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*
Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia, Australia; and
Department of Molecular Biology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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fH binds C3b and controls the alternative pathway of C in two ways. Firstly, it accelerates the spontaneous slow decay of the alternative pathway C3 convertase (C3bBb) by displacing factor B (decay-acceleration) (5, 6). Second, it acts as a cofactor for the factor I-mediated cleavage of C3b to iC3b, which is unable to participate in the alternative pathway amplification loop (cofactor activity) (7). A C3b binding site has been localized to the N-terminal segment of fH, with at least three SCRs including SCRs 2 and 3 required for full cofactor activity (8, 9) and SCRs 1 through 4 for decay-acceleration (10). Additional C3b binding sites have been identified in fH, but their functional significance is unknown (11).
The C-regulatory functions of fH are modified by its binding to polyanions such as heparin and sialic acid (12, 13, 14). Surfaces rich in sialic acid such as sheep erythrocytes do not normally activate C, but can be converted to activators by the removal of sialic acid with neuraminidase (15). The ratio of fH to factor B (fB) binding has been shown to determine whether or not the alternative pathway is activated, and the fH-to-fB binding ratio is postulated, in turn, to be affected by surface sialic acid (16).
We have recently shown that SCR 7 of fH contains a major heparin
binding site: a recombinant protein consisting of the seven N-terminal
SCRs (H7) bound to heparin, whereas the N-terminal six-SCR protein did
not. Moreover, a nine-SCR N-terminal construct (H9) bound heparin, but
its SCR 7 deletion mutant (H9
7) did not bind. We also found that at
least one heparin binding site other than SCR 7 exists in fH, but in
contrast to an earlier report (17) we did not identify such a site in
SCR 13, as a double deletion of SCRs 7 and 13 from fH (H20713)
retained heparin binding (18).
The fH-related (FHR) proteins, present in both human and mouse sera, were initially discovered by their cross-reactivity with anti-fH Abs (19). Four FHR proteins have been identified to date, and their cDNA sequences have been published (19, 20, 21, 22, 23, 24). At present, their functions have not been fully described, but FHR-1, -2, and -4 have been found to associate with plasma lipoproteins, suggesting a general role as constituents of lipoproteins (24, 25). FHR-3 and FHR-4 each possesses five SCRs with amino acid sequences similar to SCRs 6, 7, 8, 19, and 20 and to SCRs 6, 8, 9, 19, and 20 of fH, respectively (20, 24). The heparin-binding characteristics of these proteins are therefore of great interest, as they differ in their possession of an SCR similar to SCR 7 of fH, yet both contain SCRs similar to fH SCRs 19 and 20. SCRs similar to SCRs 19 and 20 of fH are present in all of the FHR proteins, and it has been postulated that these SCRs may mediate important biologic functions (20).
In this paper, we identify the second heparin binding site in fH, using truncation and deletion mutants of fH, and determine the heparin-binding characteristics of recombinant FHR proteins. Attachment of SCR 20 to the first five SCRs of fH converted it from a nonbinding to a heparin-binding protein. The heparin-binding characteristics of FHR-3 and -4 proteins correlate with the possession of an SCR similar to SCR 7 of fH.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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cDNA encoding SCRs 1 through 15 (H15) and an SCR 7 deletion of
full-length fH (H207) were prepared and cloned into the BSR
EN
eukaryotic expression vector as previously described (18).
XhoI and XbaI restriction sites were incorporated
at the 5' and 3' terminus, respectively.
C-terminal truncated proteins from which SCR 7 had been deleted were
produced utilizing a unique PpuMI site at nucleotide 2072 of
the fH sequence. Digesting BSREN-H207 with PpuMI and
XbaI released a fragment corresponding to SCRs 11–20. When
this was replaced with truncated cDNA encoding SCRs 11–15, 11–18, or
11–19, cDNA encoding H157, H187 or H197, respectively, was
produced. cDNA encoding SCRs 11–15 originated from BSREN-H15
digested with PpuMI and XbaI. cDNA encoding SCRs
11–18 and 11–19 was prepared by PCR using Vent polymerase (New
England Biolabs, Beverly, MA) from the BSREN-H20 template. The
forward primer was designed to anneal to nucleotides 2021–2040 in SCR
11, 5' to the PpuMI site. Reverse primers annealed to the
C-termini of SCRs 18 and 19 and were selected on the basis of the SCR
junction analysis by Zipfel and Skerka (20). These primers also
incorporated stop and XbaI sites. After digestion with
PpuMI and XbaI, the PCR products were then cloned
into BSREN-H207.
