Sixth Component of the Human Complement System. IDENTIFICATION OF THE C5b-BINDING DOMAIN IN COMPLEMENT. C6*. (Received for publication, May ...
Vol. 264, No. 30, Issue of October 25. pp. 18041-18051,1969 Prrnted in U.S.A.
THE JOURNAL OF BIOLOGICAL CHEMISTRY 8 1989 by The American Society for Biochemistry and Molecular Biology, Inc
Complete Primary Structureand Functional Characterization of the Sixth Component of the Human Complement System IDENTIFICATIONOF
T H E C5b-BINDINGDOMAININCOMPLEMENT
C6* (Received for publication, May 30, 1989)
Jacques-Antoine Haefliger, JiirgTschopp, Nathalie Vial, and Dieter E. JenneS From the Institute of Biochemistgv, University of Lausanne, Epalinges sur Lausanne CH-1066,Switzerland
Complement C6 is one of five plasma proteins that are incorporated into the lytic terminal complement complex on lipid membranes (C5b-9m) upon activation of the complement cascade. Oligonucleotide probes derived from partial amino acid sequences of purified C6 were used to isolate cDNA clones from a human liver cDNA library. The complete polypeptide structure of mature C 6 deduced from the cDNA sequence consists of 913 amino acid residues preceded by a typical 2 1residue signal peptide. C6 is most similar in structure to complement C7, sharing 33.5% identical residues with C7 including all 5 6 cysteine residues. The low density lipoprotein receptor class A and B modules, the thrombospondin type I module at the carboxyl terminus, and the two short consensus repeat modules are arranged in the same way as in C7. In contrast to C7 and other terminal complement proteins, the thrombospondin type I module at the amino terminus occurs as a tandem repeat in C6. The last tandem repeat at the carboxyl terminus of C 6 and C7has been identified as a new distinct module (factor I module), which is closely related to a segment in theheavy chain of complement control factor I. Binding studies with filter-bound C6 fragments generated by proteolysis showed that the C5b-binding domain of C 6 was located in the 34-kDa carboxyl terminal fragment consisting of two short consensus repeats and two factor I modules. Complement activationoccurs either via the classical or the alternative pathway through sequential conversion of several plasma proteins into biologically active components, resulting in two distinct multimolecular enzymatic complexes, the socalled convertases of the classical or alternative pathway. Both pathways then enter into common the terminal cytolytic pathway (1-3), which depends strictly on the sequential interaction of five soluble plasma proteins, C5b,’ C6, C7, C8 (a heterotrimer between C8a, CSP, and C8-y), and C9. Cleavage *This work was supported by a grant from the Swiss National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTMfEMBL Data Bank with accession number(s)505063 and 505064. $ To whom correspondence should be addressed. The abbreviations used are: C5, C6, C7, C8, and C9, the fifth, sixth, seventh, eighth, and ninth components, respectively, of the complement system; C5b-9m and C5b-9s, the membrane and soluble forms, respectively, of the terminal complement complex; SCR, short consensus repeat; T S P I, thrombospondin type I repeat; FIM, complement control factor I module; LDL, low density lipoprotein; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s); V8 protease, V8 S. aureus protease; bp, base pair(s).
of C5 between residues 74 and 75 into C5a and C5b by either
convertase triggers binding of plasma C6 to C5b. The resulting water-soluble C5b-6 complex subsequently reacts withC7. On thesurface of plasmamembranes,the assembly reaction terminates in the formationof an integral membrane protein complex, the cytolytic C5b-9m complex, which generates a transmembranous hydrophilic channel across the lipid bilayer (1-3). Apart fromlipid membranes,inthe fluid phase, a cytolytically inactive water-soluble complement complex, the C5b-9scomplex, is assembled. The latter contains at least three additional serum proteins: vitronectin (S-protein) (4, 5), cytolysis inhibitor (6, 7), and modified antithrombin I11 (8). Nascent C5b-6 complexes are not able to integrate into lipid bilayers prior to C7 binding, whereas the trimolecular C5b-7 complex, when attached to lipid bilayers, resembles a typical integral membrane protein(1). During the pastfive years, all components participating in the assembly of the C5b-9m complex except C6 have been completely characterized at the molecular genetic level. Sequence homologies havebeen established between C5 and other members of the a2-macroglobulins(9, 10) and between C7 ( l l ) ,C8a (12), C8p (13, 14), C9 (15-17), and the membrane-inserting pore-forming proteinof cytotoxic T lymphocytes, perforin (18-21). In this study, we have analyzed the complete molecular structure of complement C6 as derived fromthe cDNAsequence andthe molecularbasis of its interaction with nascentC5b. EXPERIMENTALPROCEDURES
Materials-All reagents used for the protein purificationand cDNA sequencing were purchased from Pharmacia LKB Biotechnology Inc. when not otherwise stated. [CY-~’SS]~ATP (1000 Cifmmol) and [y-”P] dATP (5000 Ci/mmol) were ordered from Amersham Corp. The 17mer universal sequencing primer for M13 and Sequenase, a modified T7 DNA polymerase (22), were supplied by United States Biochemical. C6 Purification and Sequencing-Complement component C6 was purified according to a published procedure (23). Protein concentrations were measured by a dye-binding assay (Bio-Rad) according to Bradford (24). To generate fragments of complement C6, 50 pgof purified C6 was incubated with 5 pgofV8 Staphylococcus aureus protease (Sigma) for 2 h at 37 “C in a total volume of 50 pl. The enzyme was inactivated by boiling the sample in Laemmli sample buffer (25). The resultant fragments were separated by SDS-PAGE under nonreducing conditions and blotted onto polyvinylidene difluoride membranes (Immobilon, Millipore) (26). The area of the filter containing the protein fragments was cut out and reduced in 100 p1 of a buffer consisting of0.25 M Tris-HC1, pH 8.5, 6 M guanidine hydrochloride, 1 mM EDTA, 0.5 volume % 2-mercaptoethanol for 2 h under argon. Thereafter, the sample was sulfur pyridylethylated 2 h under argon by adding 4 pl of 4-vinylpyridine (Sigma). Direct in situ aminoacid sequencing of blotted peptideswas done by automated Edman degradation (27) in an Applied Biosystems model 470A gasphase sequencer in the presence of Polybrene. Phenylthiohydantoinamino acids from each cycle were analyzed on-line by using a 120A
