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* Department of Nephrology,
Immunology Research Centre, and
University of Melbourne Department of Medicine, St. Vincent’s Hospital Melbourne, Fitzroy, Victoria, Australia;
Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, Australia; and¶
St. Vincent’s Institute, Fitzroy, Australia
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
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60 aa with 2 overlapping disulfide bonds. Regions of fH responsible for binding glycosaminoglycans such as heparin have previously been mapped to SCRs 7, 20, and possibly 12–14 (3, 4, 5, 6), whereas C-reactive protein (CRP)-binding sites have been localized to SCRs 7 and 8–11 (7). C3b-binding sites are within SCRs 2–3, 6–10, and/or 12–14 and 19–20 (8, 9), and complement-regulatory activities reside within SCRs 1–4 (10, 11, 12). Little is known about the function of FHR-1 and FHR-2, which contain 5 and 4 SCR domains respectively, but both are present in lipoprotein complexes (13, 14, 15). FHR-3 and FHR-4B are highly related and consist of five SCRs. FHR-4A contains nine SCRs and is also closely associated with human triglyceride-rich lipoproteins (16, 17). Despite their sequence similarity, only FHR-3 possesses heparin-binding activity. This is the likely consequence of FHR-3 possessing an SCR with homology to SCR 7 of fH (3). FHR-3 also binds streptococcal M protein, an interaction also mediated via SCR 7 of fH (18). Both FHR-3 and FHR-4B bind to the C3d region of C3b and have been reported to have weak factor I-mediated cofactor activity, although this activity is not independent of fH and remains controversial (19).
FHR-5 is a 65-kDa protein first identified as a component of pathological human glomerular preparations. Unlike the other FHR proteins, which contain 4 or 5 SCRs, FHR-5 consists of 9 SCR domains. SCRs 1 and 2 are homologous to corresponding SCRs from FHR-1 and FHR-2, whereas SCRs 3–7 and 8–9 share significant homology with SCRs 10–14 and 19–20 of fH (1). The gene encoding FHR-5 has been localized to human chromosome 1q32 within the regulators of complement activation (RCA) gene cluster. It is closely linked to the other fH family genes and is situated between FHR-2 and factor XIIIb. Each SCR of FHR-5 is encoded by an individual exon (20).
Although the exact role of FHR-5 in complement regulation is unknown, the protein has been shown to colocalize with C3 in vivo and to bind C3b in vitro in a dose-dependent manner (1). Its deposition in vivo appears widespread in complement-containing glomerular immune deposits and extrarenal sites of complement deposition (21). A prospective study of 100 renal biopsies showed the pattern of FHR-5 deposition to be similar to that of C3 and C5b-9 (22). More recently, FHR-5 and all other FHR proteins have been detected at high levels in middle ear effusion fluid from patients with otitis media (23).
Given the similarity of FHR-5 with fH and its association with complement deposits in vivo, we investigated whether recombinant FHR-5 (rFHR-5) exhibited functions associated with complement regulation, and shared properties with other FHR proteins such as circulation with lipoprotein complexes.
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Materials and Methods |
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E. coli strain DH5
(Bethesda Research Laboratories, Gaithersburg, MD) harboring various SCR plasmid constructs in pPICZ A (Invitrogen, Carlsbad, CA) was propagated in low salt Luria-Bertani broth containing 25 µg/ml Zeocin (Invitrogen), with aeration at 37°C.
FH was purified from plasma as previously described (24), with an additional immunoaffinity chromatography step. Functional activity of fH was confirmed using factor I-mediated cofactor assays as previously described (25).
rFHR-5 was expressed using the baculovirus/insect cell expression system and affinity purified as previously described (1). C3 was isolated from fresh human plasma (26) and C3b was generated by proteolytic cleavage with trypsin as described (27). C3i, also known as C3(H2O), was generated by five freeze/thawing cycles of purified C3.
