G2Cdb::Gene report

Gene id
G00000352
Gene symbol
Cd59a (MGI)
Species
Mus musculus
Description
CD59a antigen
Orthologue
G00001601 (Homo sapiens)

Databases (6)

Curated Gene
OTTMUSG00000014930 (Vega mouse gene)
Gene
ENSMUSG00000032679 (Ensembl mouse gene)
688 (G2Cdb plasticity & disease)
Gene Expression
NM_007652 (Allen Brain Atlas)
Marker Symbol
MGI:109177 (MGI)
Protein Sequence
O55186 (UniProt)

Synonyms (2)

  • Cd59
  • protectin

Literature (49)

Pubmed - other

  • Human CD59 inhibitor sensitizes rituximab-resistant lymphoma cells to complement-mediated cytolysis.

    Hu W, Ge X, You T, Xu T, Zhang J, Wu G, Peng Z, Chorev M, Aktas BH, Halperin JA, Brown JR and Qin X

    Department of Medicine, Brigham and Women's Hospital, and Laboratory for Translational Research, Harvard Medical School, Cambridge, Massachusetts 02139, USA. weiguo_hu@hms.harvard.edu

    Rituximab efficacy in cancer therapy depends in part on induction of complement-dependent cytotoxicity (CDC). Human CD59 (hCD59) is a key complement regulatory protein that restricts the formation of the membrane attack complex, thereby inhibiting induction of CDC. hCD59 is highly expressed in B-cell non-Hodgkin's lymphoma (NHL), and upregulation of hCD59 is an important determinant of the sensitivity of NHL cells to rituximab treatment. Here, we report that the potent hCD59 inhibitor rILYd4 enhances CDC in vitro and in vivo, thereby sensitizing rituximab-resistant lymphoma cells and primary chronic lymphocytic leukemia cells (CLL) to rituximab treatment. By defining pharmcokinetic/pharmacodynamic profiles of rILYd4 in mice, we showed that by itself rILYd4 does not adversely mediate in vivo hemolysis of hCD59-expressing erythrocytes. Increasing expression levels of the complement regulators CD59 and CD55 in rituximab-resistant cells occur due to selection of preexisting clones rather than de novo induction of these proteins. Moreover, lymphoma cells overexpressing CD59 were directly responsible for the resistance to rituximab-mediated CDC therapy. Our results rationalize the use of rILYd4 as a therapeutic adjuvant for rituximab treatment of rituximab-resistant lymphoma and CLL. Furthermore, they suggest that preemptive elimination of CD59-overexpressing subpopulations along with rituximab treatment may be a useful approach to ablate or conquer rituximab resistance.

    Funded by: NCI NIH HHS: R21 CA141324, R21 CA141324-01A1, R21CA141324; NIAID NIH HHS: R01 AI061174, R01AI061174

    Cancer research 2011;71;6;2298-307

  • A high-resolution anatomical atlas of the transcriptome in the mouse embryo.

    Diez-Roux G, Banfi S, Sultan M, Geffers L, Anand S, Rozado D, Magen A, Canidio E, Pagani M, Peluso I, Lin-Marq N, Koch M, Bilio M, Cantiello I, Verde R, De Masi C, Bianchi SA, Cicchini J, Perroud E, Mehmeti S, Dagand E, Schrinner S, Nürnberger A, Schmidt K, Metz K, Zwingmann C, Brieske N, Springer C, Hernandez AM, Herzog S, Grabbe F, Sieverding C, Fischer B, Schrader K, Brockmeyer M, Dettmer S, Helbig C, Alunni V, Battaini MA, Mura C, Henrichsen CN, Garcia-Lopez R, Echevarria D, Puelles E, Garcia-Calero E, Kruse S, Uhr M, Kauck C, Feng G, Milyaev N, Ong CK, Kumar L, Lam M, Semple CA, Gyenesei A, Mundlos S, Radelof U, Lehrach H, Sarmientos P, Reymond A, Davidson DR, Dollé P, Antonarakis SE, Yaspo ML, Martinez S, Baldock RA, Eichele G and Ballabio A

    Telethon Institute of Genetics and Medicine, Naples, Italy.

    Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease.

