (C3b) the molecule can establish a covalent bond with other molecules via a glutamic acid residue in the a’-chain, a reaction which is of crucial importance for the activation of the terminal components of the complement system [for a review see Lachmann and Peters (1982)]. As a result of conformational changes in the molecule C3b will lose this binding capacity within 60 psec (Bokisch et al.,

1975; Sim et al., 1981) but it will still be able to bind to cellular receptors for C3 and to factor B. C3b is degraded to smaller fragments by factors I and H and yet undefined proteolytic enzymes occurring in the

tPresent address: Department of Clinical Chemistry, Malmij General Hospital, S-214 01 Malmii, Sweden.

5Abbreviations: SDS-PAGE, polyacrylamide gel electrophoresis in sodium dodecyl sulfate; RCM, reduced and carboxymethylated; HPLC, high-pressure liquid chromatography; DITC, p-phenyiene-diisothjocyanate; PMSF, phenylmethvl sulfonvl fluoride; PBS. phosohate- buffered saline; C&z, a-chain of human -C3;- C3/?, p-chain of human C3; HRP, horseradish peroxidase. Enzymes: TPCK-trypsin from bovine pancreas (EC 3.4.21.4). Peroxidase from horseradish (EC 1.11.1.7).

blood stream. Characterisation of the degradation process and of the resulting fragments have recently been performed (Lachmann et al., 1982; Davis et al.,

1984). Limited proteolysis of C3 has been performed with

trypsin (Bokisch et al., 1975; Molenaar et al., 1975; Fontaine and Rivat, 1979) or elastase (Taylor et al.,

1977) with the aim of investigating its structure. However, no thorough characterization of the tryptic fragments has been undertaken and conflicting re- ports have appeared in the literature concerning the structure of these fragments, particularly C3c. The purpose of the present study was to carefully in- vestigate trypsin digestion of human C3 and to characterize the available fragments.

MATERIALS AND METHODS

Sephadex G-25, Sepharose 4B, DEAE-Sepharose and blue dextran were purchased from Pharmacia Fine Chemicals, Uppsala, Sweden. Guanidine hydrochloride (GuHCI) was obtained from Fluka AG, Switzerland, and nitrocellulose blotting paper (type GSWP 00010) was a product from Millipore Cor- poration, Bedford, U.S.A. [‘4C]iodoacetamide was obtained from the Radiochemical Centre, Am- ersham, U.K., and Aquasol” and Enhancer’” from New England Nuclear, Boston, MA. TPCK-trypsin (209 Ujmg) from bovine pancreas was purchased from Worthington Biochemical Corporation, Free-

hold, NJ Controlled pore glass (CPG 10) was from Serva Feinbiochemica. PMSF,§ dithiothreitol

833

834 GGSTA EGGERTSEN et al

and carbazole were from Sigma Chemical Co. St. Louis, MO. DITC was obtained from Rathburn Chemicals, Walkerburn, U.K.

Humun complement factor C3

C3 was prepared from fresh-frozen human plasma according to the method of Lundwall et al. (1984).

Antisera

Antisera against human C3, try-C3c, tryC3d and the C3p were raised in rabbits essentially as described by Eggertsen et a/. (1981). Rabbit antiserum against the C3c( was kindly provided by Dr Ulf Nilsson, The Blood Center, University Hospital, Uppsala, Sweden. The serum also contained a small amount of antibodies to the p-chain of C3 (Dr Ulf Nilsson, personal communication). Rabbit antisera against the individual polypeptide chains of human try-C3c was prepared by injecting 200-300 pg of antigen (see below) emulsified in Freund’s complete adjuvant into the inguinal lymph nodes, followed 34 weeks later by a booster injection of antigen in Freund’s incomplete adjuvant administered subcutanously. Rat monoclonal antibodies against the human C3g determinant (clone 9) (Lachmann et al., 1982) were kindly provided by Dr Lachmann of the MRC Immunological Unit, Cambridge, U.K. Anti-rabbit and anti-mouse immunoglobulin conjugated with HRP was obtained from Dakopatts a/s, Glostrup, Denmark. Rabbit antiserum against human C3a was obtained from

Behringwerke AG, Marburg-Lahn, F.R.G.

SDS-PAGE

This was performed in gradient gels as described by Blobel and Dobberstein (1975). After mixing with sample buffer the specimens were incubated at

37°C for 30min. Reduction of specimens were ac- complished by the presence of 0.025 M dithiothreitol in the sample buffer. The gels were stained for protein

with Coomassie brilliant blue. Gels containing “C-labelled proteins were immersed in Enhancer”, after which they were dried between dialysis mem-

branes and exposed to Fuji RX X-ray film at - 70°C. For determination of M,, reduced proteins of known M, were used as standards.