Construction of rFHR-5 protein fragments
Coding regions comprising SCRs 1–4 and 3–7 of FHR-5 were amplified by PCR and cloned into the yeast expression vector pPICZ A (Invitrogen) downstream of the AOX1 methanol-inducible promoter. The construct designs were based on the reported SCR intron/exon boundaries of FHR-5 (20). PCR, cloning, and transformation into Escherichia coli were conducted according to standard protocols (28). SCRs 1–4 were amplified using primers FHR-5 SCR 1 F (EcoRI) 5'-GGGAATTCGAAGGAACACTTTGTGATT-3' and FHR-5 SCR 4 R (KpnI) 5'-GCGGTACCTCAAACACAAGTGGGTAAAGTTG-3'. SCRs 3–7 were amplified using primers FHR-5 SCR 3 F (PmlI) 5'-GGCACGTGGAAAGGAGAATGTCATGTTCC-3' and FHR-5 SCR 7 R (KpnI) 5'-GCGGTACCTCAAACACAGCGTGGTAATGAT-3'. All PCR-amplified SCR constructs were digested with the appropriate restriction enzymes, cloned into the multiple cloning site of pPICZ A, and transformed into E. coli DH5. Sequence analysis confirmed each construct had the correct sequence.
Expression in Pichia pastoris
Up to 10 µg of SacI-digested pPICZ A DNA containing the various SCR constructs was electroporated into P. pastoris strain X33, and transformants were selected with 100 µg/ml Zeocin. Expression of recombinant proteins was induced for 2–4 days by twice daily addition of 2% methanol, according to the manufacturer’s instructions (Invitrogen).
Purification of recombinant proteins
SCR proteins expressed using the P. pastoris system were purified by immunoaffinity chromatography on CNBr-activated Sepharose (Amersham Biosciences) coupled with rabbit anti-human FHR-5 IgG, prepared by caprylic acid precipitation (29). Briefly, 50–100 ml of P. pastoris supernatant were passed over 5-ml affinity columns several times before extensive washing in PBS, pH 7.4, and elution using 3 M potassium thiocyanate. Eluates were immediately dialyzed against PBS and concentrated by ultrafiltration, and the concentration of proteins was estimated against an FHR-5 standard curve generated by ELISA.
Production of anti-FHR-5 polyclonal Abs
Rabbits were immunized as previously described (30). In brief, 200 µg of purified rFHR-5 in 0.5 ml PBS was emulsified in 1.5 ml CFA and injected s.c. at four to five sites. Animals were boosted twice at 3-wk intervals with 100 µg of rFHR-5 in IFA. One week after the final boost, rabbits were ear bled, and the sera were tested by ELISA and Western blot for activity against rFHR-5 and normal human serum.
Western blot analysis
Recombinant FHR-5 SCR proteins and lipoprotein fractions were separated on 10–12% SDS-PAGE and transferred onto Hybond C+ nitrocellulose (Amersham Biosciences). Native and rFHR-5 proteins were detected using rabbit anti-FHR-5 antiserum (1/2000 v/v) or mouse anti-FHR-5 (K2.254) mAb (Ref. 21 ; 1/2000 v/v) followed by HRP-conjugated goat anti-rabbit IgG (Silenus) or HRP-conjugated sheep anti-mouse IgG (Silenus) (1/2000 v/v), respectively. Proteins were visualized using ECL and exposure to Hyper Film ECL (Amersham Biosciences). To detect human clusterin in lipoprotein complexes, membranes were stripped by washing in 62 mM Tris, 2% SDS, 50 mM 2-ME at 42°C for 20 min, washed in PBS, and reprobed with mouse anti-human clusterin (K2.2G7) mAb (Ref. 21 ; 1/2000 v/v) followed by HRP-conjugated sheep anti-mouse IgG as described above.
