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Bioscience Reports, Vol. 6, No. 1, 1986 Partial Characterization of Human Complement Factor H by Protein and cDNA Sequencing: Homology with Other Complement and Non-Complement Proteins J. Ripoche, 1 A. J. Day, 1 A. C. Willis, 1 K. T. Belt, 1 R. D. Campbell, 1 and R. B. Sire 1'1 Received November 13, 1985 KEY WORDS: complement; factor H; C4b-binding protein, C2, factor B; ]~2-glycoprotein I; interleukin-2 receptor; sequence homology; eDNA. Factor H, a control protein of the human complement system, is closely related in functional activity to two other complement control proteins, C4b-binding protein (C4bp) and complement receptor type 1 (CR1). C4bp is known to have an unusual primary structure consisting of eight homologous units each about 60 amino acids long. Such units also occur in the N-terminal regions of the complement proteins C2 and factor B, and in the non-complement serum glycoprotein P2I. Amino acid sequencing, and sequencing of a factor H cDNA clone, show that factor H also contains internal repeating units, and is homologous to the proteins listed above. INTRODUCTION Factor H is an abundant serum glycoprotein of about 155,000 mol. wt. (Sire and DiScipio, 1982). Its principal function is to regulate the activation of the major complement protein, C3. During complement activation, C3 is activated by proteolysis to form C3b, which in turn forms a complex with the complement protease factor B. The C3bB complex is activated by proteolysis to form an active complex enzyme, C3bBb, which will in turn cleave and activate more C3. This amplification mechanism 1 M.R.C. Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. 2 To whom correspondence should be addressed. 65 0144-8463/86/0100-0065505.00/0 .(() 1986 Plenum Publishing Corporation 66 Ripoche, Day, Willis,Belt,Campbell,and Sim for C3 turnover is regulated in a number of ways, and the principal route is via proteolytic destruction ofC3b. C3b is destroyed by the complement protease factor I. This reaction requires a protein cofactor, which forms a complex with C3b. Only C3b in the C3b-cofactor complex is cleaved by factor I. (For review, see Reid, 1983; Sim et al., 1986.J Factor H is the major plasma cofactor for this reaction. Two membrane glycoprotems, complement receptor type 1 (CRI) (Fearon, 1979), and a protein termed "membrane cofactor protein" or "'gp 45-70" (Seya et al., 1985; Holers et al.. 1985), also possess cofactor activity for this reaction. A further serum protein, C4b-binding protein (C4bp), has a similar function as co factor for the factor I-mediated breakdown ofC4b, a homologue of C3b. The close functional similarity between CR1, C4bp and factor H provided an early indication that these proteins might have limited sequence homology (for discussion see Sim and DiScipio, !982; Sire and Sim, 1983: Holers ef al.. 1985) and this possibility was strengthened by the finding that the structural genes for C4bp, CR 1 and factor H are closely linked (Rodriguez de Cordoba et al,. 19851 Information on the structure of the proteins coded by this major gene linkage group is now becoming available and further comparisons can be made. The structure of the 549-residue long polypeptide chain of C4bp has been determined, and shown to consist of eight unusual consecutive repeat units, each about 60 amino acids long (Chung er aI.. 1985a, b). These repeating units are homologous to each other, and contain invariant cysteine, proline. tryptophane and glycine residues. It was noted that three of these repeating units homologous to those in C4bp are also present in the N-terminal region of complement factor B (Morley and Campbell. 1984), and in the N-terminal region of complement component C2 (Bentley and Campbell. 