    Funded by: Medical Research Council: MC_U127527203; Telethon: TGM11S03

    PLoS biology 2011;9;1;e1000582

  • The alternative and terminal pathways of complement mediate post-traumatic spinal cord inflammation and injury.

    Qiao F, Atkinson C, Kindy MS, Shunmugavel A, Morgan BP, Song H and Tomlinson S

    Department of Microbiology and Immunology, Children's Research Institute, Medical University of South Carolina, Charleston, SC 29425, USA.

    Complement is implicated in the inflammatory response and the secondary neuronal damage that occurs after traumatic spinal cord injury (SCI). Complement can be activated by the classical, lectin, or alternative pathways, all of which share a common terminal pathway that culminates in formation of the cytolytic membrane attack complex (MAC). Here, we investigated the role of the alternative and terminal complement pathways in SCI. Mice deficient in the alternative pathway protein factor B (fB) were protected from traumatic SCI in terms of reduced tissue damage and demyelination, reduced inflammatory cell infiltrate, and improved functional recovery. In a clinically relevant paradigm, treatment of mice with an anti-fB mAb resulted in similarly improved outcomes. These improvements were associated with decreased C3 and fB deposition. On the other hand, deficiency of CD59, an inhibitor of the membrane attack complex, resulted in significantly increased injury and impaired functional recovery compared to wild-type mice. Increased injury in CD59-deficient mice was associated with increased MAC deposition, while levels of C3 and fB were unaffected. These data indicate key roles for the alternative and terminal complement pathways in the pathophysiology of SCI. Considering a previous study demonstrating an important role for the classical pathway in promoting SCI, it is likely that the alternative pathway plays a critical role in amplifying classical pathway initiated complement activation.

    Funded by: NINDS NIH HHS: NS050452, R01 NS050452

    The American journal of pathology 2010;177;6;3061-70

  • The membrane attack complex of complement drives the progression of atherosclerosis in apolipoprotein E knockout mice.

    Lewis RD, Jackson CL, Morgan BP and Hughes TR

    Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, UK.

    Aims: To examine the roles of the membrane attack complex of complement and its sole membrane regulator, CD59, in atherosclerosis.

    Methods: C6 (C6(-/-)) deficient and CD59a (Cd59a(-/-)) knockout mice were separately crossed onto the apolipoprotein E knockout (apoE(-/-)) background. The double knockout mice were fed high-fat diet in order to study the effects of absence of C6 or CD59a on the progression of atherosclerosis.

    Results: C6 deficiency significantly reduced plaque area and disease severity. CD59a had the opposite effect in that deficiency was associated with a significant increase in plaque area, correlating with increased membrane attack complex (MAC) deposition in the plaque and increased smooth muscle cell proliferation in early plaques.

    Conclusions: Our results demonstrate that the MAC contributes to the development of atherosclerosis, C6 deficiency being protective and CD59a deficiency exacerbating disease.

    Funded by: British Heart Foundation: FS/05/087/19466, PG/10/015/28232; Wellcome Trust: 068590

    Molecular immunology 2010;47;5;1098-105

  • Targeted disruption of Zfp36l2, encoding a CCCH tandem zinc finger RNA-binding protein, results in defective hematopoiesis.

    Stumpo DJ, Broxmeyer HE, Ward T, Cooper S, Hangoc G, Chung YJ, Shelley WC, Richfield EK, Ray MK, Yoder MC, Aplan PD and Blackshear PJ

    Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.

    Members of the tristetraprolin family of tandem CCCH finger proteins can bind to AU-rich elements in the 3'-untranslated region of mRNAs, leading to their deadenylation and subsequent degradation. Partial deficiency of 1 of the 4 mouse tristetraprolin family members, Zfp36l2, resulted in complete female infertility because of early embryo death. We have now generated mice completely deficient in the ZFP36L2 protein. Homozygous Zfp36l2 knockout (KO) mice died within approximately 2 weeks of birth, apparently from intestinal or other hemorrhage. Analysis of peripheral blood from KO mice showed a decrease in red and white cells, hemoglobin, hematocrit, and platelets. Yolk sacs from embryonic day 11.5 (E11.5) Zfp36l2 KO mice and fetal livers from E14.5 KO mice gave rise to markedly reduced numbers of definitive multilineage and lineage-committed hematopoietic progenitors. Competitive reconstitution experiments demonstrated that Zfp36l2 KO fetal liver hematopoietic stem cells were unable to adequately reconstitute the hematopoietic system of lethally irradiated recipients. These data establish Zfp36l2 as a critical modulator of definitive hematopoiesis and suggest a novel regulatory pathway involving control of mRNA stability in the life cycle of hematopoietic stem and progenitor cells.