Immunological techniques

Immunodiffusion was carried out on microscopic slides in 1 “A agarose (Indubiose, Pharmindustrie, Clichy, France) in 10 mM Tris-acetate buffer containing 1 mM EDTA, pH 8.6. Blotting of separated proteins from SDS-PAGE was carried out according to Towbin et al. (1979), except that a voltage gradient of 18 V/cm was used. Quenching of the nitrocellulose sheets was performed with 10% (v/v) pig serum in 0.01 M TrissHCl with 0.14M NaCl, pH 7.2. For rinsing and dilution of reagents the same buffer was utilized, in addition containing 0.1% Tween 20, 20 mM EDTA and 1 mM s-aminocaproic acid. Incu- bations was carried out with 0.3 ml of liquid/cm2 of

nitrocellulose sheet for 16 hr at 4°C with gentle rocking. The nitrocellulose sheets were first treated with the appropriate rabbit antisera (see above), and then with HRP-conjugated anti-rabbit immunoglobulin (diluted l/1000). When rat monoclonal antibodies against the C3g determinant were used as a first-layer reagent, HRP-conjugated anti-mouse immunoglobulin was utilized. Staining was performed with carbazol solubilized in dimethylsulfoxide, prepared as follows. To 100 ml of a 0.1 M sodium acetate buffer, pH 5, 1 ml of a 4% (w/v) stock solution of carbazol in dimethylsulfoxide was added dropwise during continuous stirring. Finally 28 ~1 of H,O, [35% (v/v) in H,O] was added.

Radioactive measurements

Screening for radioactivity in chromatograms was done in a Packard Tricarb 300C liquid scintillation counter. Samples were prepared from 255100~1 of column effluent and 3 ml of Aquasol’“‘. For analysing samples in 6 M GuHCl 5 ml of Aquasol” were used.

Carbohydrate determination

This was performed by gas chromatography according to the method of Sawardeker et al. (1965) with the modification that the hydrolysis was done with 0.5 M trifluoroacetic acid for 18 hr at 100°C. The identity of the peaks was confirmed by mass spectrometry (Jansson et al., 1976). The method allowed detection of neutral sugars up to hexoses including galactosamine and glucosamine, but not sialic acid. To remove contaminating carbohydrate before analysis, the specimens were chromatographed on hydroxylapatite in 5mM potassium phosphate, pH 6.9, and eluted with 0.25 M potassium phosphate.

Amino acid composition and sequence unalysis

Amino acid analysis was performed on a Beckman 121 M amino acid analyser essentially as described previously (Lundwall et al., 1981). Automatic Edman degradation was carried out in an LKB 4020 solid- phase peptide sequencer. Peptides (30-100 nmoles) were immobilized on aminopropyl glass, prepared as

described by Robinson et al. (1971) by the DITC method (Laursen et al., 1972) and the coupling yield was determined by amino acid analysis. The PTH amino acids were identified by HPLC on a Nucleosil ODS column (4 x 300 mm) in 6 mM sodium acetate, pH 4.9, using a gradient of acetonitrile.

Miscellaneous

Reduction and carboxymethylation of proteins were performed essentially as previously described (Eggertsen et al., 1981). Apparent M, were calculated after gel filtration of polypeptide chains in 6 M GuHCl (Porath, 1963). HPLC of RCM try-C3c was carried out under various conditions on a TSK 3000 column, measuring 8 x 600 mm, at a flow rate of 0.5ml/min, and eluting proteins were monitored at 278 nm. Extinction coefficients were determined by

Tryptic fragments of human C3 835

measuring the absorbance at 280 nm of sample sol- utions, aliquots of which were subjected to amino acid analysis to determine the total protein concn.

Isolation of tryptic C3 fragments

Up to 400 mg of human C3 was methylaminated and the exposed SH group was subsequently carboxymethylated with [‘4C]iodoacetamide essentially as described by Lundwall et al. (1982). The material was then dialysed against 0.1 h4 KH, PO,, pH 7.4. The protein concn was adjusted to 2 mg/ml, and digestion was performed with TPCK-trypsin (4 U of trypsin/mg C3) for 3 hr at +37”C. The digestion was stopped by the addition of PMSF (25 mg/ml in isopropanol), after which the buffer was changed to 20 mM Tris-HCl containing 25 mM

NH,Cl, pH 8.0, on a 5 x 15 cm column of Sephadex G-25. The protein peak was pooled and applied on a 1.6 x 25 cm column of DEAE-Sepharose equili- brated in the same buffer. Following a lOO-ml wash with buffer the column was eluted with an SOO-ml linear salt gradient with a final NH,Cl concn of 0.2 M.