A mutant fH protein consisting of SCRs 16–20 (H16–20) was prepared by
ligating cDNA encoding the fH signal peptide via a MluI
restriction site. This was achieved by incorporating an XhoI
site into the fH signal forward primer, a MluI site into the
fH signal reverse and the H16–20 forward primers, and an
XbaI site in the SCR 20 reverse primer. The PCR product
encoding the signal peptide was restricted with XhoI and
MluI and the product encoding SCRs 16–20 with
MluI and XbaI. The digested PCR products were
then ligated into the XhoI and XbaI sites of
BSREN in a three-way ligation reaction.
cDNA encoding SCRs 18–20 was amplified using a forward primer that
annealed to the beginning of SCR 18 and introduced a MluI
restriction site. The same SCR 20 reverse primer described above was
used, and the PCR product was cloned into the MluI and
XbaI sites of BSREN-H16–20.
SCR 20 was attached to SCRs 1–5 (H5 + 20) by splice overlap extension PCR. cDNA encoding SCRs 1–5 and SCR 20 was amplified in the first and second primary reactions, respectively. The forward primer for the second primary reaction contained nucleotides complementary to the last 15 nucleotides of SCR 5 adjacent to the first 18 nucleotides of SCR 20. The secondary reaction contained the primary PCR products and the forward and reverse primers used to amplify full-length fH, containing XhoI and XbaI restriction sites. cDNA constructs were confirmed by DNA sequencing.
Chinese hamster ovary cell-expressed proteins
CHO cells were transfected with 5 µg cDNA, 35 µl Lipofectamine (Life Technologies, Gaithersburg, MD), and 1.5 ml Opti-MEM serum-free medium (Life Technologies) per 25-cm2 cell culture dish. Ham’s F12 medium (Trace Biosciences, Castle Hill, New South Wales, Australia) supplemented with 0.5% FCS and 250 µg/ml G418 (Life Technologies) was used for selection and maintenance of stable transfectants. Cell supernatants were harvested twice weekly, clarified by centrifugation, and stored at -70°C.
Baculovirus-expressed proteins
Recombinant human FHR-3 and -4 and SCRs 19–20 of fH (H19–20) were prepared in the pBSV-8His baculovirus expression system as previously described (26). Eight C-terminal histidine residues were included and used to purify expressed proteins by Ni2+-NTA-agarose affinity chromatography. An enterokinase site incorporated immediately N terminal to these His residues allowed their removal with EnterokinaseMax (Invitrogen, San Diego, CA). The His residues were removed because it was found that His tagging weakly affected heparin-binding characteristics (data not shown).
Western blotting
The correct identity of expressed proteins was confirmed by SDS-PAGE and Western blotting. Proteins were electrophoresed under nonreducing conditions and transferred to nitrocellulose. Polyclonal goat Ab to human fH (Calbiochem, San Diego, CA) followed by donkey anti-goat Ab conjugated to horseradish peroxidase (Silenus, Hawthorn, Australia) was used to detect most proteins. FHR-3 and FHR-4 were detected by polyclonal Ab produced in rabbits, followed by sheep anti-rabbit Ab conjugated to horseradish peroxidase (Silenus). Proteins were detected with the enhanced chemiluminescence system (Amersham, Buckinghamshire, U.K.). All proteins migrated on SDS-PAGE according to their predicted m.w. and degree of glycosylation.