19861. C2 and factor B have some functional similarity with C4bp, since these proteins also bind to C4b or its homologue C3b. However. a further, non-complement glycoprotein,/321 ILozier er al., 19841, is also made up of five repeating units of the type found in C4bp Ifor discussion see Chung et al.. 1985b. Sire et al.. 1986). This protein is not known to have any functional similarity to C4bp, C2 or factor B. We report here amino acid and cDNA sequencing studies on human factor H, which demonstrate that factor H is also homologous to C4bp, and contains the same type of internal repeat unit. METHODS Protein Sequencing Factor H was isolated from human plasma as described before (Sim and DiScipio, 1982). Factor H was completely reduced in denaturing conditions and alkylated with iodo-[2-aH]acetic acid (Johnson et al., 1980). Factor H was then succinylated (Koide et al., 1978) and digested twice for 2 hr at 37~ with 2% w/w trypsin. The trypsin digest from approximately 100 nmol factor H was separated into 17 pools by gel filtration on a column (100 cm • 2 cm diameter) of Sephadex G75 in 0.1 M NaHCO3. Peptide pools were separated further by ion exchange chromatography on DEAE-Sephacel (Christie and Gagnon, 1982) or by high-pressure liquid chromatography in an NH4HCO3/ CH 3CN solvent system (Christie and Gagnon, 1982). Suitable peptides were sequenced in a Beckman 890C Sequencer (Christie and Gagnon, !982). Sequence Homologyof Complement Factor H 67 Synthesis of Oligonucleotide probe The N-terminal amino acid sequence of one tryptic peptide, TR-3-2, was determined as SPYEMF(3DEEVMC. A mixed 17-base long oligonucleotide probe, complementary to the mRNA for the amino acid sequence MFGDEE, was synthesized by the solid phase phosphotriester method (Sproat and Bannworth, 1983) using a Cruachem Manual Module with synthesis reagents supplied by Cruachem (Livingstone, Scotland). The probe consisted of a mixture of 32 sequences of the form: Y-T.C-Py-T-C-Pu-T-C-N-C-C-Pu-A-A-C-A-T-Y where N = A, G, C, or T, Pu = A or (3, Py = C or T. The oligonucleotide mixture was 5' labelled with [7-32p]ATP (Amersham International, Amersham, Bucks) and T4 polynucleotide kinase (Maxam and Gilbert, 1980). Isolation of eDNA Clones The human liver cDNA library of Belt et al. (1984) was plated on nitrocellulose filters, and replica filters on nitrocellulose were made (Grosveld et al., 1981). After lysis of colonies (Grosveld et al., 1981), replica filters were prehybridized for 16 hr at 42~ in 0.9 M NaC1-0.09 M Tris/HC!, pH 7.4-0.006 M EDTA-0.1% (w/v) Ficoll-0.1% (w/v) polyvinylpyrrolidone-0.1% (w/v) bovine serum albumin-0.5% (w/v) SDS-0.05% (w/v) sodium pyrophosphate--5% (w/v) dextran sulphate-100pg/ml boiled sonicated salmon sperm DNA-100 ktg/ml tRNA, then hybridized for 48 hr at 42~ in the same solution containing the labelled probe (approximately 1 ng/ml, specific activity 1.5 x 106 dpm/ng). Filters were washed as described by Woods et al. (1982), the temperature of the 15 min high-stringency wash being 54~ Positive colonies were identified after autoradiography of dried filters. Isolation and Analysis of DNA Plasmid DNA was prepared from bacterial colonies (Birnboim and Doly, 1979), and the cloned cDNA inserts excised from the pAT 153/PvuII/8 plasmid by BamH1/Clal or Mspl/Clal double restriction endonuclease digests, cDNA inserts were analysed by standard electrophoresis and Southern blotting methods. Sequence analysis of a restriction fragment was performed after subcloning the fragment into the M13 rap9 vector (Messing and Vieira, 1978). DNA was sequenced by the dideoxynucleotide chain termination method (Sanger et al., !980; Biggin et al., 1983). Northern Blot Analysis Northern blot analysis of human liver mRNA was done as described by Chung et at. (1985b). RESULTS Identification of Factor H Specific eDNA Clones Twenty thousand colonies of the human liver eDNA library were screened by the colony hybridization method, using the mixed oligonucleotide probe. Two positive 68 Ripoche, Day, Willis, Belt, Campbell, and Sim (a) C4BP H C48P H 1 10 20 30 40 50 M TG_R H-_ A_KLUNL~P-.