RESULTS

Analysis of the tryptic digest

After digestion for 3 hr only weak remnants of the a- and b-chains were left (Fig. 1, tracks Al-8). Four main polypeptide fragments were observed, desig-

nated FI (M, 64,000) FII (Mr 40,000), FIII (M, 34,000) and FIV (Mr 23,000). The latter, and some- times FII, appeared as a double band. During the digestion two other fragments were observed, FV (M, 78,000) and FVI (M, 28,000), the latter appearing as

two closely spaced bands. These two fragments were

most prominent in the early phases of the digestion, and vanished slowly during the observation time. A few weaker bands of different M, were also observed. The appearance of FI was well correlated with the fading of the C3b band, and this fragment probably emanated from the B-chain (designated /?/-chain).

This was confirmed by immunoblotting, where it was found that besides interacting with C3B, anti-C3p only reacted with this fragment (Fig. 1, tracks Cl-3).

Anti-C3a was found to interact with C3a, C3cr’, FII, FIV, FV and FVI, suggesting that all these fragments were derived from the a-chain (Fig. 1, tracks Bl-3). Only weak reactivity was displayed with FIII. Anti-

FIII interacted in immunoblotting with FIII, FV and C3a (Fig. 1, tracks Dl-3). Autoradiography showed that the radioactive label incorporated in the cysteine residue in the thiolester of the cc-chain was present in the intact a-chain and in fragments FIII and FV (not shown). Since FIII was found to contain the labile binding site and its M, was in accordance with that reported for tryptic C3d (Thomas et al., 1982) the fragment was regarded as try-C3d (Fig. 1, tracks A2-8). Monoclonal anti-C3g interacted with C3cr, C3c(’ and FV but not with any other fragment during the course of digestion (Fig. 1, tracks Fl-3). Oc- casionally a prominent C3c(’ band was detected in

methylaminated C3 (0 min) on the blots, contrary to the Coomassie-stained gels, where only intact cc-chain

was present. The significance of this band remains

obscure. Immunoblotting of the trypsinized C3 with anti-

C3a did not result in staining of any of the bands in the tryptic digest, and the antiserum interacted only with C3a (Fig. 1, tracks El-3). In immunodiffusion

A B C D E F 12345678 123 12 3 123 123 123

FI

FJIItTry

Fig. 1. Tracks Al-8: SDS-PAGE of tryptic digestion of human methylaminated C3 at different time intervals. Gradient gel 10-15°/0 polyacrylamide. The picture represents reduced material stained with Coomassie brilliant blue. Tracks B-F: immunoblotting of reduced specimens. Gradient gels 4-20x polyacrylamide. Trypsin digestion of human C3 for 0,60 and 180 min respectively against anti-C3a (Bl-3) anti-C3p (Cl-3), anti-try-C3d (Dl-3) anti-C3a (El-3) and rat monoclonal antibody against the C3g

determinant (Fl-3). The antisera were applied at dilutions varying between l/l000 and l/50,000.

-FP

836 G&TA EGGERTSEN et al.

6000

0 250 500 750 ml

“t I

“0 1

0 250 500 750 ml

Fig. 2. (A) Chromatography of trypsin-digested methylaminated C3 on DEAE-Sepharose. The SH group in the thiolester was labelled with [3 Hliodoacetamide. Approximately 150 mg of digested C3 were applied on the column. G denotes the start of the gradient. Absorbance at 280nm (0-O); radioactivity (0-O); specific conductivity ( x - x ). Insert: SDS-PAGE of specimens from the chromatography stained with Coomassie brilliant blue. Tracks 1-3 and 4: reduced aliquots; 2a, 3a and 4a: nonreduced aliquots. The numbers of l-4 refer to the corresponding numbers of the chromatogram. (B) Chromatography of RCM try-C3c on Sepharose 4B in 6 M GuHCl. V, denotes void vol and V, total vol. Approximately 70 mg of try-C3c was applied on the column. Absorbance at 280 nm (0-O); radioactivity (e-0).

anti-C3a precipitated with intact C3 and methylaminated C3, but not after the digestion had begun (data not given).