Silver staining
12% SDS-PAGE gels were fixed in 30% ethanol, 10% acetic acid solution for 30 min, and rinsed in 20% ethanol and then water for 10 min each. Following sensitization in 0.02% sodium thiosulfate for 1 min and rinsing twice in water, 0.2% silver nitrate was added for 30 min. After rinsing, developer (30 g/L sodium carbonate, 10 mg/L sodium thiosulfate, 0.02% formaldehyde) was added, and the reaction was stopped with a 5% Tris, 2.5% acetic acid solution.
Cofactor assay
The cofactor activity for recombinant FHR-5 vs fH was performed as previously described (25). Briefly, 250 ng of C3b, 1 µg of factor I (Calbiochem-Novabiochem), and varying amounts of fH or rFHR-5 ranging from 100 ng to 10 µg were added in 20 mM phosphate buffer, pH 6.0, to a final volume of 30 µl. Samples with or without fH/rFHR-5 or factor I were included as controls. Reactions were incubated at 37°C for 3 h and then separated under reducing conditions on SDS, 7.5% PAGE and subjected to Western blot analysis using a goat anti-C3c Ab (1/2000 v/v) (Silenus) and HRP-conjugated donkey anti-goat IgG (Silenus).
C3 convertase activity and decay acceleration assays
The effect of rFHR-5 and fH on the alternative pathway C3 convertase was assessed by two methods, a solid phase ELISA-based method developed by Hourcade et al. (31), in which the C3 convertase is generated on a microtiter plate surface, and a fluid phase method, in which C3 convertase activity is monitored by C3a generation in solution (32).
Solid phase method. Microtiter plate wells were coated with 250 ng of purified C3b. Generation of the C3bBb(Ni2+) complex (alternative pathway C3 convertase) was achieved by addition of factor B (400 ng; Calbiochem-Novabiochem) and factor D (25 ng; Calbiochem-Novabiochem) in the presence of 2 mM NiCl2 and 25 mM NaCl in a final volume of 100 µl. Mixtures were incubated for 2 h at 37°C. rFHR-5 or fH was then added, and dissociation of the complexes was monitored at further time points during 30 min. Intact complexes (which had not decayed) on the ELISA plates were detected using goat anti-human factor B Ab (1/5000 v/v) (DiaSorin) followed by HRP-conjugated rabbit anti-goat Ab (1/2000 v/v) (Dako). Sigma Fast OPD substrate (Sigma-Aldrich) was added, and the OD492 was determined.
Fluid phase method. The AP C3 convertase was assembled in PBS by the addition of the following purified complement components: 50 ng C3i; 2 µg of factor B; 10 µg C3; and 12 µl of 0.1 M MgCl2. Dilutions of rFHR-5 or fH were added followed by the addition of 200 ng of factor D (the enzyme required to activate the convertase) to a final volume of 125 µl, and the mixture was incubated at 37°C for 30 min. Controls of purified complement components only (positive control), purified complement components without factor D (negative control), and purified complement components plus BSA (Sigma-Aldrich; irrelevant protein control) were included. C3a generation was measured by ELISA using the Quidel C3a Enzyme Immunoassay kit (Quidel). To measure C3a at time zero, a sample was taken just before the addition of factor D.
Heparin-BSA ELISA
A heparin-binding ELISA was performed by coating 1 µg of purified heparin-BSA (Sigma-Aldrich) or BSA only on Maxisorb ELISA microtiter plate wells (Nunc) overnight at 4°C in the presence of 100 mM bicarbonate buffer, pH 9.5. The samples were blocked with 5% (w/v) skim milk in PBS for 1 h at room temperature (RT) and washed three times in 50 mM phosphate buffer, pH 7.4. Samples (100 µl) of rFHR-5 fragments, rFHR-5 fragments, or negative control protein (BSA) were then added at 10 µg/ml in phosphate buffer and incubated at RT for 1 h. Samples were washed then incubated sequentially for 1 h with mouse anti-FHR-5 (1/2000 v/v) and HRP-conjugated sheep anti-mouse IgG (Silenus) at 1:2000 v/v in 1% skim milk-PBS for 1 h RT. Following washing, Sigma Fast OPD substrate (Sigma-Aldrich) was added, and the OD492 was determined.