    Funded by: Intramural NIH HHS; NHLBI NIH HHS: R01 HL056416, R01 HL063169, R01 HL067384, R01 HL56416, R01 HL63169, R01 HL67384; NIAID NIH HHS: R01 AI080759, R01 AI080759-01

    Blood 2009;114;12;2401-10

  • Expression and modulation of RPE cell membrane complement regulatory proteins.

    Yang P, Tyrrell J, Han I and Jaffe GJ

    Department of Ophthalmology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Purpose: Complement, inflammation, and oxidant injury contribute to age-related macular degeneration (AMD). Membrane complement regulatory proteins (mCRPs) such as CD46, CD55, and CD59, protect host cells from complement attack. The factors that regulate RPE mCRP expression are not well understood. In this study, the authors sought to determine whether cytokines and hydroquinone (HQ) affect mCRP expression in cultured human RPE (hRPE) and cultured mouse RPE (mRPE) cells.

    Methods: Cultured hRPE and mRPE cells were stimulated with cytokines for various times or with HQ for multiple 6-hour periods. mRNA and protein expression of mCRPs in cultured hRPE cells from 10 donors, native hRPE, and mouse eyecups and native mRPE cells were evaluated by real-time RT-PCR, Western blot analysis, and flow cytometry, respectively.

    Results: Three mCRPs were expressed in cultured hRPE cells (CD59>CD46>CD55). CD46 and CD59 protein were detected in native hRPE cells. CD59 protein levels in cultured hRPE cells were higher than in native hRPE cells. CD46 protein polymorphisms were observed in cultured hRPE cells. Cultured hRPE cell mCRP expression was upregulated by TNF-alpha, IL-1beta, and a repetitive nonlethal dose of HQ. CD59a levels were higher in mouse eyecups than in nonocular tissues. Mouse mCRP mRNA and protein were detected in native mRPE cells. Responsiveness to cytokines in cultured mRPE cells differed from that in cultured hRPE cells.

    Conclusions: Human and mouse RPE cell mCRPs are upregulated by inflammatory cytokines and repetitive nonlethal oxidant exposure in a species-specific manner. Increased cell mCRPs may help to protect RPE cells from complement- and oxidant-mediated injury in diseases such as AMD.

    Funded by: NEI NIH HHS: 930EY05722

    Investigative ophthalmology & visual science 2009;50;7;3473-81

  • CD59 but not DAF deficiency accelerates atherosclerosis in female ApoE knockout mice.

    An G, Miwa T, Song WL, Lawson JA, Rader DJ, Zhang Y and Song WC

    Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Room 1254 BRBII/III, 421 Curie Blvd, Philadelphia, PA 19104, USA.

    Although the complement system has been implicated in atherosclerosis, the influence of membrane-bound complement regulators in this process has not been well understood. We studied the role of two membrane complement regulators, decay-accelerating factor (DAF) and CD59, in a murine model of atherosclerosis. DAF(-/-) and CD59(-/-) mice were crossed with apolipoprotein E (ApoE)-deficient mice to generate DAF(-/-)ApoE(-/-) and CD59(-/-)ApoE(-/-) mice. Mice were fed a high fat diet (HFD) for 8 or 16 weeks. En face analysis showed that CD59 deficiency led to more extensive lesions in female ApoE(-/-) mice both at 8 weeks (2.07+/-0.27% vs.1.34+/-0.21%, P=0.06) and 16 weeks (17.13+/-1.14% vs. 9.72+/-1.14%, P<0.001). Similarly, lesions measured by aortic root sectioning were larger in female CD59(-/-)ApoE(-/-) mice than in controls at 8 weeks of HFD feeding (20.74+/-1.33% vs. 13.12+/-1.46%, P<0.005). On the other hand, DAF deficiency did not significantly influence atherosclerosis in ApoE(-/-) mice. Immunohistochemistry revealed more abundant membrane attack complex (MAC) deposition and more collagen staining in the aortic roots of CD59(-/-)ApoE(-/-) mice. Unexpectedly, total plasma cholesterol levels in female CD59(-/-)ApoE(-/-) mice were found to be elevated compared with CD59(+/+)ApoE(-/-) mice. We conclude that CD59 but not DAF offered protection in atherosclerosis in the context of ApoE deficiency. The protective role of CD59 was gender-biased and most likely involved prevention of MAC-mediated vascular injury, with possible contribution from an undefined effect on plasma cholesterol homeostasis.