The tryptic digest was chromatographed on DEAE-Sepharose (Fig. 2A). No. u.v.-positive material was detected (O.D. 280nm) in the break- through fraction, whereas two peaks emerged with the gradient elution. Most of the radioactive label was confined to the first peak (peak l), and only a minor part was present in the second one (peak 2). Peak 1 displayed prominent trailing. On SDS-PAGE this peak was found to contain the try-C3d (Fig. 2A, tracks 1-3, 2a and 3a) Three tightly spaced bands with M, ranging between 33,000 and 35,000 were

seen. The band in the intermediate position was present all through the peak, while the most anodally migrating band was found in the early-eluting part and the most cathodally one in the part that eluted later. Radioactive label was found in all bands, and the migration pattern was not altered after reduction (Fig. 2A, tracks 2a and 3a). In addition to small amounts of intact C3 (or C3b), peak 2 contained a component which migrated slightly further than intact C3, and which dissociated in three main bands after reduction, corresponding to FI, FII and FIV (Fig. 2A, tracks 4 and 4a). This material is referred to as try-C3c. Small amounts of intact C3a’ and C3/I, as well as of FV and FVI, were also seen. Radioactive


label was detected in C3cr’ and FV, which probably accounted for the radioactivity in peak 2. Generally 27&350 mg of try-C3c and 3&40 mg of try-C3d were obtained from digestion of 35&400mg of methylaminated C3.

Characterization of try-C3c

To characterize the molecule further, it was necess- ary to isolate the polypeptide chains. Different con-

ditions for separation of the chains were evaluated by running small specimens of RCM try-C3c on TSK- 3000 by HPLC. The individual chains were sufficiently well separated in 6 A4 GuHCI, whereas chromatography in other media (50% propionic acid; 0.2 M formic acid with 6 M urea; 0.2% SDS with 4 M urea; 3 M GuHCl) was inadequate for separating the subunits of tryC3c. Seventy-five milligrams of RCM try-C3c were then applied on a 2.5 x 137 cm column of Sepharose 4B in 6 M GuHCl, which was run with upward flow at 7ml/hr. Blue dextran was used to determine the void vol. The chromatogram is shown in Fig. 2B. Radioactive label was found in the earliest eluting part of the first peak. SDS-PAGE revealed that this peak contained FI, the second peak fragment FII and the third peak fragment FIV. FI and FII were fairly well separated from each other, while there was a slight overlap between FII and FIV. Minor amounts of FV and C3c(’ were found in the earliest eluting part of peak 1. The M, of the three components were calculated to be 62,000, 34,000 and 23,000 for FI, FII and FIV respectively. The amino acid compositions are given in Table 1. The extinction coefficients (Ey&,) in 1 M acetic acid were calculated to be 8.2 (FI), 12.0 (FII) and 10.2 (FIV). Automatic Edman degradation revealed that FIV made up the N-terminal part of the a/-chain of C3, since the sequence was in good accordance with the one reported by Tack et al. (1977) except in position 12, where we obtained asparagine instead of aspartic acid, an observation which has also been made by

Residue no 1 5

Table 1. Amino acid composition of tryptic fragments of human C3

Residue FIII (try-C3d) FI (C38’) FII FIV

Trp” 3.4 2.1 2.6 2.9 LYS 21.2 35.2 28.9 14.7 His 4.6 7.1 5.8 4.1 Arg 12.7 26.3 15.8 10.7 Cys” 3.4 3.9 15.1 2.8 ASX 28.8 47.4 46.0 19.9 Thf 16.0 40.5 18.6 10.2 Ser’ 15.1 44.8 22.5 16.0 Glx 39.4 72.1 52.1 26.7 Pro 12.6 35.2 13.9 12.6 GlY 20.5 43.1 18.9 7.5 Ala 27.4 27.5 16.4 8.3 Vald 21.4 58.1 19.2 20.9 Met 6.9 9.8 9.1 3.4 Il.& 12.0 28.2 17.9 13.8 Leu 38.0 53.2 29.8 19.1 TY~ 12.4 23.5 17.2 6.3 Phe 13.2 22.4 13.7 8.2 No. of residues: 309 581 364 208

“Determined after hydrolysis in 3 M toluene-p-sulfonic acid. ‘Determined as carboxymethylcysteine. ‘Determined after extrapolation to zero times of values from 24-

and 72-hr hydrolyses. ‘Determined from 72-hr hydrolysis.

Table 2. Carbohydrate [% (w/w)] analysis of human C3 and its trwtic fragments

Carbohydrate c3 Try-Cb

Mannose 1.00 0.86 Glucose 0.21 0.13

Try-C3d

0.90 Galactose Fucose N-acetyl-glucosamine N-acetvl-ealactosamine

0.03

0.18 0.18

Davis and Harrison (1982). The N-terminal sequences of FIV and the other two fragments are illustrated in Fig. 3. None of the latter sequences corresponded to any sequence data published concerning the primary structure of human C3. Material from the top fractions of each peak were used for preparation of antiserum to FI, FII and FIV. Carbo- hydrate analyses of try-C3c and, for comparison, of C3 are shown in Table 2.