Heparin-agarose chromatography
Heparin-agarose chromatography was conducted as previously described (3) with some modifications. Approximately 7.5 µg of rFHR-5, rFHR-5 constructs or fH in 1 ml of 50 mM phosphate buffer, pH 7.4, were applied to a 1-ml heparin-agarose column (Pierce), and the flowthrough was collected and reapplied five times. The column was then washed extensively in phosphate buffer, and bound protein was eluted using a linear salt gradient to 1 M NaCl (1 ml fractions). Initial, flowthrough, wash, and elution samples were analyzed by SDS-PAGE and Western blots using mouse anti-FHR-5, rabbit anti-FHR-5, or sheep anti-fH.
CRP-binding ELISA
One microgram of purified CRP (kindly provided by Dr. Carolyn Mold, University of New Mexico) or gelatin (May and Baker) was coated on Maxisorb ELISA microtiter plates) overnight at 4°C in the presence of gelatin-Veronal buffer (GVB; 5 mM barbitone sodium, 145 mM NaCl, 0.1% gelatin). The wells were washed twice with GVB containing 0.02% v/v Tween 20 and then blocked for 2 h at RT with 1% BSA in GVB. Wells were washed three times, and samples of rFHR-5 or rFHR-5 fragments (100 µl at 0–20 µg/ml) in GVB were added for 1 h at RT. Samples were washed and incubated sequentially for 1 h with rabbit anti-FHR-5 IgG (1/2000 v/v) and HRP-conjugated sheep anti-rabbit IgG (1/2000 v/v) (Chemicon International). Following washing, substrate was added, and the OD492 was determined. Experiments were repeated twice in duplicate, and the mean and SDs were determined.
Preparation and analysis of human lipoprotein complexes
Low density lipoprotein (LDL), very low density lipoprotein (VLDL), high density lipoprotein (HDL), and plasma proteins were isolated from normal human plasma of a fasting individual. LDL and VLDL were isolated by single-spin ultracentrifugation. The plasma was adjusted to a density of 1.21 g/ml with KBr, underlaid below density buffer (0.4 mM EDTA, 0.01% NaN3, 1 mM benzamidine-HCl (pH 7.4), and KBr to give a final density of 1.019 g/ml) and centrifuged at 50,000 rpm in a VTi 50 rotor (Beckman Instruments) for 2 h at 4°C. After centrifugation, LDL (a distinct single band in the midsection of the tube), VLDL (faint band at the top of the tube) and HDL and plasma proteins (the lower most fraction) were removed by needle aspiration. The HDL and plasma protein fraction was then adjusted to 1.21 g/ml and spun in a Ti 90 rotor at 90,000 rpm for 5 h. HDL was aspirated from the top of the tube, and plasma proteins were collected from the bottom. Lipoproteins were run on a native 1% agarose gel (Paragon Electrophoresis System; Beckman Instruments) and lipid stained per the manufacturer’s instructions. Association of FHR-5 or human clusterin (as a positive control protein) with lipoprotein complexes was analyzed following separation of 10% whole plasma, 10% plasma protein fraction, and 50 µg of each lipoprotein fraction (delipidated (32) and nondelipidated) by SDS-PAGE under nonreducing conditions and by Western blot using the anti-FHR-5 mAb or anti-human clusterin mAb as described above.
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Results |
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Like fH, rFHR-5 exhibited cofactor activity for factor I-dependent cleavage of C3b to iC3b, as evident by the cleavage of the C3b '-chain into two fragments of 67 kDa and 43 kDa (Fig. 1). Cofactor activity was dose dependent; and at the highest concentration (10 µg) of rFHR-5, a further cleavage product of 40 kDa (probably part of C3c) was observed.