Heparin-agarose affinity chromatography
All heparin-binding experiments used 50 mM phosphate buffer, pH 7.4, for dialysis or dilution of samples and equilibration and washing of the columns (27). Ten to twenty milliliters of transfected CHO cell culture supernatant or 5 to 10 µg of diluted baculovirus-expressed protein was applied to 1 x 1 cm heparin-agarose columns (Pierce, Rockford, IL). The sample was passed over the column at least five times and a sample of the fall-through collected. The column was washed with >40 ml of buffer, and 2.5-ml fractions were collected for testing. Bound protein was then eluted by a linear salt gradient to 300 mM sodium chloride-phosphate buffer with 2-ml fractions collected. An automated fraction collector and a flow rate of 1 ml/min was used for all experiments. Conductivity was measured at 24°C, using a portable meter (Activon model 301, Sydney, Australia), and a consistent salt gradient was obtained for all experiments. Columns were reconstituted by washing afterward with 1 M NaCl, then stored in 50 mM phosphate buffer with 0.02 M sodium azide.
All experiments were performed at least three times, and reproducible results were obtained in all cases.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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7 and H157
SCR 7 was shown in a previous report to contain a heparin binding
site (18). However, an SCR 7 deletion mutant of fH (H207) still
bound to heparin and eluted in a linear salt gradient at approximately
150 mM (Fig. 1
). As H15 also binds to
heparin (18), we first prepared a deletion of SCR 7 from H15 (H157)
to assess whether a second heparin binding site existed in SCRs
1–15.
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7 expressed well and migrated slightly faster on SDS-PAGE than
H15 did (data not shown). H157 did not bind heparin, with a similar
amount of protein seen in the start and fall-through fractions.
Moreover, the small amount of protein remaining associated with the
column was washed off in 50 mM of phosphate equilibration buffer. No
protein eluted from the column in the linear salt gradient. Figure 2 shows the heparin-binding and elution
profiles of H15 and H157, demonstrating the clear difference in
behavior of the two proteins. These results show that SCR 7 contains
the only heparin binding site within SCRs 1–15.
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The binding of H207 to heparin and the lack of binding of
H157 together indicate that a second heparin binding site is located
in SCRs 16–20 of human fH. To investigate this possibility, we
prepared a construct in which the fH signal sequence was attached to
cDNA encoding SCRs 16–20. An 
40-kDa protein was identified by
Western blot in supernatants of transfected CHO cells. Heparin binding
analysis showed that H16–20 bound to immobilized heparin and was not
washed off in 50 mM phosphate buffer. H16–20 eluted in a lower
concentration of salt than H157 (Fig. 3). These results indicate that a second
heparin binding site resides within SCRs 16–20, and that this site may
be of lower affinity than that contained in SCR 7.
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To further localize the second heparin binding site, we next
produced SCR 7 deletion mutant proteins of SCRs 1–18 and 1–19
(H187 and H197).
Expression of the proteins was determined by Western blot with both
proteins migrating on SDS-PAGE at their predicted m.w. In contrast to
H207, neither H187 nor H197 demonstrated significant binding
to heparin. A small amount of protein associated with the
heparin-agarose affinity column, but was washed off in 50 mM of
phosphate buffer (Fig. 4).
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The above results indicate that SCR 20 is an essential component
of the second heparin binding site, but do not address whether SCR 20
alone is sufficient for heparin binding. Recombinant proteins
consisting of SCRs 19 and 20 (H19–20) and SCRs 18–20 (H18–20) were
prepared. H19–20 did not bind heparin, because protein was present in
the fall-through and wash fractions and none was eluted (Fig. 6). In contrast, H18–20 was not detected
in the fall-through or wash fractions, and bound protein eluted in the
salt gradient (Fig. 6). Similar results were found for a protein
containing SCRs 17–20 (data not shown). Therefore, while SCR 20 is an
essential component of the second heparin binding site, at least two
adjacent SCRs are required. The adjacent SCRs may be nonspecific, since
H5 + 20 also bound to heparin.