10 15

X-Arg-AsN-Tyr-Phe-Val-Thr- X -Glu-Ala-Thr 1. FI (C3$

2. FII X-Pro-Gln-Asp-Ala-Lys-AsN-Thr-Met-Ile-Le~~-Glu-Ile-Val-~.ys-Arq-Le~~-Tyr

l

3. FIII (try-C3d) X-Leu-Ile-Val-Thr-Pro-Ser-Gly-Cys-Gly-G1~~-Glu-AsN-Met-Ile-Gly-Met

4. FIV

Fig. 3. N-terminal amino acid sequence of different fragments of trypsin-digested C3: (1) FI (C3b’), (2) FII, (3) FIII (try-C3d) (*denotes the glutamic acid residues taking part in the thiolester), and, (4) FIV. Since the first residues of the peptides will not be determined when DITC coupling is utilized in solid-phase

sequencing, they have been denoted with an X.

838 G&TA EGGERTSEN et al.

Characterization of try-C3d

The amino acid composition is given in Table 1.

Specimens analysed from the early- and late-eluting parts of peak 1 (Fig. 2A) did not differ significantly in their composition. Carboxymethyl-Cys in nonreduced carboxymethylated C3d equalled 0.7-0.9 residues/mole, and in completely RCM C3d three residues of CM-Cys were found, suggesting that the number of residues is three. Carbohydrate analysis was negative except for a small quantity of glucose (0.9x), which probably represented contamination from the Sepharose gel. E& in PBS was calculated to

be 17.0/male. Automatic N-terminal sequence analysis revealed a homogeneous N-terminal sequence (Fig. 3). This sequence corresponded perfectly with the data reported by Tack et al. (1980) from a tryptic fragment of C3d.

Immunologic analysis of the tryptic C3 fragments

Rabbit antisera raised against FI and FII produced a single precipitation line on immunodiffusion against human C3, whereas no reaction occurred with antiserum against FIV (not shown). On immunoblotting, anti-F1 was found to interact with C3p (Fig. 4, track 9) and FI (data not given). Anti-F11 reacted with FII and C3c( and C3a’ (Fig. 4, tracks l-3), while anti-FIV was found to interact with FIV,

FV and FVI, C3c( and C3c(’ (Fig. 4, tracks 4-6, 11

and 16). When intact C3 was applied on the gel, two weak bands were observed in the same location as C3b with anti-F11 (Fig. 4, track 10). However, with rnethylaminated C3 only one of the bands was found

(Fig. 4, track 15), suggesting that the other component was representing the larger autolytic fragment of C3c(, probably released by interaction of native C3 with SDS (Howard, 1980; Sim and Sim, 1981). This fragment, with M, of 74,000 and derived from the C-terminal end of the cc-chain, migrates at almost the same velocity as C3/3. The origin of the other weak component remains unexplained. The second autolytic fragment (M, 46,000) was only visualized after boiling native C3 in SDS solution and interacted with anti-FIV, anti-C3 and anti-C3a (data not shown). Antiserum directed against C3 and try-C3c interacted with all components in try-C3c, although anti-C3 showed very weak reactivity towards FIV (Fig. 4, tracks 7 and 8). Anti-C3c reacted with all components in try-C3c and the intact chains of C3 (Fig. 4, tracks 7 and 12) but not with try-C3d, neither on immunoblotting or immunodiffusion.

DISCUSSION

This report describes the digestion of human methylaminated C3 with trypsin and characterization

of the obtained fragments C3c and C3d. No other digestion products were isolated, although low-

C3a C3a

123 45 6

-FP

78

._ -c3p

“WI -FI(C3p’)

FLl

-FYI -FIY

- -FII

-FIX

910111213 141516

I

Fig. 4. Immunoblotting of reduced specimens. Gradient gels 420% polyacrylamide. Tracks 1-6: trypsin digestion of human methylaminated C3 for 0 (1 and 4), 60 (2 and 5) and 180 min (3 and 6) against anti-F11 (l-3) and anti-FIV (46). (7) Try-C3c/antLtry C3c. (8) Try-C3c/anti-C3. (9) Native C3/anti-FI. (10) Native C3/anti-FII. (11) Native C3/anti-FIV. (12) Native C3/anti-C3c. (13) Native C3/anti-C3. (14) Methyl- aminated C3/anti-FI. (15) Methylaminated C3/anti-FII. (16) Methylaminated C3/anti-FIV. The antisera

were applied at dilutions varying between l/l000 and l/50,000.