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S t r u c t u r e of Complement C6
phenylthiohydantoin-amino acid analyzer (Applied Biosystems) using the manufacturer's program.An average repetitive yield of%% of derivatized amino acids was obtained from 200 pmol of protein or peptide fragments in each degradationcycle. Cloning and cDNA Sequencing-All methods relating to the cloning and sequencing of complement C6 were performed as described (28). Oligonucleotides encoding amino-terminal residues of C6 were synthesized in an Applied Biosystems model 380B DNA synthesizer and purified on an NAP-25 column (Pharmacia LKB). To obtain a radioactive hybridization probe, oligomers were labeled at the 5' end using [y-32P]ATP(5000 Ci/mmol, Amersham) according to standard procedures (28). Initially, a mixed pool of synthetic oligonucleotides corresponding to residues 2-7 of intact C6 (see "Results") was used to screen a human liver cDNA library in pGEM4 (Amersham),which was kindly provided by K. K. Stanley, European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany. Ten pmol of labeled oligonucleotides was diluted in 150 ml of hybridization buffer containing 6 X S s c (0.15 M NaCl, 0.15 M sodium citrate, pH 7.3), 5 X Denhardt's solution, 0.1% SDS, and 50 Fg/ml sheared and denatured herring sperm DNA. After incubating the filtersat 42 "C for 4 h, the filters were extensively washed in 2 X SSC a t 35 "C. When the cDNA library was screened a second time, a nonredundant 25-mer corresponding to the 3' cDNA sequence of clone 11 (5"A GGG AAA CGC T G T GAG GGG GAG AAG-3') was chosen as a probe. The hybridization and washing temperatures were 45 and 40 "C, respectively. The cDNAsequence of C6 was assembledfrom random M13 subclones that were generated by ligating sonicated size-selected cDNA fragments (29) into an SmaI-cut dephosphorylated M13mp8 vector (Amersham).Single-stranded M13mp8clones and doublestrandedpGEM4plasmids were sequenced by the dideoxy chain termination procedure (30) usingSequenase (22) according to the manufacturer's protocols (United States Biochemicals). The nucleotide sequence of C6 was determined on both strands of the cDNA in multiple overlapping clones. The Taq DNApolymerase chain reaction (31) wasperformed essentially asrecommended by the manufacturer (Perkin-Elmer Cetus). One Fgof human genomic DNA (Pharmacia LKB) was heat denatured at 98 "C for 9 min in the absence of the enzyme. After the enzyme was added,two specific 18-mer primers were annealed a t 61 "C for 2 min, then extended a t 72 "C for 3 min, and the newly synthesized double-stranded DNA was melted a t 93 "C for 1.5 min. The last three consecutive steps (one cycle) were repeated 25 times. The sequences for the specific primers were derived from the cDNA sequence ofC6: 5"GT GAG AAA CAG TCT CCA G-3' (positions 1810-1827 in Fig. 2); 5'-ATCACA GGT ACTCCA GGA-3' (complementary to the sequence from position 1872 to 1889). Computer Analyses-The cDNA sequence of complement C6 was assembled and analyzed using the UWGCG program package (32) implemented on a Digital VAX computer. Multiple protein sequence alignments were performed with the use of a program package called CLUSTAL (33), which essentially follows the strategy of Feng and Doolittle (34) for the alignment of multiple protein sequences. Gap penalties of 10 and penalties of 10 for every item ina gap were found most satisfactory for these alignments. The statistical significance of sequence similarities was tested with the Alignment Score Program of the Protein Identification Resource (35). For similarity searches in thesequence databases, the programs FASTA and TFASTA of the National Biomedical Research Foundation (36) were used. Electron Microscopy-Cryoelectron microscopy of vitrified specimens was performed essentiallyas described (37,38). A drop of native C6 solution was mounted on a perforated carbon film on a 200-mesh grid. Most of the drop was removed by blotting, and theresidual film was vitrified by plunging into liquid ethane.The specimen was transferred intoa Gatan 626 cryospecimen holder (Gatan, Pittsburgh, PA)and observed in an electronmicroscope (PhilipsCM12) a t 42 000-fold magnification under minimal beam conditions and a t a temperature of -170 "C. Negative staining of C6 on carbon-coated grids was performed as described (39). Binding of C5b to C6 and Fragments of CG-SDS-PAGE followed by electroblotting was performed as described (25, 40). After electrotransfer of proteins, thenitrocellulose strips were saturated with 0.1% bovine serum albumin in incubationbuffer (1% gelatin, 1mM EDTA, 20 mM Tris-HCl, pH7.4, 150 mM NaC1) for 2 h a t room temperature. Eachstrip was then placedindividually in apolypropylene tube containing 1 ml of either plasma plus 20 mM EDTA, whole human serum, plus 10 mg/ml zymosan (w/v), or C6-deficient serum plus 10 mg/ml zymosan. The contents of the tube were shaken for 3 h at
37 "C; thereafter, the filter strips were washed five times for 5 min in washing buffer (incubation buffer without gelatin). Ascites fluid of murine monoclonal antibodiesagainsthumancomplement C5b, named 558 (411, or C9 neoantigen, named MCbC5 (42), was diluted 1000 times for overnight incubations. Binding of the first antibody was detected with peroxidase-labeled anti-mouse IgG using hydrogen peroxide and 4-chloro-1-naphthol (Merck, Darmstadt, F.R. G.). RESULTS
Amino Acid Sequencing of C6-Previous aminoacid sequence data published for complement C6 were not suitable for designing specific oligonucleotide probes (43). Human C6 was therefore purified fromhumanplasma according to publishedprocedures(23). Tenamino acids fromthe amino terminus of C6 were determined, and residues 2-7 were selected to of intact C6 (Phe-Cys-Asp-His-Tyr-Ala) synthesizethe following mixed pool of 17-mers: 5'-TTTTGTGATCATTATGC-3'.