    Funded by: NIAID NIH HHS: AI-44970, AI-49344, AI-62388, R01 AI044970, R01 AI044970-09, R01 AI049344, R01 AI049344-08, R01 AI063288, R01 AI063288-05, R21 AI049344, UC1 AI062388

    Molecular immunology 2009;46;8-9;1702-9

  • Deficiency of the complement regulator CD59a exacerbates Wallerian degeneration.

    Ramaglia V, King RH, Morgan BP and Baas F

    Neurogenetics Laboratory, University of Amsterdam, Amsterdam, The Netherlands.

    The complement system is implicated in Wallerian degeneration (WD). We have previously shown that the membrane attack complex (MAC), the terminal activation product of the complement cascade, mediates rapid axonal degradation and myelin clearance during WD after peripheral nerve injury. In this study we analyzed the contribution of CD59a, a cell membrane negative regulator of the MAC, to WD. Following injury, the level of MAC deposition was higher in the CD59a deficient mice than wildtypes whereas the residual axonal content was lower in CD59a deficient mice than wildtypes, strongly implicating MAC as a determinant of axonal damage during WD. The number of endoneurial macrophages was significantly higher in CD59a deficient mice compared to wildtypes at 1 day post-injury. These findings are relevant to the understanding of the mechanisms of axon loss in injury and disease.

    Funded by: Wellcome Trust: 068590

    Molecular immunology 2009;46;8-9;1892-6

  • Focal and segmental glomerulosclerosis induced in mice lacking decay-accelerating factor in T cells.

    Bao L, Haas M, Pippin J, Wang Y, Miwa T, Chang A, Minto AW, Petkova M, Qiao G, Song WC, Alpers CE, Zhang J, Shankland SJ and Quigg RJ

    University of Chicago, Illinois, 60637, USA. lbao@medicine.bsd.uchicago.edu

    Heritable and acquired diseases of podocytes can result in focal and segmental glomerulosclerosis (FSGS). We modeled FSGS by passively transferring mouse podocyte-specific sheep Abs into BALB/c mice. BALB/c mice deficient in the key complement regulator, decay-accelerating factor (DAF), but not WT or CD59-deficient BALB/c mice developed histological and ultrastructural features of FSGS, marked albuminuria, periglomerular monocytic and T cell inflammation, and enhanced T cell reactivity to sheep IgG. All of these findings, which are characteristic of FSGS, were substantially reduced by depleting CD4+ T cells from Daf(-/-) mice. Furthermore, WT kidneys transplanted into Daf(-/-) recipients and kidneys of DAF-sufficient but T cell-deficient Balb/(cnu/nu) mice reconstituted with Daf(-/-) T cells developed FSGS. In contrast, DAF-deficient kidneys in WT hosts and Balb/(cnu/nu) mice reconstituted with DAF-sufficient T cells did not develop FSGS. Thus, we have described what we believe to be a novel mouse model of FSGS attributable to DAF-deficient T cell immune responses. These findings add to growing evidence that complement-derived signals shape T cell responses, since T cells that recognize sheep Abs bound to podocytes can lead to cellular injury and development of FSGS.

    Funded by: NIAID NIH HHS: AI044970, AI049344, AI063288, R01 AI044970, R01 AI049344, R01 AI063288, R21 AI049344; NIAMS NIH HHS: AR049775, R01 AR049775; NIDDK NIH HHS: DK051096, DK056799, DK066802, R01 DK041873, R01 DK051096, R01 DK055357, R01 DK056799, R01 DK066802, R56 DK041873

    The Journal of clinical investigation 2009;119;5;1264-74

  • Mouse CD4+ CD25+ T regulatory cells are protected from autologous complement mediated injury by Crry and CD59.