M, components (< 10,000) were observed on 0 4

SDS-PAGE. No definite fragments, except C3c and 127 KW)

C3d, were found even after direct gel filtration of the tryptic digest in 6 M GuHCl on Sepharose 4B (data not given). Antiserum against human C3a did not interact with any of the components in the tryptic

SK 118 Kb’) digest. (C3a)

Our tryptic C3c contained three SS-linked polypeptide chains, the largest emanating from the /?-chain and the others from the a-chain. This is the same principal structure as has been reported for C3c obtained from digestion of C3b with elastase (Taylor et al., 1977) kallikrein (Meuth et al., 1983) trypsin (Fontaine and Rivat, 1979) or factors H and I plus trypsin or plasmin (Nagasawa and Stroud, 1977; Davis et al., 1984) with the exception that elastase and kallikrein do not seem to cleave C3/?. The smallest fragment of our try-C3c (FIV) was found to have the same amino terminal sequence as C3a’. The cleavage of the cr’-chain in C3b by factor I was shown to generate a 68,000-M, fragment which also con-

I ,

FV 70 K F 11 40K

Trp Trp

I I 1 , TOP

F VI 28 K -10 i.34 Kbry-C3d) -6 K

I, TOP

+ -+-, tained the N-terminal part of the cc/-chain (Davis and F IV 23K -5 K

Harrison, 1982). This part could be further cleaved by trypsin or plasmin into two pieces with M, of

Fig. 5. Proposed scheme of the trypsin cleavage of the cc-chain of human C3. Fragments which were not identified

27,000 and 43,000, of which the former represented are represented by dashed lines.

the amino terminal end and the latter the C-terminal part of the fragment (Lachmann et al., 1982; Davis et a/., 1984). gestion, namely ctl (M, 36,000) and a3 (25,000)

Antibodies directed against FIV also reacted on (Taylor et al., 1977). Furthermore, the amino acid immunoblotting with fragments FVI and FV, of compositions of FII and a 1, and FIV and a 3 are very which the latter contained the radioactive label incor- similar. Most probably these fragments are equiv- porated in the cysteine residue taking part in thiol- alent to each other, even though their N-terminal ester. These fragments would then comprise the amino acids are different. N-terminal part of C3r’, and FV also the C3d It was found that the M, of FI was 11,000 less than fragment. The sum of the M, of FVI and C3d would that of the intact p-chain. The iv-terminal sequence equal 62,000, thus creating a difference of 16,000 did not correspond either to the amino terminal compared to the M, of FV (78,000). Part of this sequence reported for C3p by Tack et al. (1977) or to discrepancy could correspond to the C3g fragment the sequences of cyanogen bromide fragments of C3b (M, approx. 10,000) defined by Lachmann et al. which cover 61 of the amino terminal residues of the (1982) located immediately N-terminal to C3d in the intact p-chain (Lundwall et al., 1984). Plasmin has cc-chain. We could not identify this polypeptide, and earlier been reported to release a fragment with an M, it might have been degraded to smaller peptides of 17,000 from C3p (Nagasawa and Stroud, 1977) during the digestion. The existence of such a fragment but at present we cannot state whether trypsin cleaves was recently verified during the investigations of the only in the amino terminal end or if it also produces effect of kallikrein on human C3 (Meuth et al., 1982). a split at the C-terminus. An interesting observation

According to our findings trypsin initially cleaves is that FI still contains the same number of cysteine the a’-chain into two pieces: an N-terminal part residues as the non-degraded chain. with M, of 78,000 (FV) (containing the C3d part) and Try-C3d has the same M, as the corresponding

a C-terminal part with M, of 40,000 (FII). The molecule obtained by elastase (Thomas et al., 1982) N-terminal sequences of FII and the 43,000-M, frag- but the latter has an additional 14 amino acids in the ment of C3c obtained on digestion of human C3d amino terminal end. The analogous physiological

with factors I and H do not agree (Davis and product [C3dg (M, 40,000)] contains the g- Harrison, 1982). Since FII is smaller in size, the polypeptide in its amino terminal end (Davis et al.,

trypsin split is probably located further to the C- 1984). One strange feature of our try-C3d is the

terminal end. Accordingly, FV would then be made charge heterogeneity demonstrated by its elution

up of FVI, C3g, try-C3d and a further 5&60 amino from DEAE-Sepharose, and the microheterogeneity

acids in the C-terminal end. The trypsin splitting of on SDS-PAGE. Like other authors we found that

