Download Partial characterization of human complement factor H by protein

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.
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