C - Bioscience Reports

779

Bioscience Reports i, 779-784(1981) Printed in Great Britain

C I r and C l s s u b c o m p o n e n t s of h u m a n c o m p l e m e n t : two serine proteinases lacking the 'histidine-loop' d i s u l p h i d e bridge

Gerard 3. ARLAUD ~ and 3ean GAGNONT Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OXl 3QU, U.K. (Received 25 August 1981)

The N-terminal amino acid sequence of human CTs b chain has been extended to 52 residues. The histidine residue involved in the charge-relay system is located at position 38, whereas the 'histidine-loop' disulphide bridge is missing. So far, human complement subc o m p o n e n t s CIr and Cls are the only known mammalian serine proteinases lacking this disulphide bridge.

C1, the f i r s t component of human complement, is a calciumdependent complex containing three types of subcomponent: Clq, Clr, and Cls. In the native form of C1, C l r and C l s are proenzymes of serine proteinases which become activated on binding oi the complex, through its Clq subcomponent, to activators such as antigen-antibody aggregates. Activation of both C l r and Cls is by the splitting of one bond in the polypeptide chain. In the case of Clr, this is probably due to an autocatalytic mechanism mediated by the proenzyme itself (Dodds et al., 1978; Arlaud et al., 1980a)~ whereas activation of Cls is by activated C l r (CTr). Once activated, C~s then cleaves and activates complement components C2 and C4 (see review by Reid & Porter, 1981). In non-denaturing conditions, C l r is a non-covalently-linked dimer of tool. wt. 166 000-18g 000 and Cls a calcium-dependent dimer of mol. wt. 170 000-176 000; each C l r and C l s monomer is, in the proenzyme form, a single polypeptide chain of mol. wt. 83 000-95 000, which splits during activation into two disulphide-linked polypeptide chains a and b of respective tool. wts. 56 000-60 000 and 27 00036 000 (De Bracco & Stroud, 1971; Valet & Cooper, 1974a, b; Ziccardi & Cooper, 1976a,b; Sire et al., 1977; Arlaud et al., 1977; Tschopp et el., 1980). The c-terminal b chain contains the di-isopropyl fluorophosphate-reactive site (Barkas et al., 1973; Takahashi et al., 1975a; Sire & Porter, 1976), whereas inter-monomer interactions are thought to be mediated by the N-terminal a chain (Arlaud et al., 1980b). *Present address: D.R.F./Biologie Moleculaire C.E.N.G. 85X, 38041 Grenoble-cedex, France. correspondence should be addressed

et Cellulaire, %To whom all

9 1981 The Biochemical Society

780

ARLAUD & GAGNON

Limited amino acid sequence data for C l r and Cls have been obtained (Takahashi et al., 1975a,b; Sim et al., 1977). Recent studies (Arlaud et al., 19gl) have extended the N-terminal sequence of Clr b chain to 60 residues and revealed that this chain lacks the 'histidine loop', a disulphide bond invariant in all other known mammalian serine proteinases (Young et al., 1978). The present work shows that this characteristic is shared by CTs b chain.

Methods Purification of C~s and separation of Cls chains

CTs was purified from human serum as described previously (Arlaud et al., 1979), dialysed at #~ against 1% (v/v) acetic acid, and freeze-dried. The p r o t e i n (250 nmol) was dissolved in 6 M guanidine / HCI / 0.# M Tris/HCl / 2 mM EDTA, pH 8.0 (#.5 ml), and reduced by 20 mM dithiothreitol, then alkylated by iodo 2[3H] acetic acid (250 pCi) as described by 3ohnson et al. (19g0). The labelled protein was dialysed at # ~ against 1% (v/v) acetic acid and freeze-dried. Reduced and alkylated CTs was dissolved in 6 M urea / 0.2 M f o r m i c acid (2.5 ml), and Cls a and b chains were isolated by high-pressure gel-permeation chromatography on a 7.5-mm x 600-mm TSK-G3000 column (Tokyo Soda Manufacturing Ltd., Tokyo, 3apan), as described for CIr chains (Arlaud et a l , 1981). The isolated chains were dialysed against 1% acetic acid and freeze-dried. Amino acid analysis and automatic sequencing

Reduced and alkylated samples were hydrolysed under vacuum at I10~ in c o n s t a n t boiling HCI containing 0.1% (v/v) 2-mercaptoethanol and # mM phenol. Half-cystine was determined as S-carboxymethyl cysteine, and methionine was measured as methionine sulphone after performic acid oxidation (Hirs, 1967). Analyses were carried out on a Durrum DB00 analyser. N-terminal amino acid sequence of CTs b chain was determined in a B e c k m a n g g 0 c s e q u e n c e r as described by 3ohnson et al. (1980). Amino acid phenylthiohydantoin derivatives were identified by highpressure liquid chromatography as described by Bridgen et al. (1976). Radioactivity released at each step of the sequence run was measured by liquid scintillation counting of a 200-pl portion of the butyl chloride wash.