    Li Q, Nacion K, Bu H and Lin F

    Department of Pathology, Sichuan University, Chengdu, China.

    Self cells depend on surface complement regulators to protect them from autologous complement mediated attack. CD4(+)CD25(+)foxp3(+) T regulatory (Treg) cells are critical in maintaining immune homeostasis, however, which complement regulators are expressed on them and how they are protected from autologous complement attack remains unknown. We report here that mouse Treg cells express virtually no DAF or CR1. Instead, all of them express Crry and approximately half of them express CD59. Both Crry(-/-) and CD59(-/-) Treg cells exhibit greater complement mediated injury than WT Treg cells. These results clarify the status of cell surface complement regulators on mouse Treg cells and indicate that both Crry and CD59 are required to protect Treg cells from autologous complement mediated injury. Additionally, these data also argue that different from previous assumption, at least in mice, CD4(+)CD25(+)foxp3(+) Treg cells are not homogenous and could be further divided into subgroups based on CD59 expression.

    Funded by: NINDS NIH HHS: NS052471, R01 NS052471, R01 NS052471-04

    Biochemical and biophysical research communications 2009;382;1;223-6

  • Crry deficiency in complement sufficient mice: C3 consumption occurs without associated renal injury.

    Ruseva MM, Hughes TR, Donev RM, Sivasankar B, Pickering MC, Wu X, Harris CL and Morgan BP

    Complement Biology Group, Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.

    The rodent-specific complement regulator complement receptor 1-related gene/protein-y (Crry) is critical for complement homeostasis. Gene deletion is 100% embryonically lethal; Crry-deficient (Crry(-/-)) mice were rescued by back-crossing onto C3 deficiency, confirming that embryo loss was complement mediated. In order to rescue viable Crry(-/-) mice without deleting C3, we have tested inhibition of C5 during gestation. Crry(+/-) females were given neutralizing anti-C5 mAb immediately prior to mating with Crry(+/-) males and C5 inhibition maintained through pregnancy. A single, healthy Crry(-/-) female was obtained and mating with Crry(+/-) males yielded healthy litters containing equal numbers of Crry(+/-) and Crry(-/-) pups. Inter-crossing Crry(-/-) mice yielded healthy litters of expected size. Although the mice were not anemic, exposure of Crry(-/-) erythrocytes to normal mouse serum caused C3 deposition and lysis, while transfusion into normal or C6(-/-) mice resulted in rapid clearance. Complement activity and C3 levels in Crry(-/-) mice were markedly reduced. Comparison with factor H deficient (CfH(-/-)) mice revealed similar levels of residual C3; however, unlike the CfH(-/-) mice, Crry(-/-) mice showed no evidence of renal injury, demonstrating distinct roles for these regulators in protecting the kidney.

    Funded by: Medical Research Council: G0700102; Wellcome Trust: 068823, 079115

    Molecular immunology 2009;46;5;803-11

  • Generation and phenotyping of mCd59a and mCd59b double-knockout mice.

    Qin X, Hu W, Song W, Grubissich L, Hu X, Wu G, Ferris S, Dobarro M and Halperin JA

    Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts, USA. xuebin_qin@hms.harvard.edu

    CD59 is a membrane protein inhibitor of the membrane attack complex (MAC) of complement. Humans express only one, whereas mice express two CD59 genes. We previously reported the targeted deletion of the mCd59b gene in which absence of mCd59b together with an unintended down regulation of mCd59a caused hemolytic anemia with spontaneous platelet activation. To confirm the complement role in the hemolytic anemia caused by abrogation of mCd59 function, we have developed a mCd59a and mCd59b double knock out mice and analyzed its phenotype in complement sufficient and deficient (C3(-/-)). We report here that total abrogation of mCd59 function in mCd59ab(-/-) mice results in complement-mediated hemolytic anemia that is rescued by the deficiency of C3 in compound mCd59ab(-/-)/C3(-/-) mice.