c c c c c
Moreover, V8 protease was used
to cleave native C6 into two fragments of about 65 and 34 kDa. Thesefragments, which arenotlinked by disulfide bridges, were separated by SDS-PAGE withoutreduction and blottedonto Immobilon membranes for protein sequence analysis. In this way, a second internal amino acid sequence of C6 was obtained (see Fig. 2, underlined). Isolation and Characterization of cDNA Clones-Approximately 300,000 recombinant bacterial colonies from an adult human liver cDNA library were screened by using the in situ colony hybridization technique (28).After colonypurification, 15 clones were obtained whichhybridized to the oligomer probe under the conditions described. In order to identify authentic C6clones,allcDNA inserts were subjected to double-stranded nucleotide sequencing using the T7 and SP6 promoter primers, which anneal close to the BamHI cloning site on opposite sites of the inserts. In this way, five cDNA clones (11, 31, 35, 41, 41n) were found whose nucleotide sequences started within200 bp upstream of the coding region for mature C6 (Fig. 1B). Clone 11, withthe longest insert of about 3.0 kb, was sequenced completelyby using the shotgun procedure. Despite its large size, this clone turned out tobe incomplete at the 3' end. Sequence analysis revealed that an additional sequence of about 900 bp containing several in-frame stop codons was present in the3' half of clone 11. To isolate a cDNA clone that covers the carboxyl-terminal part of C6, a second nonredundant oligonucleotide was derived from the 3' sequence of clone ll and used to rescreen the human liver library. The additional clones lA, 9A, and 18A extended the partial C6 codingsequence of clone 11 toward the 3' end by about 3 kb. The position of clone 11 relative to clone 1A suggested that the 1.5-kb insert of clone 1A was sufficient to complete the 3'-coding region of the cDNA sequence. The 1.5-kb insert of clone 1A was therefore subjected to shotgun sequencing (Fig. 1B). Finally, thecDNA sequence of C6 was assembled from the two partially overlapping sequences of clones 11and 1A (Figs. 1B and 2). Since clone 1A also spanned the region of clone 11, which appeared to interrupt thecoding sequence of complement C6, we were able to confirm the precise boundaries of the sequence insertion in clone 11. In addition, two other independentplasmid clones, 35 and 31, were partiallysequencedfrom the 3' ends in the same region. These two cloneslacked the nucleotide insertionas well. The DNA insertion found inclone 11 (Fig. 3) commenced with the donor consensus sequence of exon-intron boundaries(-gt)after position 1839, interrupting the codon of Asn-530 after the first base, and ended with the acceptor consensus sequence (-ag)
S t r u c t u r e of Complement C6 FIG. 1. cDNA cloning of complement C 6 . A , location of oligonucleotide probes, the mixed pool of 17-mers (left verticalarrow) and the25-mer (right uertical arrow), and restriction sites of the cDNA sequence; the protein-coding region is indicated by an open bar. B , C6 cDNA clones. Clones 11,41n, 41,31,and 35 were obtained by DNA hybridization screening using the mixed pool of 17mersand clones 9A, lA,and 18A by usingthe 25-mer. The position of the 900-bp intron sequence inclone 11 is marked by a verticalarrow above the cDNA insert. Clone 18A (dashed arrow) extends 1.8 kb more to the3' end and is not depicted in its entire length. C, sequenceassemblyfor the C6 cDNA sequence. A series of overlapping random subclones in M13 generatedfromthe inserts of clone 11and 1A was sequenced by the dideoxy chain termination method (30). Clones of the coding strand are shownabove the two thick horizontal arrows, which represent the two strands of the cDNA;clones of the antisense strand are placed below.
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stream noncoding region contains further terminationcodons in all three frames but does not include a polyadenylation signal. As indicated by thelength of clone 18A, the3'untranslated region of C6 extends further by at least 2.2 kb. This portion of the 3"untranslated region has not been sequenced. Amino Acid Sequence of C6-The predicted amino acid sequence of complement C6 has the following features. After polypeptide removal of a 21-residue signalpeptide, the mature chain of C6 is composed of 913residues. The amino acid composition of the mature polypeptide agrees well with composition datapublished previously forcomplement C6 (23,43, 48). There aretwo possible sites for asparagine-linked glycosylation at positions 303 and 834 (49). The calculated molecular weight of C6 amounts to 102,425 without carbohydrates. Considering the carbohydrate content of C6, which was reported to be in the range of 4-9% (43, 48), the calculated molecular weight of C6 compares well with the reportedvalue of 104,800, which was determined by sedimentation equilibrium ultracentrifugation (43). Two regions of the derived amino acid sequence were confirmed by protein sequencing in this study. The cDNA-derived amino acid sequence of the amino terminus is identicalwith the proteinsequence determined for intact C6 and with amino acid sequencing data reportedpreviously (43,50). In addition, the