Results

and D i s c u s s i o n

High-pressure gel-permeation chromatography allowed a rapid and complete separation of CTs~ a and b chains, which were obtained in pure form9 as judged by SDS-polyacrylamide-gel electrophoresis. The amino acid composition of CTs b chain (Table 1) is in agreement with that reported previously (Sim et a l , 1977).

/C-TERMINAL SEQUENCES OF Ci'r AND Cis b CHAINS

Table i.

781

Amino acid compositions of Cls and C~r b chains

Amino acid composition of C~s b chain was calculated from triplicate 24-h~ 48-h~ and 72-h HCI hydrolysates~ except methionine~ which was determined as methionine sulphone from triplicate 24-h HCI hydrolysates of samples after performic acid oxidation. Destruction of serine and threonine was corrected for by extrapolation to zero hydrolysis time. Half-cystine was estimated as S-carboxymethylcysteine. _Tryptophan was not determined. The amino acid composition of Clr b chain is taken from Arlaud et al. (1981). Amino acid composition (residues/100 residues) Amino Acid

Cls b chain

CTr b chain

Asx Thr Ser Glx Pro Gly Ala Val Cys Met lie Leu Tyr Phe His Lys Arg

10.95 6.24 5.96 7.63 6.59 9.44 7.08 8.74 2.32 2.92 3.72 6.96 3.30 3.29 1.45 7.71 5.46

12.55 3.98 5.21 9.98 4.21 10.94 5.45 7.19 2.21 2.63 4.19 9.56 3.35 5.23 3.32 4.52 5.47

Automated sequence determination performed on the whole C~s b chain gave a single sequence extending to 52 residues (Fig. i). The identification of residues at positions 1-20 agreed exactly with the results previously reported by Takahashi et al. (1975b) and_by Sim et al. (1977). Comparison of the N-terminal sequence of Cls b chain with the c o r r e s p o n d i n g p a r t of other serine proteinases (Fig. 1) showed that the histidine residue involved in the charge-relay system (His-57 in the chymotrypsinogen numbering) is located at position 3g and that, among the 30 typically conserved residues in this part of serine proteinases (Young et al., 1978), 19 are also conserved in C l s b chain. Between them~ the b chains of Cis and C l r have 22 homologies along the N-terminal 52 residues sequenced. Moreover, "Cls b chain is found to share with CTr b chain the same striking structural feature, i.e. the absence of two half-cystine residues invariant in all other known serine proteinases. These residues, located at positions 30 and #6 in Fig. 1, are normally linked to form the 'histidine loop', one o~ the two disulphide bonds invariant in some 20 other serine proteinases (Young et al., 197g).

782

ARLAUD & GAGNON

lO

1

20

30

Elastase (pig)

V V G GTEAQRNSWP

SQ I S L Q Y R S G S S W A H T C

Chymotrypsin A (cow)

I V N GEEAVPGSWP

WQVSLQDKTG---FHFC

Trypsin (cow)

I V G GYTCGANTVP

Y Q VS L N - -

Plasmin (human)

V V G GCVAHPHSWP

WQVSLRTRFG---MHFC

Factor X (cow)

I V G GRDCAEGECP

WQALLVNEEN---EGFC

Thrombin (cow)

I V E G QDAEVGLSP_

WQVMLFRKSPQ--ELLC

conserved residues

I V GG

C~r b chain

I I G _GQ KA K M G N F P W Q V F T N I H G R G

Cls b chain

I I GGSDADIKNF

A

GS

P WQVSL

YHFC_

SG

HFC ......

PWQVFFDNPWA .......

40

Elastase (pig)

SG---

50

60

G GTLIRQNWVMTAAHCVDRELT---FR---

Chymotrypsin A (cow) G G S L I N E N W V V T A A H C G V T T S D

........

Trypsin (cow)

G GSLINSQWVVSAAHCYKSGIQ

........