~V QNAT . I V~RQ. . MS K . P S E . R~RLR_~QL~RSL~-~]E 51 60 70 P T T ~ P TT~MI~- QPC~LR W : ~ : ~ : ~ MF G E- - - L N'G_ WT E (b) v K1COpF P S R N20F NH- L NAK I - P-T Q AT I V S R- QMS K PS GERV RY Q B21 H "M B21 H 5SILD~P~E-qI-:~: K L N ~ A ; ~ S Q ~ E~ FL~DELL~V L E - - Y P A -3KOp T L - Y - - K ? K A T F G H D S (c) Be i RW~-Q I0 20 TA I C D~G A G Y C S . ~ - I 30 40 5O P I G~- - - ~ K V G[~Q~IR L E D SF~TF~Hr~-[~R 70 H ~YEEF D E E V ML~' " -L~"L~- - TE_UPWQW Fig. 1. Homology of factor H with C4bp, #zI, and factor B. A segment of factor H sequence is shown aligned with regions of C4bp,/~2 I, and the Ba fragment of factor B. Since each of these proteins contains internal repeat homology units, the comparison of the factor H segment can be made with several regions of C4bp,/~zI, or Ba. The C4bp, #zI, and Ba segments shown are chosen randomly, Gaps have been inserted to maximize homology. Identical residues are boxed, and conservative replacements (Dayhoff groupings) are underlined. The C4bp sequence shown begins at residue 246 in the numbering ofChung et al. (1985a), that of /~2I begins at residue 179 (Lozier et al., 1984), and that of Ba at residue 125 (Morley and Campbell, 1984). clones, R1 and R2, were identified, and isolated after a second screening with the same probe. The corresponding cDNA inserts were excised from the vector by Cla 1/BamH 1 digestion, and sized. R 1 contained an insert of about 1 kb, while the R2 insert size is about 2.2 kb. R1 is contained within R2. Analysis by Southern blotting of HinF 1 fragments of R 1 indicated that an internal HinF 1 fragment of 195bp hybridized with the original oligonucleotide probe. Sequence analysis of this fragment confirmed that the DNA sequence corresponded to that of the original probe, and the derived amino acid sequence contained the rest of the amino acid sequence determined for tryptic peptide TR-3-2. The derived amino acid sequence of this region is shown in Fig. 1. The sequence of peptide TR-3-2 corresponds to residues 46-61 in Fig. la. Northern Blot Analysis Northern blot analysis of human liver messenger RNA using the 32P-labelled 195bp HinF1 fragment, or the complete 2.2 kb R2 fragment, as hybridization probes, resulted in detection of 2 mRNA species. One species, of 4.7-4.8 kb, is of approximately the expected size for factor H mRNA, since the coding sequence for factor H, a polypeptide of about 1280 amino acids (Sire and DiScipio, 1982), would be about 69 Sequence Homologyof Complement Factor H 3.9 kb. The other species detected was 5.7-5.8 kb. The presence of more than one mRNA species for one protein has been reported before (see, e.g. Dozin et al., 1985). Two mRNA species of 9 and 11 kb have also been reported for the related protein CR1 (Wong et al., 1985). F a c t o r H and CR1 both exist in membrane bound and soluble forms (Malhotra and Sire, 1985; Yoon and Fearon, 1985), and it is possible that this phenomenon may be associated with the appearance of 2 mRNA species, or different mRNA intermediates for the two proteins. Sequence Homology The amino acid sequence derived from the DNA sequence of the 195bp HinF1 fragment is homologous to the internal repeat units of C4bp (Fig. la), the /~2 glycoprotein I (Fig. lb) and to the three internal repeat units of the Ba fragment of factor B (Fig. lc). In each comparison, the degree of homology is 24~27% based on identity, or 39-42~ based on conservative replacement. This degree of homology is very similar to that found when making comparisons between individual repeat units within C4bp, within/~z I, or within the Ba fragment of factor B. The N-terminal region of complement component C2 also shares sequence homology with this group of proteins, and a similar comparison can be made between C2 and factor H (Bentley and Campbell, 1986; Bentley, 1986). Each of the proteins with which factor H has been compared (Fig. 1) contains repetitive internal sequence. This is also the case for factor H. Alignment of randomlygenerated amino acid sequence data from tryptic peptides, and the derived sequence shown in Fig. 1, reveals extensive repetitive sequence (Fig. 2). Other brief reports of amino acid or cDNA sequencing of human factor H (Kristensen et al., 1985a; Schulz er V45 I Hinf 1 TR-8-4 30 10 20 SQESYAH-GYxL_~YTC 40 50 60 ~ PTVQNATIV~RQ~PS ~R~Y ~ ] ~ E M F - ~ ~M( KKDQ~]KV TR-3-4B TR-8-6 (vq KSID~~Px~IALF(K~QTT TR-14-3 TR-8-2 TR-4-5A TR-8-3 D KS-Y F~~-~~A-M( -- i-( rE TR-8-3 EKS TR-8-8 Fig. 2. Internal homology within factor H. Amino acid sequences of peptides, and the amino acid sequence derived from a cDNA HinF1 fragment (discussed in the text), are aligned to illustrateinternal homologies.The alignmentis based on the pattern of the repeat unit of about 60 amino acids found in C4bp (Chunget al., 1985a,b). Conservedresiduesare boxed or underlined, as in Fig. 1. Unidentified residues are shown as "x". 