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Human FHR-3 and -4 are both composed of five SCRs, with each SCR similar to certain SCRs of fH. SCRs 1–3 of FHR-3 and FHR-4 share partial identity with fH SCRs 6, 7, and 8 and SCRs 6, 8, and 9, respectively. SCRs 4 and 5 of both FHR-3 and FHR-4 are almost identical to each other and have partial similarity with SCRs 19 and 20 of fH (20, 24). Therefore, we used these proteins as "natural mutants" of fH and examined their heparin-binding characteristics. These results were then correlated with the amino acid sequences of SCRs 7 and 20 in an attempt to identify potential heparin binding sites. FHR-3 was expected to bind heparin because its third SCR is so similar to SCR 7 of fH. Binding of FHR-4 to heparin would most likely depend on whether there is sufficient similarity between SCR 5 of FHR-4 and SCR 20 of fH to preserve the heparin binding site.
The heparin-binding characteristics of FHR-3 and FHR-4 are shown in
Figure 7. As expected, FHR-3 binds to
heparin. FHR-4 does not bind, indicating that its SCR 5 is not
sufficiently similar to SCR 20 of fH to result in heparin binding.
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A schematic representation of the proteins produced and analyzed
in this study is shown in Figure 8. SCRs
are numbered in the figure on the basis of similarity to native human
fH. The heparin-binding properties of each construct are also
shown.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A series of recombinant mutant proteins was initially expressed in transfected CHO cells. Recombinant fH (H20) produced in this system has full cofactor activity and migrates on SDS-PAGE in a fashion identical to the native protein (18). This suggests that transfected CHO cells produce correctly glycosylated recombinant proteins.
Deleting SCR 7 from H15 (H157) resulted in the loss of heparin
binding, while H207 retained its binding to heparin (Figs. 1
and 2).
These findings identified a second heparin-binding domain and localized
it to SCRs 16–20. H16–20 was produced by attaching the fH signal
sequence to cDNA encoding SCRs 16–20 in the expression vector
BSREN. This approach has been used previously in our laboratory to
produce recombinant N-terminal truncations of fH (8), domains of the
HIV gp120 glycoprotein, and recombinant rubella virus proteins
(unpublished data). H16–20 bound heparin, but eluted earlier in a salt
gradient (Fig. 3). Therefore, it is possible that the binding site in
this region is of lower affinity.
We next prepared truncations of the C-terminal SCRs from H207 to
determine the location of the second heparin binding site. In contrast
to H207, neither H187 nor H197 bound to heparin (Fig. 4).
These findings indicate that SCR 20 contains an essential component of
the second heparin binding site of fH. To confirm that it contains a
heparin binding site, SCR 20 was attached to a non-heparin-binding
protein. The addition of SCR 20 to the N-terminal five SCRs (H5+20)
generated a heparin-binding protein (Fig. 5), conclusively
demonstrating that SCR 20 contains a heparin binding site.
These results indicate that SCR 20 is an essential part of the second
heparin binding site, but the role of adjacent SCRs remained to be
determined. Therefore, the heparin-binding characteristics of SCRs 19
and 20 construct (H19–20) and SCRs 18–20 (H18–20) were examined
(Fig. 6). H19–20 did not bind heparin, whereas H18–20 did. Therefore,
adjacent SCRs in spacer or structural roles are required for heparin
binding. SCR 20 will mediate heparin binding even when attached to
nonspecific SCRs, as shown by the binding of H5 + 20. This
indicates that the second heparin binding site consists of SCR 20 and
at least another two nonspecific SCRs. Three to four SCRs are also
necessary for maximal C3 regulatory activity of RCA proteins including
CR1 (31) and fH (8, 9). Within the three- to four-SCR unit, one to two
SCRs are essential, with the flanking SCRs required for full functional
activity.
We took advantage of the similarity of the FHR proteins FHR-3 and
FHR-4, both of which were cloned and expressed in a baculovirus system.
FHR-3 and FHR-4 contain SCRs similar to particular SCRs of fH. They
differ in that only FHR-3 contains an SCR similar to SCR 7 of fH; SCR 2
of FHR-3 has 88% amino acid identity to SCR 7 of fH. SCRs 4 and 5 of
FHR-3 are almost identical to the comparable SCRs of FHR-4, with a
total of only four amino acids different between the four SCRs (24).