Plasmin (human)

GGTLISPEWVLTAAHCLEKSPRPSSYK---

Factor X (cow)

GGTILNEFYVLTAAHCLHQAKR---FT---

Thrombin (cow)

GASLISDRWVLTAAHCLLYPPWBKNFTVDD

conserved residues

GG

C~r b chain

GGALLGDRWILTAAH-TLYPKEHEAQSXAX

CTs b chain

L I

WVLTAAHC

GGALINEYWVLTAAH-VVEGNREPTMYVGS _

m

Inside the C1 complex, C l r is thought to undergo a u t o c a t a l y t i c activation (Dodds et al., 1978), in contrast to Cls, which has been shown not to be s e l f - a c t i va t a bl e (Gigli et al., 1976; Arlaud et al., 1977), and t he act i vat i on of which is mediated by C i r . Again, i s o l a t e d C~r has a very limited est erol yt i c activity, whereas Cls hydrolyses a wide range of synthetic esters (Sim, 198i). Therefore, the absence of the 'histidine-loop' disulphide bridge cannot be related to precise functional char a c t e r i s t i c s of C l r or Cls, though both differ from most other mammalian serine proteases in that in viv% they f u n c t i o n in a h i g h - m o l e c u l a r - w e i g h t c o m p l e x , not as dissociated enzymes.

IV-TERMINAL SEQUENCES OF CTr AND C]s b CHAINS

783

Fig. I. N-terminal amino acid sequences of Cls and CTr b chains; h o m o l o g y with sequences of other serine proteinases. Cls h chain (90 nmol) was subjected to 52 cycles of Edman degradation. The recovery at the first step was 50% 9 and the stepwise yield was 96%. The N-terminal sequence of CTr b chain is taken from Arlaud et al. (1981). Sequence data for elastase9 chymotrypsin~ trypsin9 plasmin 9 factor X, and thrombin are from Young et al. (1978). '-' denotes that a gap was left to give maximum homology on alignment of amino acid residues. The residue numbering indicated is arbitrary, and takes into account the maximum number of positions in all the proteinases listed. The conserved residues are from Young et al. (1978), those underlined being invariant in the known sequences (Young et al.~ 1978). Residues are in the single-letter code: A~ Ala; C 9 Cys; D 9 Asp; E9 Glu; F 9 Phe; G 9 Gly; H, His; 19 lie; K 9 Lys; L 9 Leu; M, Met; N 9 Ash; P, Pro; Q~ Gin; R, Arg; S~ Ser; T 9 Thr; V~ Val; W, Trp; X 9 unknown; Y, Tyr.

References Arlaud GJ, Reboul A, Meyer CM & Colomb MG (1977) Biochim. Biophys. Acta 485, 215-226. Arlaud GJ, Sim RB, Duplaa A-M & Colomb MG (1979) Mol. Immunol. 169 445-450. Arlaud GJ 9 Villiers CL, Chesne S & Colomb MG (1980a) Biochim. Biophys. Acta 6169 116-129. Arlaud GJ 9 Chesne S 9 Villiers CL & Colomb MG (1980b) Biochim. Biophys. Acta 6169 105-115. Arlaud GJ~ Gagnon J & Porter RR (1981) Biochem. J.~ in press. Barkas T, Scott GK & Fothergill JE (1973) Biochem. Soc. Trans. 19 1219-1220. De Bracco MME & Stroud RM (1971) J. Clin. Invest. 509 838-848. Bridgen PJ, Cross GAM & Bridgen J (1976) Nature (London) 263, 613-614. Dodds AW 9 Sim RB~ Porter RR & Kerr MA (1978) Biochem. J. 175, 383-390. Gigli I, Porter RR & Sim RB (1976) Biochem. J. 1579 541-548. Hirs CHW (1967) Methods Enzymol. 11, 197-199. Johnson DMA, Gagnon J & Reid KBM (1980) Biochem. J. 1879 863-874. Reid KBM & Porter RR (1981) Ann. Rev. Biochem. 509 433-464. Sim RB (1981) Methods Enzymol. 807 in press. Sim RB & Porter RR (1976) Biochem. Soc. Trans 4, 127-129. Sim RB, Porter RR, Reid KBM & Gigli I (1977) Biochem. J. 163, 219-227. Takahashi K, Nagasawa S & Koyama J (1975a) FEBS Lett. 55, 156160.

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Takahashi K, Nagasawa S & Koyama J (1975b) FEBS Lett. 50, 330333. Tschopp J, Villiger W, Fuchs H, Kilchherr E & Engel J (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7014-7018. Valet G & Cooper NR (1974a) J. Immunol. 112, 339-350. Valet G & Cooper NR (1974b) J. Immunol. 112, 1667-1673. Young CL, Barker WC, Tomaselli CM & Dayhoff MO (1978) in At2as of Protein Seguence and Structure, vol 5, suppl 3 (Dayhoff MO, ed)~ pp 73-93~ Natl. Biomed. Res. Found. Washington~ D.C. ziccardi RJ & Cooper NR (1976a) J. Immunol. I16, 496-503. Ziccardi RJ & Cooper NR (1976b) J. Immunol. I16~ 504-509.