70 Ripoche, Day, Willis, Belt, Campbell, and Sire I i0 20 30 40 50 60 . . . . . G. . . . . , . . . . * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * . . . . P'C* Factor H /4 Y yF-*c**c Ba ***C G C4bp ***C--P C 7 F 7 ~ ~u?j ~***RTC***G*WS--~*C* C W***KP)*C* Fig. 3. Consensus sequences for the internal homology units of factor H, C4bp, Ba, and fi21. These sequences show the residues which are most strongly conserved in comparisons of internal repeat units. Data for factor H is taken from Fig. 2. Data for C4bp, fi2I, and Ba are from complete Sequences, as discussed in the text. Residues shown in brackets are present in most, but not all, repeat units. Other residues shown are completely conserved. Asterisks, in the factor H and other sequences, indicate residues which are not highly conserved. The spacing of conserved residues in~C4bp, fl2I, and Ba is slightly variable, as is the total length of the repeat units (approximately 60~71 residues). Lines indicate non-conserved regions of variable length. al., 1985) confirm that factor H contains repetitive structure of the ty.pe found in C4bp, An extensive structural study on mouse factor H, reported as an abstract (Kristensen et al., 1985b), indicates that mouse factor H contains 20 repeat units of the type found in C4bp. The alignment shown in Fig. 2 is based on repeat units of C4bp (Chung et al., 1985a~ b), and shows the same pattern of conserved cysteine, proline, glycine and tryptophane residues. A consensus sequence for factor H internal homology, based on the data in Fig, 2, is shown in Fig. 3, and compared with consensus sequences for the internal homology units of C4bp, Ba, and/32I. DISCUSSION On the basis of work reported here, and of brief reports elsewhere (Kristensen et al., 1985a, b; Schulz et al., 1985), factor H is made up of repeating units of amino acid sequence, based on a framework of four cysteine residues, together with highly conserved tryptophane, giycine, and proline residues. The same units of sequence occur in the functionally related proteins C4bp (Chung et al., 1985a, b), and it is now known that a similar repetitive structure occurs in CR1 (Klickstein et al., 1985). The Nterminal regions of complement components C2 and factor B (Morley and Campbell, 1984; Bentley, 1986; Bentley and Campbell, 1986) also each contain 3 units of this repeat structure. All of these five proteins interact with complement fragments C4b or C3b, and it was initially thought that homology between these proteins might be associated with this common function, However, in C4bp, H and CR1, the repetitive structure is extensive, and is likely to involve much larger regions of the molecule than those required for interaction with C3b or C4b. Further, these repeat units of about 60 amino acids long are also found in the functionally unrelated protein/~2I, and there are also two such units in the interleukin-2 receptor (Nikaido et al., 1984), although in this case the units are non-contiguous. Sequence Homology of Complement Factor H 71 The presence of these cysteine-rich units in functionally unrelated proteins suggests strongly that the repeat unit represents a c o m m o n basic structural element, such as, e.g., the immunoglobulin fold. It is o f interest that C4bp, factor H and f121 have very elongated structures, and factor H and fl2I have circular dichroism spectra which indicate unusual secondary structure (for discussion see Sire et al, 1986). These features have not been examined for the other proteins discussed above, but it is possible that the repeated sequence units m a y be associated