Therefore, finding that FHR-3 binds to heparin and that FHR-4 does not
(Fig. 7) suggests that SCR 2 of FHR-3 is likely to be responsible for
the binding of FHR-3. The highly conserved amino acid sequences of SCR
7 of human fH and SCR 3 of human FHR-3 make it difficult to identify
putative heparin-binding domains within these SCRs. Using
two-dimensional nuclear magnetic resonance (2D NMR), a "hypervariable
loop" within each SCR was postulated to be the region responsible for
specific ligand binding (4, 33, 34, 35). The hypervariable loop of SCR 7 of
fH consists of the sequence (H/Y)GRK (the first amino acid is the site
of H/Y polymorphism) (36, 37) with YGRK in SCR 3 of human FHR-3 (20).
Despite the fact that this site contains two or three basic amino
acids, it would be surprising if it alone is responsible for heparin
binding, because in other proteins the heparin-binding domain may be
spread over a discontinuous sequence of many amino acids (38). It would
also be interesting to examine whether a heparin-binding domain exists
in SCR 7 of murine and bovine fH, as each shares only 57% identity
with the human counterpart. It may then be possible to identify likely
amino acid sequences responsible for heparin binding and to determine
the functional significance of the hypervariable loop of SCR 7. The
removal of SCRs 7 and 20 from fH completely abrogates its capacity to
bind heparin, which rules out a significant contribution from SCR 13 in
our assay system. It is surprising that no heparin-binding activity was
observed for SCR 13 of fH, because based on the model of Barlow et al.
(3), the hypervariable loop of SCR 13 contains many basic amino acids
(HLKNKKEF). This suggests that the hypervariable loop model may not be
applicable to every SCR.
As demonstrated by the lack of binding of FHR-4, the heparin binding site in SCR 20 of fH is not conserved in SCR 5 of FHR-4. The amino acid sequence of SCR 20 of fH (37) was analyzed for clusters of basic residues not present in SCR 5 of FHR-4 (24). No such areas were apparent, making it difficult to identify putative heparin binding sites within SCR 20 of fH.
We plan to use H197 to establish the role that fH plays in
regulating C activation by sialic acid-rich bacteria and cells.
Important pathogens that may utilize fH binding as a way of avoiding
alternative pathway activation include Streptococcus
agalactiae (39, 40), Neisseria meningitidis,
Neisseria gonorrhoeae (41, 42, 43, 44), and Escherichia
coli K1 (45). By comparing the protective effects against C
deposition of H197 and H20, it will be possible to test the
hypothesis that the heparin/sialic acid-binding capacity of fH is
essential to self/nonself recognition by the alternative pathway. C
activation is controlled on host cells to which fH is bound (17), and
the degree of fH binding to cell surfaces is related to the amount of
membrane-associated sialic acid (12, 13, 14). Sheep erythrocytes, which are
rich in sialic acid, may be converted by neuraminidase treatment from
nonactivators to activators of the alternative pathway, accompanied by
a reduction in fH binding (15). Therefore, it would be expected that
H197 added to an alternative pathway lacking fH would not protect
sheep erythrocytes from C-mediated lysis. Such an approach would
examine the possibility that other regulators, such as those found on
sheep erythrocytes (46), are also playing an important
C-regulatory role.
In summary, we have identified the second heparin binding site of human
fH and produced a mutant fH containing all SCRs except those required
for heparin binding. This molecule (H197) will be invaluable for
studying the significance of polyanion binding to the C-regulatory
functions of fH.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Timothy K. Blackmore, at his current address, The Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia 5042. E-mail address:
3 Abbreviations used in this paper: fH, factor H; CHO, Chinese hamster ovary; FHR-3 and -4, factor H-related proteins 3 and 4; H20, recombinant factor H; HT, N-terminal tryptic fragment of fH; RCA, regulators of complement activation; SCR, short consensus repeat.