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The effect of rFHR-5 on alternative pathway C3 convertase activity was determined in fluid and solid phase assays and compared with factor H activity. In the fluid phase, rFHR-5 exhibited significant inhibition of C3 convertase activity as shown by the dose-dependent reduction in C3a generation. Potency of rFHR-5 was lower than fH on a weight-for-weight basis (Fig. 2A). However, no dissociation of the convertase was detected during 30 min in the solid phase assay following addition of up to 10 µg of FHR-5, whereas significant decay of C3bBb(Ni2+) was observed following the addition of 100 ng of fH (Fig. 2B).
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The binding of rFHR-5 to heparin was examined in two assay systems. A heparin-BSA ELISA demonstrated binding of rFHR-5 to heparin-BSA but not to BSA alone (Fig. 3A). Heparin-agarose chromatography was used to confirm this observation and to estimate the relative affinities of rFHR-5 and fH for heparin. A mixture of rFHR-5 and fH was applied to a heparin-agarose column, and the flowthrough, washes, and gradient salt elutions were collected and analyzed by Western blot (Fig. 3B). Both rFHR-5 and fH bound to the heparin agarose column, with neither protein detected in the flowthrough or wash fractions. However, rFHR-5 was eluted later at a higher salt concentration (300 mM), suggesting a higher affinity for heparin than fH, which eluted in 150 mM NaCl.
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To localize domains responsible for particular functions, constructs containing SCR 1–4 or SCR 3–7 of FHR-5 (Fig. 4A) were produced and expressed using P. pastoris. Affinity-purified rFHR-5 protein fragments were subjected to SDS-PAGE and Western blotting using mouse anti-FHR-5 mAb (K2.254) (Fig. 4B). The mAb recognized both SCR1–4 and SCR3–7 fragments, indicating that the epitope recognized by the anti-FHR-5 mAb is located within SCR3–4. The corresponding silver stain shows high purity of both SCR fragments.
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To determine the SCR domains of FHR-5 responsible for heparin binding, SCRs 1–4 and 3–7 were tested by heparin-agarose chromatography. SCR 1–4 did not bind heparin, with all protein being detected in the flowthrough and wash fractions (Fig. 5A). However, SCR 3–7 bound to the heparin column and eluted from 250 mM NaCl (Fig. 5B). Combined, these data indicate that there is a region within SCRs 5–7 that is essential for heparin binding. The involvement of SCRs 3 and 4 in heparin binding cannot be ruled out, but alone they are not sufficient for heparin binding.
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An ELISA was developed to study rFHR-5 binding to CRP. rFHR-5 binding to CRP was dose dependent and saturable (Fig. 6). To determine the region of FHR-5 responsible for CRP binding, rFHR-5 fragments 1–4 and 3–7 were analyzed in the binding assay. The SCR 3–7 fragment also bound in a dose-dependent manner, although with a lower affinity than the complete rFHR-5. The SCR 1–4 fragment, however, showed no specific binding. Thus, FHR-5 binds directly to CRP and binding is partly mediated by SCRs 3–7 with SCRs 5–7 likely to contain the major binding region. This region also mediated heparin binding, suggesting a common or closely related binding site for both ligands. This was further supported by the observation that FHR-5 binding to CRP was totally inhibited by 50 IU of heparin (data not shown).
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To determine whether FHR-5 is associated with lipoprotein complexes, lipoprotein-containing fractions were isolated from normal human plasma and analyzed by Western blot. Fig. 7A demonstrates the purity of lipoprotein fractions following separation by ultracentrifugation. Western blot analysis using anti-human FHR-5 mAb (K2.254) (Fig. 7B) showed that like human clusterin (Fig. 7C), FHR-5 was present in whole plasma, the plasma protein fraction, and the HDL-purified preparation. FHR-5 was not detected in the LDL- or VLDL-purified preparations. This suggests that some FHR-5, like FHR-1, -2, and -4, associates with lipoprotein particles in normal plasma, although the majority probably exists as a free protein.