cDNA-derived amino acid sequence between residues612 and 619 is identical with the protein sequence determined for the amino terminus of the 34-kDa V8 fragment of C6. Furthermore, our cDNA sequence includes a recently described 1539-bp cDNA fragment of C6 (51). nucleotides in that consensus sequence, a purine nucleotide Complement C6 contains a large number of cysteine resiat position -3 and a guanine at position +4, are present (46). dues, 64 intotal, which areclusteredintheamino-and In addition, the first21 residues of the following open reading carboxyl-terminal portionsof the polypeptide chain. Interestframe have the typical features of a signal peptide, as one ingly, the amino-terminal position of the mature protein is would expect for a secreted protein. A 13-residue stretch of occupied by a cysteine residue. mainly hydrophobic residues is flanked by positively charged Structural Organization of C6"Following the terminology residues on either side. According to von Heijne's rules (47), of Traut (52, 53), we can identify and delineate nine distinct this signal sequence is most likely cleaved after the alanine structural units in C6 which have the characteristic features residue. The resulting mature protein would therefore start of modules (Figs. 4 and 5): three TSP I modules (54), one with the proteinsequence determined for the amino terminus LDL receptorclass A( A )and classB ( B )module, respectively of mature C6. The open reading frame is terminated by a (55-58), two SCR modules (59,60), andtwo FIMs (see below). TAG stop codon after nucleotide position 2957. The down- These cysteine-rich modules are exclusively located in the for intron-exon boundaries (44). T o confirm our suggestion that clone 11was derivedfrom anincompletely splicedmRNA molecule of human liver rather than from an alternatively spliced mRNA molecule, we amplified total human genomic DNA and purified plasmid DNA of clone 11 with the use of two 18-mers flanking the intervening sequence in clone 11. Electrophoreticexamination of the Taq DNA polymerase chain reaction products revealed a single homogeneous band of about 900 bp in both cases under optimized reaction conditions (data not shown). Therefore, we conclude that clone 11 actually containsa complete nonspliced intron. T h e Nucleotide Sequence of C6-Fig. IC shows the relative position of overlapping M13subclones from which the entire nucleotide sequence was assembled. Most areas are covered by several different M13 clones on both DNA strands. One short area was sequenced only in one orientation, and the cDNAsequences in that direction were unambiguous and identical in multiple overlapping clones. Our sequencing results established a continuous cDNAsequence of 3303 bp from the 5' end of clone 11 to the 3' end of clone 1A. The region beyond the 3' end of clone lA, which is covered by clone MA, was not further sequenced since clone 1A already contained 347 bp of 3'-noncoding region. Examination of the cDNA sequence revealed a single large open reading frame of 934 amino acids which commenced at the first available ATC; codon at position 157. The sequence flanking the initiationcodon ATG in theC6 cDNA (AAGGC ATG G) does not strictly agree with Kozak's consensus seA quence CCGCC ATGG (45). However, the most critical
18044 99
Structure of Complement C6
1 ~ G C C T T G T G T T A G C T A G C T A A G A A A A G ~ G C T T n ; T T c AGCTTAGGTCCGAGGACACCC~CTCTGC~~GGGCCTGGAGGCTCTC~GGCA~GCCAGAC~TCTGTC~GTAC~CATCCTGCTG~T~TCTGATC~C~G~CCAA -
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GAACAGATTTGCAGCAAGCAGGAGACTAGAG~TGTAACTGGC~GATGCCCCA~~CT~CTCC~G~GAT~T~ACCA~G~AGAC~T~CCCT~TATT E Q I C S K Q E T R E C N W Q R C P I N C L L G D F G P W S D C D P C I E K Q S AARGTTAGATCTGTCTTGCGTCCCAGTCAGT~G~G~CAGCCAT~A~GCGCCTCTGGTAGCCT~C~CCATGCATTCCA~T~G~C~CAAAATT~GAGGCTGAC K V R S V L R P S Q F G G Q P C T A P L V A F Q P C I P S K L C K I E E A D C K
AATAAATTTCGCTGTGACAGTGGCCGCTGCT~A~GCCA~GTTAG~TGCAATGGAG~TGACTGTGGAGACAATTAGAT~GGGAC~T~GAGGACRAAGCTTTGGCAGTA~ N K F R C D S G R C I A R K L E C N G E N D C G D N S D E R D C G R T K A V C T
CGGAAGTATAACTTCCCATCCCTAGTGTACAGTTGATGGGCAATG~T~CATT~C~GCAG~GAGCCCA~~A~GTCCTTGAT~CTCT~CACTGGA~AATATGTAAAACTGT R K Y N P I P S V Q L M G N G F H F L A G E P R G E V L D N S F T G G I C K T V AAAAGCAGTAGGACAAGTAATCCATACCGTGTTCCGGCCAATC~G~TG~G~~GAGGTAC~~GCA~GATGAC~G~CAGAT~CTAC~GGAT~AACT K S S R T S N P Y R V P A N L E N V G F E V Q T A E D D L K T D F Y K D L T S L
GGACACAATGAAAATCAACAGGCTCATTCTCAAGTCAGG~G~AGCTCTTTCAGTGTACCTT~ATTC~RAAGCTTTGAGAAGT~TATCAACCAT~TTCT~C~C~CAA G H N E N Q Q G S F S S Q G G S S F S V P I F Y S S K R S E N I N H N S A F K Q GCCATTCARGCCTCTCACGATTCTAGTT~A~A~ATCCAT~G~A~~G~TT~CT~AC~CG~~TAAAGATCTGCACCTT~TGAT A I Q A S H K K D S S F I R I H K V M K V L N F T T K A K D L H L S D V F L K A
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1299 A G C A G T G A G G A A C T A A A G A A C T C A G G T T T A A C C G A G G ~ G ~ G C C ~ C A C T G T G T A G G A T G ~ ~ G ~ C G C G T T T A ~ T ~ T A A G A A A A C ~ G T G ~ C A T A G G T G C A C C 3 61 S S E E L K N S G L T E E E A K H C V R I E T K K R V L F A K K T K V E H R C T 1419 4 01