Received for publication May 2, 1997. Accepted for publication December 1, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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J. L. McRae, T. G. Duthy, K. M. Griggs, R. J. Ormsby, P. J. Cowan, B. A. Cromer, W. J. McKinstry, M. W. Parker, B. F. Murphy, and D. L. Gordon Human Factor H-Related Protein 5 Has Cofactor Activity, Inhibits C3 Convertase Activity, Binds Heparin and C-Reactive Protein, and Associates with Lipoprotein J. Immunol., May 15, 2005; 174(10): 6250 - 6256. [Abstract] [Full Text] [PDF]
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 Z.-Z. Cheng, M. J. Corey, M. Parepalo, S. Majno, J. Hellwage, P. F. Zipfel, R. J. Kinders, M. Raitanen, S. Meri, and T. S. Jokiranta Complement Factor H as a Marker for Detection of Bladder Cancer Clin. Chem., May 1, 2005; 51(5): 856 - 863. [Abstract] [Full Text] [PDF]
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 A. Saito and H. Munakata Factor H Is a Dermatan Sulfate-Binding Protein: Identification of a Dermatan Sulfate-Mediated Protease That Cleaves Factor H J. Biochem., February 1, 2005; 137(2): 225 - 233. [Abstract] [Full Text] [PDF]
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 V. K. Ganesh, S. A. Smith, G. J. Kotwal, and K. H. M. Murthy Structure of vaccinia complement protein in complex with heparin and potential implications for complement regulation PNAS, June 15, 2004; 101(24): 8924 - 8929. [Abstract] [Full Text] [PDF]
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A. Alitalo, T. Meri, T. Chen, H. Lankinen, Z.-Z. Cheng, T. S. Jokiranta, I. J. T. Seppala, P. Lahdenne, P. S. Hefty, D. R. Akins, et al. Lysine-Dependent Multipoint Binding of the Borrelia burgdorferi Virulence Factor Outer Surface Protein E to the C Terminus of Factor H J. Immunol., May 15, 2004; 172(10): 6195 - 6201. [Abstract] [Full Text] [PDF]
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M.-A. Dragon-Durey, V. Fremeaux-Bacchi, C. Loirat, J. Blouin, P. Niaudet, G. Deschenes, P. Coppo, W. Herman Fridman, and L. Weiss Heterozygous and Homozygous Factor H Deficiencies Associated with Hemolytic Uremic Syndrome or Membranoproliferative Glomerulonephritis: Report and Genetic Analysis of 16 Cases J. Am. Soc. Nephrol., March 1, 2004; 15(3): 787 - 795. [Abstract] [Full Text] [PDF]
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H. Jarva, J. Hellwage, T. S. Jokiranta, M. J. Lehtinen, P. F. Zipfel, and S. Meri The Group B Streptococcal {beta} and Pneumococcal Hic Proteins Are Structurally Related Immune Evasion Molecules That Bind the Complement Inhibitor Factor H in an Analogous Fashion J. Immunol., March 1, 2004; 172(5): 3111 - 3118. [Abstract] [Full Text] [PDF]
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A. Richards, E. J. Kemp, M. K. Liszewski, J. A. Goodship, A. K. Lampe, R. Decorte, M. H. Muslumanogglu, S. Kavukcu, G. Filler, Y. Pirson, et al. Mutations in human complement regulator, membrane cofactor protein (CD46), predispose to development of familial hemolytic uremic syndrome PNAS, October 28, 2003; 100(22): 12966 - 12971. [Abstract] [Full Text] [PDF]
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 R. Oksjoki, H. Jarva, P. T. Kovanen, P. Laine, S. Meri, and M. O. Pentikainen Association Between Complement Factor H and Proteoglycans in Early Human Coronary Atherosclerotic Lesions: Implications for Local Regulation of Complement Activation Arterioscler Thromb Vasc Biol, April 1, 2003; 23(4): 630 - 636. [Abstract] [Full Text] [PDF]
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J. Hellwage, T. S. Jokiranta, M. A. Friese, T. U. Wolk, E. Kampen, P. F. Zipfel, and S. Meri Complement C3b/C3d and Cell Surface Polyanions Are Recognized by Overlapping Binding Sites on the Most Carboxyl-Terminal Domain of Complement Factor H J. Immunol., December 15, 2002; 169(12): 6935 - 6944. [Abstract] [Full Text] [PDF]