with abnormally elongated regions of proteins. CR1, C4bp, and factor H are encoded by linked structural genes, likely to be on c h r o m o s o m e 1 (Rodriguez de C o r d o b a et al., 1985; Klickstein et al., 1985), F a c t o r B and C2 are encoded on c h r o m o s o m e 6 (Campbell et al, 1984), while the IL-2 receptor is on c h r o m o s o m e 10 (Leonard et al., 1985), It is k n o w n in factor B that each of the three repeat units present are encoded by separate exons (Campbell et ai., 1984). The occurrence of this h o m o l o g y unit in proteins encoded on three c h r o m o s o m e s m a y be an early indication of the wide distribution Of a c o m m o n structural element. ACKNOWLEDGMENTS We are very grateful to Drs. K. B. M. Reid, D . R . Bentley, B.F. Tack, and T. Kristensen for helpful discussion. J. Ripoche is an E M B O Fellow. REFERENCES Belt, K. T., Carroll, M. C, and Porter, R. R. (1984). Cell 36:907-914. Bentley, D. R. (1986). EMBO J., in press~ Bentley, D~ R., and Campbell, R~ D. (1986). Biochem. Soc. Syrup. 51, in press. Biggin, M. D., Gibson, T. J., and Hong, G. F, (1983). ProC. Natl. Acad. Sci. USA 80:3963-3965. Birnboim, H. C., and Doly, J. (1979). Nuc. Acid Res. 7:1521-1534. Campbell, R. D., Bentley, D, R., and Morley~ B. J. (1984). Phil, Trans. R. Soc. Lond. B 306:367-378. Christie, D. L., and Gagnon, J. (1982). Biochem. J. 201:555-567. Chung, L. P,, Gagnon, J., and Reid, K. B. M. (1985a). Molec. lmmunol. 22:427-435, Chung, L. P., Bentley, D. R., and Reid, K. B. M, (1985b). Biochem. J. 230:133-141. Dozin, B., Magnuson, M. A., and Nikodem, V. M. (1985). Biochemistry 24: 5581-5586. Fearon, D. T~ (1979). Proc. Natl. Aead. Sci. USA "/6:5867-5871. Grosveld, F. G., Dahl, H. M., Boer, E. D., and Flavell, R. A. (1981), Gene 31:227 237, Holers, V. M., Cole, J. L., Lubiln, D. M:, Seya, T., and Atkinson, J. P. (1985). ImmunolOgy Today 6:188=192. Johnson, D. M. A., Gagnon, J., and Reid, K. B. M. (1980). Biochem. J. 187:863 874. Kliekstein, L. B., Wong, W. W, Smith, J, A. Morton, C, Fearon, D. T, and Weis,J. H. (1985). Complement 2: 44-45 (abstract). Koide, A., Titani, K., Ericsson, L~H., Kumar, S., Neurath, H., and Walsh, K. A. (1978). BiochemiStry 17:5657 5672~ Kristensen, T., Wetsel, R. A., and Tack, B. F. (1985a). Fed. Proc. 44:1531 (abstract). Kristensen, T., D'Eustachio, P., and Tack, B. F. (i985b)~ Complement 2i46-47 (abstract). Leonard, W. J., Donlon, T. A., Lebo, R. V., and Greene, W. C. (1985). Science 228:1547 1548. Lozier, L, Takahashi, N~, and Putnam~ F. W. (t984). Proc. Natl. Acad. Sci. USA 81:3640-3644. Malhotra, V., and Sim, R. B. (1985). Eur. J. Immunol. 15"935-94l. Morley, B. J., and Campbell, R. D. (1984). EMBO 3.3:153 158. Maxam, A. M., and Gilbert, W. (1980). Methods Enzymol~ 65:499-560. Messing, J., and Vieira, J. (1978)~ Gene 19:269=276. Nikaido, T., Shimizu, A., Ishida, N.. Sabe, H., Teshigawara, K., Maeda, M., Uchiyama, T.~ Yodoi, J., and Honjo, T, (1984). Nature 3ih631-635~ Reid, K. B. M. (1983). Biochem. Soc. Trans. i1:1-12. 72 Ripoche, Day, Willis, Belt, Campbell, and Sim Rodriguez de Cordoba, S., Lublin, D. M., Rubinstein, P., and Atkinson, J. P. (1985). J. Exp. Med. 161:11891195. Sanger, F., Coulson, A. R., Barrell, B. G., Smith, A. J. H., and Roe, B. A. (1980). J. Mol. Biol. 143:161-178. Schulz, T. F., Schw/ible, W., Stanley, K. K., Weiss, E., and Dierich, M. P. (1985). Complement 2:71 (abstract). Seya, T., Turner, J., and Atkinson, J. P. (1985). Complement 2:72 (Abs.). Sire, E., and Sire, R. B. (1983). Biochem. J. 210:567-576. Sire, R. B,, and DiScipio, R. G. (1982). Biochem. J. 205:285-293. S hn, R. B., Malhotra, V., Ripoche, J., Day, A.J., Micklem, K. J., and Sim, E. (1986). Biochem. Soc. Syrup. 51, in press. Sproat, B. S., and Bannworth, W. (1983). Tetrahedron Letts. 24:5771-5774. Wong, W, W., Klickstein, L. B., Smith, J.A., Weis, J. H., and Fearon, D. T. (1985). Complement 2:87-88 (abstract). Woods, D. E., Markham, A. F., Ricker, A. T., Goldberger, G., and Colten, H. R. (1982). Proc. Natl. Acad. Sci. USA 79:5661-5665. Yoon, S. H., and Fearon, D. T. (1985). J. Immunol. 134:3332-3338.