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The cofactor experiments showed that FHR-5, like fH, possesses fI-dependent cofactor activity cleaving C3b and generating C3' chain fragments of 67 and 43 kDa. In addition, FHR-5 significantly inhibited the activity of the fluid phase C3 convertase; however, FHR-5 displayed no decay acceleration activity in solid phase experiments using C3bBb(Ni2+) convertase complexes constructed on the surface of microtiter plates. This was unlikely to be due to differences in assay sensitivity because fH caused similar decay in both systems. Whether the inhibitory effect of FHR-5 on C3 convertase activity in this fluid phase system is directly due to decay acceleration of the C3bBb complex requires clarification, although the similarity in response to that of fH would suggest dissociation of the C3 convertase is a likely mechanism.
It is conceivable that the C3bBb complexes in the fluid and solid phases are conformationally distinct and that fH with its multiple C3b-binding sites is capable of disrupting either form, whereas FHR-5 is able only to dissociate the fluid phase complex. Alternatively, the solid phase in vitro system may not accurately reflect the conditions in vivo where the presence of polyanions on the cell surface may influence binding of fH family regulators (34, 35, 36, 37).
Although the immunohistological data (22) and the results described here support a complement regulatory role for FHR-5, sequence analysis of its SCRs does not predict the domains involved. To date, all RCA proteins with complement regulatory ability contain SCRs homologous to the four N-terminal SCRs of fH or C4bp -chains (38). Our finding that FHR-5 exhibits cofactor and possible decay accelerating activity despite lacking such domains suggests that there is a novel complement-regulatory site in FHR-5. Localization of these domains requires analysis of additional FHR-5 constructs.
The ability of fH to distinguish between foreign and host cells and regulate alternative pathway activation on the appropriate cell surface is influenced by cell surface molecules. Polyanions such as sialic acid and heparin are abundant on host cell membranes and enhance the binding (34, 35, 36, 37) and subsequent activity of fH (39, 40). As with fH and factor H-like protein 1, the ability of FHR-5 to bind polyanions may assist in positioning it at the cell surface where it can use its complement-regulatory functions. We localized a heparin-binding site in FHR-5 to SCRs 5–7, which show highest homology to SCRs 12–14 of fH. Of the 9 SCRs in FHR-5, SCR 2 shows the closest homology (33%) to SCR 7 of fH, but the FHR-5 SCR 1–4 construct was unable to bind to heparin. The possibility of a heparin-binding site in SCR 9 of FHR-5, which shows 42% homology with SCR 20 of fH, was not investigated in this study.
FH plays an essential role in regulating complement activation at sites of tissue damage. This function appears to be mediated in part by specific binding of fH to the acute phase protein CRP (7). The main biological function of CRP is to recognize pathogens and damaged host cells and to activate the classical pathway of complement, which results in their uptake by phagocytosis (41, 42). A rapid increase in CRP serum levels (up to 1000-fold) is observed within 6 h after exposure to such stimuli (43). CRP can recruit fH, which in turn is capable of inhibiting the formation of the AP convertase and the C5 convertases. Thus, CRP may act to prevent complement-mediated damage to self-tissues by limiting the inflammatory response (7, 41, 42). We demonstrated that FHR-5 contains at least one CRP binding site within the SCR 5–7 region, which does not correspond to any of the known CRP-binding sites of fH. Nevertheless, as proposed for fH, CRP may recruit FHR-5 to sites of tissue damage, where it could have a role in regulating complement activation. Recent immunofluorescence analysis of lupus nephritis biopsies demonstrated consistent glomerular deposition of CRP in the mesangial and peripheral capillary wall (44), similar to the distribution of FHR-5 that we observed in all lupus nephritis cases examined (22). In contrast, CRP was rarely detected in immune deposits of nonlupus glomerulonephropathies, which invariably contained FHR-5. This suggests that although CRP may play a role in recruiting FHR-5 to sites of complement activation and tissue injury in some circumstances, other mechanisms may also be involved. For example, in vitro and in vivo studies by Ren et al. (45) have demonstrated that rat FHR mRNA (analogous to human FHR-5) is up-regulated in cultured glomerular epithelial cells subjected to complement attack and in models of membranous nephropathy.