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M. K. Pangburn Cutting Edge: Localization of the Host Recognition Functions of Complement Factor H at the Carboxyl-Terminal: Implications for Hemolytic Uremic Syndrome J. Immunol., November 1, 2002; 169(9): 4702 - 4706. [Abstract] [Full Text] [PDF]
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T. G. Duthy, R. J. Ormsby, E. Giannakis, A. D. Ogunniyi, U. H. Stroeher, J. C. Paton, and D. L. Gordon The Human Complement Regulator Factor H Binds Pneumococcal Surface Protein PspC via Short Consensus Repeats 13 to 15 Infect. Immun., October 1, 2002; 70(10): 5604 - 5611. [Abstract] [Full Text] [PDF]
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T. Areschoug, M. Stalhammar-Carlemalm, I. Karlsson, and G. Lindahl Streptococcal beta Protein Has Separate Binding Sites for Human Factor H and IgA-Fc J. Biol. Chem., April 5, 2002; 277(15): 12642 - 12648. [Abstract] [Full Text] [PDF]
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H. Jarva, R. Janulczyk, J. Hellwage, P. F. Zipfel, L. Bjorck, and S. Meri Streptococcus pneumoniae Evades Complement Attack and Opsonophagocytosis by Expressing the pspC Locus-Encoded Hic Protein That Binds to Short Consensus Repeats 8-11 of Factor H J. Immunol., February 15, 2002; 168(4): 1886 - 1894. [Abstract] [Full Text] [PDF]
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J. CAPRIOLI, P. BETTINAGLIO, P. F. ZIPFEL, B. AMADEI, E. DAINA, S. GAMBA, C. SKERKA, N. MARZILIANO, G. REMUZZI, and M. NORIS The Molecular Basis of Familial Hemolytic Uremic Syndrome: Mutation Analysis of Factor H Gene Reveals a Hot Spot in Short Consensus Repeat 20 J. Am. Soc. Nephrol., February 1, 2001; 12(2): 297 - 307. [Abstract] [Full Text]
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 S. A. Smith, N. P. Mullin, J. Parkinson, S. N. Shchelkunov, A. V. Totmenin, V. N. Loparev, R. Srisatjaluk, D. N. Reynolds, K. L. Keeling, D. E. Justus, et al. Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense J. Virol., June 15, 2000; 74(12): 5659 - 5666. [Abstract] [Full Text]
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S. Junnikkala, T. S. Jokiranta, M. A. Friese, H. Jarva, P. F. Zipfel, and S. Meri Exceptional Resistance of Human H2 Glioblastoma Cells to Complement-Mediated Killing by Expression and Utilization of Factor H and Factor H-Like Protein 1 J. Immunol., June 1, 2000; 164(11): 6075 - 6081. [Abstract] [Full Text] [PDF]
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H. Jarva, T. S. Jokiranta, J. Hellwage, P. F. Zipfel, and S. Meri Regulation of Complement Activation by C-Reactive Protein: Targeting the Complement Inhibitory Activity of Factor H by an Interaction with Short Consensus Repeat Domains 7 and 8-11 J. Immunol., October 1, 1999; 163(7): 3957 - 3962. [Abstract] [Full Text] [PDF]
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A. M. Blom, J. Webb, B. O. Villoutreix, and B. Dahlback A Cluster of Positively Charged Amino Acids in the C4BP alpha -Chain Is Crucial for C4b Binding and Factor I Cofactor Function J. Biol. Chem., July 2, 1999; 274(27): 19237 - 19245. [Abstract] [Full Text] [PDF]
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T. S. Jokiranta, J. Hellwage, V. Koistinen, P. F. Zipfel, and S. Meri Each of the Three Binding Sites on Complement Factor H Interacts with a Distinct Site on C3b J. Biol. Chem., September 1, 2000; 275(36): 27657 - 27662. [Abstract] [Full Text] [PDF]
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R. Pio, A. Martinez, E. J. Unsworth, J. A. Kowalak, J. A. Bengoechea, P. F. Zipfel, T. H. Elsasser, and F. Cuttitta Complement Factor H Is a Serum-binding Protein for Adrenomedullin, and the Resulting Complex Modulates the Bioactivities of Both Partners J. Biol. Chem., April 6, 2001; 276(15): 12292 - 12300. [Abstract] [Full Text] [PDF]
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