    Funded by: NIAID NIH HHS: R01 AI061174; NIDDK NIH HHS: R01 DK060979; PHS HHS: R01052855

    American journal of hematology 2009;84;2;65-70

  • Microarray analysis of Foxa2 mutant mouse embryos reveals novel gene expression and inductive roles for the gastrula organizer and its derivatives.

    Tamplin OJ, Kinzel D, Cox BJ, Bell CE, Rossant J and Lickert H

    Program in Developmental and Stem Cell Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario, M5G 1X8, Canada. owen.tamplin@utoronto.ca

    Background: The Spemann/Mangold organizer is a transient tissue critical for patterning the gastrula stage vertebrate embryo and formation of the three germ layers. Despite its important role during development, there are still relatively few genes with specific expression in the organizer and its derivatives. Foxa2 is a forkhead transcription factor that is absolutely required for formation of the mammalian equivalent of the organizer, the node, the axial mesoderm and the definitive endoderm (DE). However, the targets of Foxa2 during embryogenesis, and the molecular impact of organizer loss on the gastrula embryo, have not been well defined.

    Results: To identify genes specific to the Spemann/Mangold organizer, we performed a microarray-based screen that compared wild-type and Foxa2 mutant embryos at late gastrulation stage (E7.5). We could detect genes that were consistently down-regulated in replicate pools of mutant embryos versus wild-type, and these included a number of known node and DE markers. We selected 314 genes without previously published data at E7.5 and screened for expression by whole mount in situ hybridization. We identified 10 novel expression patterns in the node and 5 in the definitive endoderm. We also found significant reduction of markers expressed in secondary tissues that require interaction with the organizer and its derivatives, such as cardiac mesoderm, vasculature, primitive streak, and anterior neuroectoderm.

    Conclusion: The genes identified in this screen represent novel Spemann/Mangold organizer genes as well as potential Foxa2 targets. Further investigation will be needed to define these genes as novel developmental regulatory factors involved in organizer formation and function. We have placed these genes in a Foxa2-dependent genetic regulatory network and we hypothesize how Foxa2 may regulate a molecular program of Spemann/Mangold organizer development. We have also shown how early loss of the organizer and its inductive properties in an otherwise normal embryo, impacts on the molecular profile of surrounding tissues.

    BMC genomics 2008;9;511

  • Brief report: accelerated atherosclerosis in low-density lipoprotein receptor-deficient mice lacking the membrane-bound complement regulator CD59.

    Yun S, Leung VW, Botto M, Boyle JJ and Haskard DO

    NHLI Cardiovascular Sciences, Imperial College, Hammersmith Hospital, Du Cane Road, London, UK.

    Objective: Whereas studies in humans and animal models have suggested a role for complement activation in atherosclerosis, there has been little analysis of the importance of complement regulators. We tested the hypothesis that the terminal pathway inhibitor CD59 plays an essential role in limiting the proinflammatory effects of complement activation.

    CD59 gene targeted mice (CD59a(-/-)) mice were crossed with low-density lipoprotein receptor-deficient (Ldlr(-/-)) mice. CD59-deficient Ldlr(-/-) mice had significantly more extensive en face Sudan IV staining of thoracoabdominal aorta than Ldlr(-/-) single knock-outs, both after a low-fat diet (6.51+/-0.36% versus 2.63+/-0.56%, P<0.001) or a high-fat diet (17.05+/-2.15% versus 7.69+/-1.17%, P<0.004). Accelerated lesion formation in CD59a(-/-)/Ldlr(-/-) mice on a high-fat diet was associated with increased lesional vascular smooth muscle cell (VSMC) number and fibrous cap formation.

    Conclusions: Our data show that CD59 deficiency accelerates the development of lesions and increases plaque VSMC composition. Assuming that the main function of CD59 is to prevent the development of C5b-9 membrane attack complexes, our observations are consistent with the terminal complement pathway having proatherogenic potential in the Ldlr(-/-) mouse model, and highlight the importance of complement regulation.

    Funded by: British Heart Foundation: FS/13/12/30037

    Arteriosclerosis, thrombosis, and vascular biology 2008;28;10;1714-6

  • Deficiency of decay-accelerating factor and complement receptor 1-related gene/protein y on murine platelets leads to complement-dependent clearance by the macrophage phagocytic receptor CRIg.