We have established that FHR-5 is present at 3–6 µg/ml in normal human serum (J. L. McRae, P. J. Cowan, and B. F. Murphy, unpublished data), which is relatively low compared with fH (46). However, the potential for recruitment and up-regulation of FHR-5 may allow it to regulate complement at sites of complement-mediated injury. Whether FHR-5 serum levels vary in response to disease is also of interest and is currently under investigation.
Although no direct link between fH and lipoprotein has been described, FHR proteins -1, -2, and -4A are present in lipoprotein complexes in human plasma. FHR-1 and FHR-2 are components of lipoprotein particles, which also contain apoA-I, LPS-binding protein, and fibrinogen, and may be involved in facilitating the adhesive response of neutrophils to lipopolysaccharides (33, 47). FHR-4A associates with the triglyceride-rich lipoproteins VLDL and chylomicrons and to a lesser extent HDL and LDL (1, 16, 17). Consequently, the use of lipoproteins as transport vehicles or a role of FHR proteins in lipid transport has been suggested (16). Detection of FHR-5 in the HDL lipoprotein fraction further demonstrates a relationship between FHR family members and lipoproteins. Furthermore, the complement regulators, C4-binding protein (48), CD59 (49), and clusterin (50) also interact with lipoprotein indicating a possible functional interaction between lipoproteins and the complement system. With lipoprotein association and complement-regulatory capability, FHR-5 lends further support to this theory.
Fig. 8 summarizes the SCRs of fH and FHR-5 known to be involved in protein binding and complement-regulatory activity. FHR-5 is unique among the FHR proteins in its ability to bind C3b, heparin and CRP, and its possession of cofactor and likely fluid phase decay-accelerating activity. These results add to our previous finding of a direct association between FHR-5 and complement activation in vivo (22), support a role for FHR-5 in complement regulation in vivo, and define the first clear complement-regulatory function among FHR proteins.
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Footnotes |
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1 This work was supported by grants from the National Health and Medical Research Council of Australia. 
2 Address correspondence and reprint requests to Corresponding author: Jennifer L. McRae, Immunology Research Centre, St. Vincent’s Hospital Melbourne, PO Box 2900, Fitzroy, Victoria, 3065, Australia., Ph: 61 3 9288 3140. Fax: 61 3 9288 3151. e-mail: Jennifer.MCRAE{at}svhm.org.au
3 Abbreviations used in this paper: FHR-5, factor H-related protein 5; fH, factor H; FHR-1, fH-related protein 1; FHR-2, fH-related protein 2; FHR-3, fH-related protein 3; FHR-4, fH-related protein 4; AP, alternative pathway; SCR, short consensus repeat; CRP, C-reactive protein; RCA, regulators of complement activation; RT, room temperature; GVB, gelatin-Veronal buffer; LDL, low density lipoprotein; VLDL, very low density lipoprotein; HDL, high density lipoprotein.
Received for publication January 14, 2004. Accepted for publication March 14, 2005.
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References |
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1H control and generation of restriction on neuraminidase-treated cells. Proc. Natl. Acad. Sci. USA 75: 2416-2420.1H-dependent decay-dissociation of amplification C3 convertase of the alternative complement pathway. Proc. Natl. Acad. Sci. USA 75: 1971-1975.1 H for cell-bound C3b. J. Immunol. 122: 75-81.
receptor type IIA alleles. Arthritis Rheum. 48: 460-470.[Medline]
1H globulin. J. Exp. Med. 144: 1147-1163.This article has been cited by other articles:
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