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ACCAACAAGCTGTCAGAGCATGAAGGTTCATTTATACAGGGAGCAGAG~TCCATATCCC~A~CGA~TffiAAGGAGTGAATATGGA~AGCT~G~ATGGGAG~GGGAGC T N K L S E K H E G S F I Q G A E K S I S L I R G G R S E Y G A A L A W E K G S TCTGGTCTGGAGGAGAAGACAT~TCTGAGTGGTTAGAATCAG~AAGG~TCCTGCTGTGA~GAC~GAGCTT~CCCCATCGTGGAC~GGTAAG~CATCCCCTGT~AGTG S G L E E K T F S E W L E S V K E N P A V I D F E L A P I V D L V R N I P C A V ACAAAACGGAACAACCTCAGGRAAGCTTTGCTTTGCAAGAGTATGCAGCCAAG~GATCCTTGCCAGTGTGCTCCA~CCCT~T~T~CCGACCCACCCTCTAGGGACT~TGTCTGTGT T K R N N L R K A L Q E Y A A K F D P C Q C A P C P N N G R P T L S G T E C L C
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TACTTGGTTGGAGAAGATGTGAAATTTCAT~C~ACTGGCTTTGRAAGCTTTCTG~GGATACCAGTACTTA~~C~ACCAGACffiGACC~GAGA~~GGATGTGGAA~C~CG Y L V G E D V E I S C L T G F E T V G Y Q Y F R C L P D G T W R Q G D V E C Q R A C G G A G T G C A T C A A G C C A G T G T G C A G G A A G T C C T G A C A T G T T G T T G C T G G G C C A
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TCAAGGTACACATGCCAGGGGAATTCCTGGACACCACCCA~T~CTCTCTACCTGTG~GATACTCT~C~~~~C~TTGT~GCTG~ACAG~CA S R Y T C Q G N S W T P P I S N S L T C E K D T L T K L K G H C Q L G Q K Q S G
TCTGAATGCAT~GTATGTCTCCAGAAGAAGACTGTAGCCATCATTCAG~GATC~TGTG~TTGACACAGAC~C~CGATTACTTTACT~ACCC~T~TAAG~T~GGCTGAG 2499 7 61 S E C I C M S P E E D C S H H S E D L C V F D T D S N D Y F T S P A C K F L A E 2619 801
AAATGTTTAAATAATCAGCAC~CATTTTCTACATA~GGTTCCT~C~GACG~C~CAGTTAGAATGG~TCTT~GGACAAGACTT~ATCCAACAGCAC~G~GA K C L N N Q Q L H F L H I G S C Q D G R Q L E W G L E R T R L S S N S T K K E S
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TCATCAACAAGTGAGAAAACATTGAACATCTGTG~GTGG~CTATAAGATGTG~CAGG~GATGG~TACTGCATCCTGG~G~T~GGCCTAGCACAATTACTGCTAGGCC S
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3
CAGCACAATGRACAGAT~ACCATCCCGAAGAACCAACTCCTACAAAGAGGTCTGAGAATTCTTGCAC~CA~A~CTGGCATGC~RAAGCTTTGTACTGAC~TTATTTTCTGTTAG~TGAG ATCATTATTCTCCCCTGACTTCCTGTTTGGGCATGTCTTATTCAG~CCAGCTCATGACGCCC~TAGCATACCCCTAGGTACCCTTCCACAGCAG~TCGT~~CTCC~TTA C A T T G T A C A A A A A T A A T G T G C ~ C T G A G G C C C T T A T G T A G C C ~ T G A ~ T T A A G C A ~ C T C G C T T A ~ T A A G A3303 AT~C
FIG. 2. Nucleotide and amino acid sequence of human C6. The deduced amino acid sequence of C6 is shown by standard one-letter symbols beneath the cDNA sequence. Nucleotide bases and amino acid residues (italicized figures)are numbered onthe left. The numbering of the nucleotide sequence starts with the first base of the cDNA insert of clone 11 and the numbering of the amino acid sequence with the first residue of mature C6. Amino acid residues -21 to -1 represent the signal peptide; the residues numbered from 1 to 913 encode the mature C6. The underlined amino acid residues in the amino-terminal region (residues 1-10) were determined hy Edman degradation of affinity-purified C6; the underlined residues from 612 to 619 were determined for a V8 protease cleavage fragment. The two predicted asparagine-linked glycosylation sites are marked by filled triangles and the stop codon at the end of the open reading frame by an asterisk.
Structure of Complement C6
18045 1827
537
GAT TATAAATCCA 1839 Asp TyrLysSerA 541
( 1) gtaagtatcaggaatctattgtgaggtagataagttttcccctccaaagagtattctaagttggtcaattaaaaagaaacaaaacttctattagcaacct (101) ccaccttgtacaggctcagagggaagataaacctgcaaaaagtgtgagtctcagctgtaacctaccaactatgtgagcttgggaaagttacccagcttcc (201) c t a t g c c t c a a t c t g t t c a t c c a t a a a a t g a g g a t a a c a c c a g t a t c t a t c t a a g g a a t a t g a a t a t a t t a t a t g c g t a t t t a t a t a t c a a g a a t a c a t a (301) t a c a a t g c t c a t a a c a t a a a a a a t c a a a c a t a a c a t c a a a t a a a a t a t a t g a t t c t g a t a t t a t a a g c c t c t t a t g t a t t g t a a t a t a g a a g g a t g g c t t (401) t g t a a t a a a a a a t a a a t t a a gccacaacca a a g t t t t c a t acaggactaa gtagtaacat agttacctca aatcctcctt agaaacaggg taaggcatgg (501) g a g t a a g a a t g t a t a c t a c t a c t t c c t t t a a a a g t g t a a t t t a a t a t g c a t t c t g t t a a g a a g a t g t t t a t a t t t a t a c a c a t a t g a g t g c a c a t t t t t a (601) a a a g g c c t c c a a a g c c a a g a a t a c a g a g g t t t c t t a a c a g t t g a a a a t a a t t c a t a a t q a t t g c c a t t t g t t g c c a t t t t a t t t a g g c t t atgggaacaa (701) a g t c t a a a a g g a a a t c a g g c a a t g t g t c a g g c c c c t t g c a c a g g t a a t t t c t t t t a a t c c t t a q a a q a a c c c t a t m t a t cccacaaagg aagttactga (801) gctaaaaagt gaqgaaacca ggttggcctc acttccaaac ccaaattcag ctgcaccatg atccatggga gtgagtcttc ctttgttcca gATGCAGTA 1847 snA1ava1 543 "
FIG. 3. Nucleotide sequence of a C6 intron. The nucleotide sequence of a sequence insertion found in one of the cDNA clones, clone 11, is depicted. The base numbers joinedwith brackets are shown on the left and start with the first nucleotide of the intron. Thetypical donor (-gt)and acceptor sequences (-ag) at the beginning and end of the intron are underlined. A few nucleotidebases (capital letters) of the coding region of C6 and the corresponding translation are added at the boundaries of the intron. The numbering of the cDNA regions is not put into brackets.
