Human Factor H-Related Protein 5 Has Cofactor Activity, Inhibits C3 Convertase Activity, Binds Heparin and C-Reactive Protein, and Associates with Lipoprotein This information is current as of October 1, 2021. Jennifer L. McRae, Thomas G. Duthy, Kim M. Griggs, Rebecca J. Ormsby, Peter J. Cowan, Brett A. Cromer, William J. McKinstry, Michael W. Parker, Brendan F. Murphy and David L. Gordon J Immunol 2005; 174:6250-6256; ; Downloaded from doi: 10.4049/jimmunol.174.10.6250 http://www.jimmunol.org/content/174/10/6250 References This article cites 48 articles, 30 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/174/10/6250.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on October 1, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Human Factor H-Related Protein 5 Has Cofactor Activity, Inhibits C3 Convertase Activity, Binds Heparin and C-Reactive Protein, and Associates with Lipoprotein1 Jennifer L. McRae,2*†‡Thomas G. Duthy,§ Kim M. Griggs,§ Rebecca J. Ormsby,§ Peter J. Cowan,†‡ Brett A. Cromer,¶ William J. McKinstry,¶ Michael W. Parker,¶ Brendan F. Murphy,*†‡ and David L. Gordon§ Factor H-related protein 5 (FHR-5) is a recently discovered member of the factor H (fH)-related protein family. FHR proteins are structurally similar to the complement regulator fH, but their biological functions remain poorly defined. FHR-5 is synthesized in the liver and consists of 9 short consensus repeats (SCRs), which display various degrees of homology to those of fH and the other FHR proteins. FHR-5 colocalizes with complement deposits in vivo and binds C3b in vitro, suggesting a role in complement Downloaded from regulation or localization. The current study examined whether rFHR-5 exhibits properties similar to those of fH, including heparin binding, CRP binding, cofactor activity for the factor I-mediated degradation of C3b and decay acceleration of the C3 convertase. rFHR-5 bound heparin-BSA and heparin-agarose and a defined series of truncations expressed in Pichia pastoris localized the heparin-binding region to within SCRs 5–7. rFHR-5 bound CRP, and this binding was also localized to SCRs 5–7. FHR-5 inhibited alternative pathway C3 convertase activity in a fluid phase assay; however, dissociation of the convertase was not observed in a solid phase assay. rFHR-5 displayed factor I-dependent cofactor activity for C3b cleavage, although it was appar- http://www.jimmunol.org/ ently less effective than fH. In addition, we demonstrate association of FHR-5 with high density lipid lipoprotein complexes in human plasma. These results demonstrate that FHR-5 shares properties of heparin and CRP binding and lipoprotein association with one or more of the other FHRs but is unique among this family of proteins in possessing independent complement-regulatory activity. The Journal of Immunology, 2005, 174: 6250–6256. actor H-related protein 5 (FHR-5)3 is a member of the Regions of fH responsible for binding glycosaminoglycans such as human factor H (fH) protein family (1). This family of heparin have previously been mapped to SCRs 7, 20, and possibly structurally and immunologically related proteins includes 12–14 (3–6), whereas C-reactive protein (CRP)-binding sites have F by guest on October 1, 2021 fH, fH-like protein 1, and five other FHR proteins denoted FHR-1, been localized to SCRs 7 and 8–11 (7). C3b-binding sites are -2, -3, -4A, and -4B. fH is a crucial negative regulator of the within SCRs 2–3, 6–10, and/or 12–14 and 19–20 (8, 9), and com- alternative pathway (AP) of complement activation and targets the plement-regulatory activities reside within SCRs 1–4 (10–12). C3 (C3bBb) and C5 (C3bBbC3b) convertases of the AP in three Little is known about the function of FHR-1 and FHR-2, which ways. It acts as a competitor for factor B binding to C3b to form contain 5 and 4 SCR domains respectively, but both are present in the convertases, is an essential cofactor for factor I-mediated deg- lipoprotein complexes (13–15). FHR-3 and FHR-4B are highly radation of C3b to iC3b, and it accelerates the decay of Bb from related and consist of five SCRs. FHR-4A contains nine SCRs and the C3/C5 convertases (for review, see Ref. 2). fH is a 150-kDa is also closely associated with human triglyceride-rich lipoproteins serum protein consisting of 20 short consensus repeats (SCRs) (16, 17). Despite their sequence similarity, only FHR-3 possesses each composed of ϳ60 aa with 2 overlapping disulfide bonds. 