C3a is schematically outlined in Fig. 5. try-C3d does not contain carbohydrate (Taylor et al.,

FII and FIV in try-C3c correspond in size to the 1977; Meuth et al., 1983) and the N-terminal se-

cc-chain fragments in C3c obtained by elastase di- quence was found to be homogeneous, suggesting


840 COSTA EGGERTSEN et al.

that the explanation for the above phenomenon should be sought for in other parts of the primary structure than the amino terminal end. The amino acid analysis did not reveal any significant differences between material eluting at different positions from the ion exchanger, but minor differences might have escaped detection. Try-C3d is similar to tryptic C4d in several respects, and charge heterogeneity in tryptic C4d has been shown to be due to differences in the

primary structure between C4 molecules ori~nating from two different gene loci (Hellman et ui., 1984; Belt et al., 1984). Human C3 seems to originate from a single locus on chromosome 19 (Whitehead et al.,

1982), but several allotypes have been recorded (Alper and Rosen, 1976). At present nothing is known about the structural basis of the genetic variation of C3, but it would not be surprising to find that at least part of the variation is located in the C3d part of the molecule.

Arknowledgements-The excellent technical assistance of Mrs Monica Ferm is greatly acknowledged. We are in great debt to Dr Per-Erik Jansson, Department of Organic Chem- istry, Arrhenius Laboratory, University of Stockholm, for performing the carbohydrate analysis. We are also in great debt to Dr Ulf Nilsson, Academic Hospital, Uppsala, for help in preparing the antisera against the chains of try-C3c, and to Dr Peter J. Lachmann at the Medical Research Council Centre, Cambridge, U.K., for providing mono- ctonal anti-C3g immunoglobulin. This project was sup- ported by the Swedish Medical Research Council (project No. 13X-2518) and by a grant from Helge Ax:son Johnson’s Stiftelse, Stockholm.

REFERENCES

Alper C. A. and Rosen F. S. (1976) Genetics of the complement system. Ado. Hum. Genet. 7, 141-188.

Belt K. T., Carroll M. C. and Porter R. R. (1984) The structural basis of the multiple forms of human complement component C4. Cell36, 907-914.

Blobel G. and Dobberstein B. (1975) Transfer of nroteins across membranes I. Presence bf proteolytically processed and improcessed nascent immunoglobulin light chains on membrane bound ribosomes of murine myeloma. 3. Cell Biol. 67, 83558.51.

Bokisch V. A., Dierich M. P. and Miiller-Eberhard H. J. (I 975) Third component of complement (C3): structural properties in relation to functions. Proc. natn. Acad. Sci. CI.S.A. 72, 1989-1993.

Davis A. E. and Harrison R. A. (1982) Structural characterization of factor I mediated cleavage of the third component of complement. Biochemistry 21, 57455749.

Davis A. E., Harrison R. A. and Lachmann P. J. (1984) Physiologic inactivation of fluid phase C3b: isolation and structural analysis of C3c, C3dg (2D), and C3g. J. Immun. 132, 1960-1966.

Eggertsen G. E., Lind P. and Sjoquist J. (1981) Molecular characterization of the complement activating protein in the venom of the Indian cobra (I?& n. siumensis). Molec.

. ” Immun. 18, 125-133. Engertsen G., Lundwall A.. Hellman U. and Sidauist J.

-0983) Antigenic relationships between human and cobra complement factors C3 and cobra venom factor (CVF) from the Indian cobra (Naju nqjcl). J: Immun. 131, 1920-1923.

Fontaine M. and Rivat C. (I 979) A study of the breakdown

of the third component of human complement (C3). Annls Immun. (Inst. P&teur) 13OC, 349-366.

Harrison R. A. and Lachmann P. J. (1980) The ohvsio- logical breakdown of the third component of human complement. ,%4olec. ~rnrn~. 17, 9-20.

Heltman U., Eggertsen G., Lundwall A., Engstriim A. and Sjiiquist J. (1984) Primary sequence differences between Chido and Rodgers variants of tryptic C4d of the human complement sysiem. FEBS L.ett. -170, 254-258.

Howard J. B. (1980) Methylamine reaction and denaturation-dependent fragmentation of complement component C3. J. biol. Chem. 255, 7082-7084.

Jansson P. E., Kenne L., Liedgren H., Lindberg B. and Liinngren J. (1976) A practical guide to the methylation analysis of carbohydrates Chem. Commun., Univ. Stockh. 8, l-75.