The lasttwo FIMs of C6 and C7 have not yetbeen described amino- and carboxyl-terminal thirds of the molecule. Three out of the five different types of modules, the TSPI, the SCR, in other proteins. Scanning all sequences in the protein semodule of C6using the and the FIM, occur in tandem form in C6. None of these is quence database against the last FASTAsearch program (36), we found onlytwo protein found as tandem repeats in C8a, C8& and C9. The amino acid sequence of C6 is most closely related to sequences with initial and optimized scores high enough to of that module. As expected, the complement C7 with respect to the percentage of residue indicatetherecurrence identities (33.5%) and to the overall organization (Figs. 4and highest optimized score of 175 was obtained for the match 5, and Table I). The only striking difference is an amino- with complement C7 (11). Surprisingly, residues 24-139 of terminal extension of 59 residues, which is a complete dupli- complement control factor I (72, 73) gave the second highest cation of a single thrombospondin-like repeat present at the optimized score of 105. In order to evaluate the statistical amino terminus of C7, C8, and C9. In addition, C6 and other significance of this sequence similarity, we calculated the terminal complement proteins except human and murine C9 scorefor an optimal alignment between the last carboxylhave another carboxyl-terminallylocated TSP I module (Fig. terminal module of C6 (FIM 2) and thehomologous region in 6). TSP I modules are also present in three copies in throm- factor I and an average score for the alignment between the factor I region and 300 randomizations of FIM 2 of C6 using bospondin (54), in six copies in properdin (611, and in one s mutation data matrix, a copy in all circumsporozoite proteins as well as in thrombo- the program ALIGN with Dayhoff spondin-related anonymous protein, which is a secondprotein matrix bias of 6, and a gap penalty of 10. The ALIGN score of malaria parasites (62). The two thrombospondin type I for the alignmentof the two real sequences was 7.65 standard repeats (residues 1-59 and 60-120) at the amino terminusof deviations above the average score obtained for the random C6 are followed by the LDL receptor class A module (residues alignments, indicating a high probability that the two pairs of sequences are evolutionarily related.Sincethis region 118-156), which is found seven times in the LDL receptor (63) and10 times ina putative apolipoproteinE receptor (64). recurs in C6 and C7 in tandem form butonly once in factor I The central third of the polypeptide chain, ranging from (Fig. 7), we would like to designate it as a factor I module residue 157 to 501, has no discernible module structure and (FIM). The recent suggestion that residues 71-136 within is, however, not supported by our is almost free of cysteine residues. This region is unique to FIM represent an SCR (73) the terminal complement proteinsC6, C7, C8a, CSP, C9, and calculations. The structural featuresof SCRs have been anato the cytolytic T lymphocyte-derived lytic protein, perforin lyzed extensively and compiled by several authors (67-70). One of the most highly conserved residues besides the cys(18, 19). No sequence homologies have been found in other proteins.Withinthis region, themost highly conserved teines is a glycine that is found atposition +3 relative to the stretch of residues across all five sequences including perforin second cysteine in SCRs.The occurrence of a glycine at and C6 lies between positions 328 and 345. The hypothesis position +4 is therefore evidence against rather than infavor of a structural relationship between this factor I region and has been put forward that this stretch may possess conformational flexibility and adopts the conformation of a mem- SCRs. Furthermore, taking residues 71-136 of factor I as a brane traversing amphipathica helix during formationof the querysequence, we have not beenable to find highscore lytic terminal complementcomplex on lipid bilayers (17).The matches to other SCR-containing proteins in the entire proB tein data base. We therefore conclude that theregion 71-136 following cysteine-rich region is an LDL receptor class (epidermal growth factor precursor) module that is linked to in factor I had inadvertently been identified as an SCR in a the thrombospondin type I module as in C7, C8a, C8& and previous study (73). trout C9 (11,65,66). Electron Microscopy of Monomeric C6-The typical ultraDistinct from the complement components C8a, C8& and structural appearance of nonaggregated well dispersed C6 is trout C9 but similar to C7, complement C6 has a carboxylshown in Fig. 8. The fully hydrated C6 (panel A in Fig. 8) has terminal extensionof 322 residues which consists of the same a rod-like appearance without discernible globular substructwo kinds of tandem repeats as in complement C7 (Fig. 5). tures, whereas the negatively stained C6 appears to be subThe first two SCR modules have been described in several divided into four distinct globular domains of about the same e.g. in size. The length of the long axis is 18 k 2 nm in side views, complement proteins of the early activation pathways, C4-binding protein, factor H, in the complement receptors and the cross-sectional diameter measured a t half-distance in CR1 andCR2, and in other noncomplement proteins (60,67- the centerof the molecule is 6 k 1nm. Theoverall dimensions 70). Only some of the multiple SCRs within these proteins of C6 and its ultrastructural shape resemble complement C7 have specific binding functions and mediate interactions withto a large extent (11). C3b and C4b (71). Identification of the C5b-binding Domain in CG-To study
Structure of Complement C6
18046
1 20 CFCDHYAWTQWTSCSKTCNSGTQSRHRQIVVDKYYQENFCEQICSKQETR QYTTSYDPELT
FIG. 9. C5b binding to the carboxyl-terminal domain of complement C6. C6 (lane 1, filled triangle) and V8 protease-cleaved C6 fragments(lane 2, open triangles)were fixed to nitrocellulose filter strips by electroblottingafterSDS-PAGE.Onthe far leftpanel (Stain), the Ponceau S staining patternof 4 pg of C6 and V8-cleaved C6 is shown. The additional band migrating between the two proteolytic fragments isan impurity. To testfor C5b and C9 binding, filter strips were incubated in zymosan-containing serum a t 37 "C (panels A and B ) . Control experiments were performed using 20 mM EDTA containingplasma (panel C). Bound C5b (panels A and C) and neoepitopes of C9 (panel B ) were detected by specific mouse monoclonal antibodies against these components and peroxidase-coupled and anti-mouse IgG second antibodies.