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 *Department of Nephrology, †Immunology Research Centre, and ‡University of Mel- binds streptococcal M protein, an interaction also mediated via bourne Department of Medicine, St. Vincent’s Hospital Melbourne, Fitzroy, Victoria, SCR 7 of fH (18). Both FHR-3 and FHR-4B bind to the C3d region Australia; §Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, Australia; and ¶St. Vincent’s Institute, Fitzroy, Australia of C3b and have been reported to have weak factor I-mediated Received for publication January 14, 2004. Accepted for publication March 14, 2005. cofactor activity, although this activity is not independent of fH The costs of publication of this article were defrayed in part by the payment of page and remains controversial (19). charges. This article must therefore be hereby marked advertisement in accordance FHR-5 is a 65-kDa protein first identified as a component of with 18 U.S.C. Section 1734 solely to indicate this fact. pathological human glomerular preparations. Unlike the other 1 This work was supported by grants from the National Health and Medical Research Council of Australia. FHR proteins, which contain 4 or 5 SCRs, FHR-5 consists of 9 SCR domains. SCRs 1 and 2 are homologous to corresponding 2 Address correspondence and reprint requests to Corresponding author: Jennifer L. McRae, Immunology Research Centre, St. Vincent’s Hospital Melbourne, PO Box SCRs from FHR-1 and FHR-2, whereas SCRs 3–7 and 8–9 share 2900, Fitzroy, Victoria, 3065, Australia., Ph: 61 3 9288 3140. Fax: 61 3 9288 3151. significant homology with SCRs 10–14 and 19–20 of fH (1). The e-mail: [email protected] gene encoding FHR-5 has been localized to human chromosome 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 1q32 within the regulators of complement activation (RCA) gene 3; FHR-4, fH-related protein 4; AP, alternative pathway; SCR, short consensus repeat; cluster. It is closely linked to the other fH family genes and is CRP, C-reactive protein; RCA, regulators of complement activation; RT, room tem- perature; GVB, gelatin-Veronal buffer; LDL, low density lipoprotein; VLDL, very situated between FHR-2 and factor XIIIb. Each SCR of FHR-5 is low density lipoprotein; HDL, high density lipoprotein. encoded by an individual exon (20). Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 6251 Although the exact role of FHR-5 in complement regulation is bled, and the sera were tested by ELISA and Western blot for activity unknown, the protein has been shown to colocalize with C3 in vivo against rFHR-5 and normal human serum. and to bind C3b in vitro in a dose-dependent manner (1). Its dep- Western blot analysis osition in vivo appears widespread in complement-containing glo- merular immune deposits and extrarenal sites of complement dep- Recombinant FHR-5 SCR proteins and lipoprotein fractions were sepa- rated on 10–12% SDS-PAGE and transferred onto Hybond Cϩ nitrocel- osition (21). A prospective study of 100 renal biopsies showed the lulose (Amersham Biosciences). Native and rFHR-5 proteins were detected pattern of FHR-5 deposition to be similar to that of C3 and C5b-9 using rabbit anti-FHR-5 antiserum (1/2000 v/v) or mouse anti-FHR-5 (22). More recently, FHR-5 and all other FHR proteins have been (K2.254) mAb (Ref. 21; 1/2000 v/v) followed by HRP-conjugated goat detected at high levels in middle ear effusion fluid from patients anti-rabbit IgG (Silenus) or HRP-conjugated sheep anti-mouse IgG (Sile- with otitis media (23). nus) (1/2000 v/v), respectively. Proteins were visualized using ECL and exposure to Hyper Film ECL (Amersham Biosciences). To detect human Given the similarity of FHR-5 with fH and its association with clusterin in lipoprotein complexes, membranes were stripped by washing in complement deposits in vivo, we investigated whether recombi- 62 mM Tris, 2% SDS, 50 mM 2-ME at 42°C for 20 min, washed in PBS, nant FHR-5 (rFHR-5) exhibited functions associated with comple- and reprobed with mouse anti-human clusterin (K2.2G7) mAb (Ref.