Lachmann P. J., Pangburn M. K. and Oldroyd R. G. (1982) Breakdown of C3 after complement activation. J. exp. Med. 156, 205-216.

Lachmann P. J. and Peters D. K. (1982) Complement. In Clinicaf Aspects of Zmmunoiogy (Edited by Lachmann P. J. and Peters D. K.), Vol. 1, 4th edition, pp. 18-49. Blackwell Scientific Publications, London.

Laursen R. A., Horn M. J. and Bonner A. G. (1972) Solid-phase Edman degradation. The use of p-phenyl diisothiocyanate to attach lysine- and arginine-containing peptides to insoluble resins. FEBS L&t. 21, 67-70.

Lundwall A., Hellman U., Eggertsen G. and Sjiiquist J. (1982) Isolation of tryptic fragments of human C4 ex- pressing Chido and Rodgers antigens. Molec. Immun. 19, 1655-1665.

Lundwall A., Hellman U., Eggertsen G. and Sjoquist J. (1984) Chemical characterization of cyanogen bromide fragments from the b-chain of human complement factor C3. FEBS L&t. 169, 57-62.

Lundwall A., Malmheden I., Stalenheim G. and Sjoquist J. (1981) Isolation of component C4 of human complement and its polypeptide chains. Eur. J. Biochem. 117, 141-146.

Meuth J. L., Morgan E. L., DiScipio R. G. and Hugh T. E. (1983) Suppression of T-lymphocyte functions by human C3 fragments. 1. Inhibition of human T-cell proliferative responses by a kalhkrein cleavage fragment of human iC3b. J. Immun. 130, 2605-2611,

Molenaar J. L., Helder A. W., Miiller M. A. C., Goris- Mulder M., Jonker L. S., Brouwer M. and Pondman K. W. (1975) Physicochemical and antigenic properties of human C3. Immunochemistry 12, 359-364.

Nagasawa S. and Stroud R. M. (1977) Mechanism of action of the C3b-inactivator: requirement for a high molecular weight cofactor (C3b-C4b INA cofactor) and nro- duction of a new C3b derivative (C3b’). ?mm~no~hem~st~y 14, 749-756.

procedures and their application to peptide and protein hormones. J. Pure appl. Chem. 6, 233-244.

Nilsson U. R., Mandle R. J. and McConnell J. A. (1975)

Robinson P. J., Dunnill P. and Lilly M. D. (1971) Porous glass as a solid support for immobilisation or affinity

Human C3 and CS: subunit structure and modifications

chromatography of enzymes. Biochim. biophys. Acta 242, 659-661.

by trypsin and C42-C423. J. Immun. 114, 815-822. Porath J. (1963) Some recentlv develooed fractionation

Sawardeker J. S., Sloneker J. H. and Jeanes A. (1965) Quantitative determination of monosaccharides as their alditol acetates by gas-liquid chromatography. Analyt. Chem. 37, 1602- 1604.

Sim R. B. and Sim E. (1981) Autolytic fragmentation of complement component C3 and C4 under denaturating conditions, a property shared with a,-macroglobulin. Biochem. J. 193, 129-141.

Sim R. B., Twose T. M., Paterson D. S. and Sim E. (1981) The covalent-binding reaction of complement component C3. Biochem. J. 193, 115-127.


Tack B. F., Harrison R. A., Janatova J., Thomas M. L. and Prahi J. W. (1980) Evidence for presence of an internal thiolester bond in the third component of human complement. Proc. nann. Acad. Sci. U.S.A. 77, 5164-5768.

Tack B. F., Morris S. C. and Prahl J. W. (1977) Third component of human complement: structural analysis of the polypeptide chains of C3 and C3b. Biochemistry 18, 1497-1503.

Taylor J. C., Crawford I. P. and Hugh T. E. (1977) Limited degradation of the third compcncnt (C3) of human leukocyte elastase (HLE): partial characterization of C3 fragments. Biochemistry 16, 339tL-3396.

Thomas M. L., Janatova J., Gray W. R. and Tack B. F. (1982) Third component of human complement: local- ization of the internal thiolester bond. Proc. natn. Acad. Sci. U.S.A. 79, 1054-1058.

Towbin H., Staehelin T. and Gordon J. (1979) Electro- phoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. mtn. Acad. Sci. U.S.A. 76, 435C4354.

Whitehead A. S., Solomon E., Chambers S., Bodmer W. F., Povey S. and Fey G. (1982) Assignment of the structural gene for the third component of human complement to chromosome 19. Proc. natn. Acad. Sci. U.S.A. 79, 5021-5025.

characterization of tryptic fragments of human complement factor c3

Documents