Structure of Complement C6
18049
it that the the case of C6 and C9 to 33.6% in the case of C8a and C8P; correcting a sequencing error4 in trout C9), follows the figures for conserved residues from eight different conser- unpaired cysteine of the carboxyl-terminal TSP I module is likely to be bonded with Cys-500. Thus, the remaining two vation groups (lower triangle) are somewhat higher and are close to 50% for eachsequence pair. Careful inspection of the equivalent cysteines, Cys-602 in C6 and Cys-616C (sequence values reveals that C ~ Cand Y C8P on one hand andC6 and C7 insertion) in C7, must be linked to Cys-478, which lies just classB module. These disulfide again on the other hand are more closely related to each other beforetheLDLreceptor linkages in C6 and C7 are supportedby the proteasecleavage than to any other complement component. The same relations were found by calculating the similarity scores with the use pattern of C6 and C7 generated by V8 protease and trypsin of CLUSTAL 1(33). Complement C6 and C7 form one cluster, digestion. whereas C8a and C8p form a second cluster at about the same There are3 unique cysteineresidues confinedto C8a,which level of sequence similarity. Thesehomology relationships are are not found in other terminal complement proteins. Cysfurther supported by the fact that C6 and C7 have the same teine residue 202L in C8a is located within a large sequence carboxyl-terminalextensionsbeyondthe common region insertion and forms an intermolecular disulfide bridge with cysteine residue 40 ofC8-y.’ The adjacent cysteine residues shared with C8a andC8P. The presented structural model of complement C6 (Fig. 5) 472 and 473 are presumed to form a disulfide cross-link in of adjacentcysteine residues that are is based on the finding that small cysteine-rich segmentsof C8a.Anotherpair C6 are also found in several other proteins outside of the missing in mouse and trout C9 as well as in other terminal complement system in different arrangements, combinations,complement proteins has been encountered in human comand sequence environments. InC6, we have distinguishedfive plement C9. The adjacent cysteineresidues 2643 and265F of different classes of modules including the new FIM, which human C9 lie within a wide sequence insertion whose boundhasnot been reportedin previous papers (72, 73). These aries show little homology tootherterminalcomplement structural units in C6 have the typical features of modules. proteins. These 2 cysteine residues of Cg are also expected to to located within Hence, C6 modules are expected to fold into stable structures, be cross-linked to each other and appear be to exert a specific binding function inat least some cases, and a large surface loop of human C9 (Fig. 4). There are at least to be potentially encoded by a single exon.This last notion is three other proteins known for which vicinal disulfide linkin accord with our finding that the carboxyl-terminalTSP I ages have been demonstrated malformin A (77), 7 subunit of module is separated from the LDL receptor class B module bovine transducin (78), and a-subunit of the acetylcholine location of vicinal half-cystinyl residues by an intron whose location and sequence were determined receptor (79). Surface disulfide bondextremely susceptible to low by cDNA cloning of a n incompletely splicedmRNA transcript makesthe amounts of reducing agents as has been shown for the CY (Fig. 3). Since the terminal complement proteins C6 (50), C7 (111, subunit of the acetylcholine receptor (79). Since mild reducing and C9 (76) have no free sulfhydryl groups, in agreement agents are released from target cells (erythrocytes) attacked with aneven number of cysteine residues,all cysteineresidues by the terminal complementcomplex, the 2 cysteine residues are assumed to be cross-linked to one another. Moreover, 2643 and 265F are likely to be involved in the cysteine bond individual modules containing an even number of conserved exchange with a second C9 molecule within the human C5bcysteine residues are supposed tohave disulfide linkages only 9 complex, resulting in the formation reported previously of within the same module, since all types of C6 modules occur disulfide-linked C9 homodimers within the human C5b-9m independently in several proteins. Eight out of the nine C6 complex (80,81). The arrangement of disulfide linkages between conserved modules have an even number of cysteine residues, 52 out of 64 cysteine residues of complement C6 are found at conserved cysteine residues within modules can only be inferred for the positions within homologous modules. A fourth unique cys- LDL receptor class B and the SCRmodules by analogy to the teine pair (Cys-1 andCys-44) is found in the amino-terminal known cysteine linkages in epidermal growth factor (82) and in &glycoprotein I (59). The 6 cysteines of the LDL receptor TSP I module of C6, and an additional fifth cysteine pair (Cys-841 and Cys-852) is present in FIM2of C6 and C7 and class B modules are connected to one another ain1to 3, 2 to in the FIMof factor I as compared with FIM1 of C6 and C7 4, and 5 to 6 pattern and the 4 cysteines of SCR modules in (Fig. 7). Bothcysteine pairs areprobably linked to each other a 1 to 3 and 2 to 4 pattern. in the respective modules. Only the TSP I module at the Since the molecular size of the modules described above carboxyl terminus of C6, C7, C8, and trout C9 has an odd appearsto be toosmalltorepresent acompleteglobular number of cysteines, namely 50. One of the 5 cysteines of this domain, as observed in the electron microscope, we assume module (Fig. 5, dashed line), therefore, must be linked to 1 of that two or more adjacent modules are combined to form one the 5 conserved cysteine residues located in themiddle portion distinct globular domain. The four modules at the carboxyl of C6, C7, C8a, C8& and troutC9. terminus of C6 probably form one domain that can be cleaved The cysteine linkages among the remaining 8 cysteines in off by V8 protease. Another distinct domain may be comprised C6 can be inferred from structure comparisons with the otherof the LDL receptor class B and the TSP I modules, since terminal complement proteins and from protease digestion one cysteine bond appears to span from the amino terminus results available for C6 (this paper),C7 (11), andC9 (16) (Fig. of the LDL receptor class B module to the TSP I module. 5). Cleavage of C9 with thrombin or chymotrypsin splits C9 The number of globular domains in terminal complement into two fragments that are not disulfide-linked, indicating proteins, however, is not known and is controversal (17, 83). first that Cys-378 is linked to Cys-399 (C6 numbering) and From our electronmicroscopical studies, onemay suggest the second that Cys-159 is linked to Cys-197 in C9. The 4 cysteine existence of four globular domains in C6. Definitive identifiresidues 159, 197, 378, and 399 are strictly conserved among cation of discrete globular domains will require more extensive all terminal complement proteins, strongly suggesting that protease digestion experiments and separation of independent the disulfide linkages are conserved as well. By comparing entities under nondenaturing conditions in order to distinhuman and mouse CB with human C8p and trout C9 (after guish cleavages within surface loops of individual domains J.-A. Haefliger, J. Ts’chopp,N. Vial, and D. E. Jenne, unpublished data.
A “C” was inserted in the cDNA sequence of trout C9 after position 1689 (EMBL identification code, SGC9R).
Structure of Complement C6
18050
from cleavages in the spacerregion between two domains. 7. Jenne, D., and Tschopp, J. (1989) Proc. Natl. Acad. Sci. U. S. A., in press In the present study we have, for the first time, identified 8. Kolb, W. P., and Kolb, L. M. (1983) Biochemistry 2 2 , 496-504 a domain in aterminal complement proteinwhich specifically 9. Lundwall, A. B., Wetsel, R. A., Kristensen, T., Whitehead, A. S., interacts with another component, C5b, within the complex. Woods, D.E., Ogden, R. C., Colten, H. R., and Tack, B. F. The isolated 34-kDadomain of C6 has the same binding (1985) J. Biol. Chem. 260,2108-2112 propertiesasintact C6 and associateswith nascent C5b. 10. Wetsel, R. A., Lemons, R. S., Le Beau, M. M., Barnum, S. R., Noack, D., and Tack, B. F. (1988) Biochemistry 2 7 , 1474-1482 When intact C6 is bound to nitrocellulose, the assembly of the terminal complement complex proceeds, where C6 has 11. DiScipio, R. G., Chakravarti, D. N., Muller-Eberhard, H. J., and Fey, G . H. (1988) J. Biol. Chem. 2 6 3 , 549-560 been blotted, and leads to the exposure of neoepitopes that 12. Rao, A. G., Howard, 0. M. Z., Ng, S. C., Whitehead, A. S., Colten, are characteristicof unfolded C9. The carboxyl-terminalfragH. R., and Sodetz, J. M. (1987) Biochemistry 26,3556-3564 ment of C6, however, fails to trigger the assembly of the full 13. Howard, 0.M. Z., Rao, A. G., and Sodetz, J. M. (1987) Biochemistry 26,3565-3570 terminal complement complex. This result is notunexpected, since stable association of C7 with nascent C5b6 complexes 14. Haefliger, J.-A., Tschopp, J.,Nardelli, D., Wahli, W., Kocher, H.-P., Tosi, M., and Stanley, K. K. (1987) Biochemistry 2 6 , presumably requires additional binding sites in other portions 3551-3556 of the C6 molecule. 15. DiScipio, R. G., Gehring, M. R., Podack, E. R., Kan, C. C., Hugli, Mapping theC5b-bindingsitetothecarboxyl-terminal T . E., and Fey, G. H. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 7298-7302 region of complement C6 is consistent with structural and 16. Stanley, K. K.,Kocher, H. P., Luzio, J. P.,Jackson, P., and biochemical features of complement C6 andtheterminal Tschopp, J. (1985) EMBO J . 4 , 375-382 complement complex. The carboxyl-terminal domain of C6 is 17. Stanley, K. K., and Herz, J. (1987) EMBO J. 6 , 1951-1957 composed of two tandem modules, i.e. two SCRs and two 18. Sbinkai, Y., Takio, K., and Okumura, K. (1988) Nature 3 3 4 , FIMs. Both typesof modules appear tobe good candidates to 525-527 function as the C5b recognition site in C6 for the following 19. Lichtenheld, M. G., Olsen, K. J., Lu, P., Lowrey, D. M., Hameed, A., Hengartner, H., and Podack, E. R. (1988) Nature 335,448reasons. SCRs have been shown to bind specifically to C5b 45 1 homologues C3b and C4b (71). On the other hand, factor I, a 20. Lowrey. D. M., Aebischer, T., Olsen, K., Lichtenheld, M., Rupp, plasma protease with high substrate specificity, is known to F., and Podack, E. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 8 6 , inactivate C3b in cooperation with factor H, which solely 247-251 consists of SCRs (69, 70). Thus, it appears possible that the 21. Kwon, B. S., Wakulchik, M., Liu, C.-C., Persechini, P. M., Trapani, J. A,, Haq, A. K., Kim, Y., and Young, J. D. E. (1989) FIM located on theheavy chain of factor I helps inrecognizing Biochem. Biophys. Res. Commun. 1 5 8 , l - 1 0 specifically the protease substrate C3b. It will be interesting 22. Tabor, S., and Richardson, C. C. (1987) Proc. Natl. Acad. Sci. U. to determine which of these two modules in the carboxylS. A . 84,4767-4771 terminal domain of C6 actually mediates binding to C5b or 23. Podack, E. R., Kolb, W. P., Esser, A. F., and Muller-Eberhard, H. J . (1979) J. Immunol. 1 2 3 , 1071-1077 whether more than one binding site exists in C6. Although both modules also occur in complement C7, the 24. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 initial binding of C6 to nascent C5b appears to be highly 25. Laemmli, U. K. (1970) Nature 227,680-685 26. Matsudaira, P. (1987) J. Biol. Chem. 2 6 2 , 10035-10038 specific. C5b-C7 complexes (data not shown) were not gen- 27. Edman, P., and Begg, G. (1967) Eur. J. Biochem. 1, 80-91 erated from zymosan-activated or C6-deficient serum in our 28. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory, assay. The presence of structurally very similar modules in Cold Spring Harbor, NY C6 and C7 obviously does not imply functional equivalence. 29. Deininger, P. L. (1983) Anal. Biochem. 129,216-223 Sequence changes within themodules may have resulted ina 30. Sanger,F.,Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. functional specialization of ancestrallyrelated modules to Acad. Sci. U. S. A. 7 4 , 5463-5467 direct the interaction between different complement compo- 31. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N. (1985) Science 230,1350-1354 nents with one another. In this way, the highly specific order of the assembly processamong terminal complement proteins32. Devereux, J., Haeberli, P., and Smithies, 0. (1984) Nucleic Acids Res. 12,387-395 may have evolved, making each componentabsolutely neces- 33. Higgins, D. G., and Sharp, P. M. (1989) CABIOS 5 , 151-153 sary for the formation of the lytic complex on lipid mem- 34. Feng, D. F., and Doolittle, R. F. (1987) J . Mol. Evol. 25,351-360 branes. 35. Davhoff, M. 0.. Barker, W. C., and Hunt, L. T. (1983) Methods.
;
Acknowledgments-We wish to thank M. Adrian and J.Dubochet for electron microscopy of vitrified C6; S. Schafer for outstanding technical assistance; Z. Freiwald for help in preparing figures; Dr. G. Kitten for readingthe manuscript;Dr. P. J. Spath, Bern,for providing C6-deficient serum; Dr. T. E. Mollnes, Oslo, andDr. D. BitterSuermann, Hannover,for the generous gift of monoclonal antibodies; Dr. C. Nager, Basel, for his advice concerning the UWGCG and ALIGN programs; and Drs. D. G. Sharp and P. M. Higgins, Dublin, for sending the CLUSTAL microcomputer software. REFERENCES 1. Muller-Eberhard, H.J. (1988) Annu. Reu. Biochem. 57,321-347 2. Tschopp, J., and Jongeneel, C . V. (1988) Biochemistry 27,26412646 3. Bhakdi, S., and Tranum-Jensen, J. (1987) Reu. Physiol. Biochem. Pharmacol. 1 0 7 , 147-223 4. Kolb, W. P., and Muller-Eberhard, H. J. (1975) J . Exp. Med. 1 4 1 , 724-735 5. Jenne, D., and Stanley, K. K. (1985) EMBO J. 4 , 3153-3157 6. Murphy, B. F., Kirszbaum, L., Walker, I. D., and d'Apice, A. J. F. (1988) J . Clin. Inuest. 8 1 , 1858-1864
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