    Kim DD, Miwa T, Kimura Y, Schwendener RA, van Lookeren Campagne M and Song WC

    Department of Pharmacology, Institute for Translational Medicine and Therapeutics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Complement activation on human platelets is known to cause platelet degranulation and activation. To evaluate how normal platelets escape complement attack in vivo, we studied the fate of murine platelets deficient in 2 membrane complement regulatory proteins using an adoptive transfer model. We show here that deficiency of either decay-accelerating factor (DAF) or complement receptor 1-related gene/protein y (Crry) on murine platelets was inconsequential, whereas DAF and Crry double deficiency led to rapid clearance of platelets from circulation in a complement- and macrophage-dependent manner. This finding contrasted with the observation on erythrocytes, where Crry deficiency alone resulted in complement susceptibility. Quantitative flow cytometry showed DAF and Crry were expressed at similar levels on platelets, whereas Crry expression was 3 times higher than DAF on erythrocytes. Antibody blocking or gene ablation of the newly identified complement receptor CRIg, but not complement receptor 3 (CR3), rescued DAF/Crry-deficient platelets from complement-dependent elimination. Surprisingly, deficiency of CRIg, CR3, and other known complement receptors failed to prevent Crry-deficient erythrocytes from complement-mediated clearance. These results show a critical but redundant role of DAF and Crry in platelet survival and suggest that complement-opsonized platelets and erythrocytes engage different complement receptors on tissue macrophages in vivo.

    Funded by: NIAID NIH HHS: AI-44970, AI-49344, AI-63288, R01 AI044970, R01 AI049344, R01 AI063288, R21 AI049344

    Blood 2008;112;4;1109-19

  • The mouse complement regulator CD59b is significantly expressed only in testis and plays roles in sperm acrosome activation and motility.

    Donev RM, Sivasankar B, Mizuno M and Morgan BP

    Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK. donevrm@cardiff.ac.uk

    In mouse, genes encoding complement regulators CD55 and CD59 have been duplicated. The first described form of CD59, CD59a, is broadly distributed in mouse tissues, while the later identified CD59b was originally described as testis specific. Subsequent studies have been contradictory, some reporting widespread and abundant expression of CD59b. Resolution of the distribution patterns of the CD59 isoforms is important for interpretation of disease studies utilising CD59 knockout mice. Here we have performed a comprehensive distribution study of the CD59 isoforms at the mRNA and protein levels. These data confirm that expression of CD59b is essentially restricted to adult testis; trace expression in other tissues is a consequence of contamination with blood cells, shown previously to express CD59b at low level. In testis, onset of expression of CD59b coincided with puberty and was predominant on the spermatozoal acrosome. Ligation of CD59b, but not CD59a, markedly reduced spermatozoal motility, suggesting a specific role in reproductive function.

    Funded by: Medical Research Council: G0700102; Wellcome Trust: 068590

    Molecular immunology 2008;45;2;534-42

  • Rapid conditional targeted ablation of cells expressing human CD59 in transgenic mice by intermedilysin.

    Hu W, Ferris SP, Tweten RK, Wu G, Radaeva S, Gao B, Bronson RT, Halperin JA and Qin X

    Department of Medicine, Brigham and Women's Hospital, 75 Francis Street, Boston, Massachusetts 02115, USA.

    Conditional targeted cell ablation is a powerful approach for investigating the pathogenesis of human diseases and in vivo cellular functions. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses human cells exclusively, owing to its receptor specificity for human CD59. We generated two transgenic mouse strains that express human CD59 either on erythrocytes (strain ThCD59(RBC)) or on endothelia (strain ThCD59(END)). Intravenous injection of ILY in ThCD59(RBC) mice induced acute intravascular hemolysis, leading to reduced nitric oxide bioavailability, increased platelet activation and rapid death. In ThCD59(END) mice, ILY induced rapid endothelial damage, leading to acute death and disseminated intravascular coagulation. Additionally, we show that human serum contains ILY-specific neutralizing antibodies not found in any other animal species. Together, these results suggest that this new rapid conditional targeted ILY-mediated cell ablation technique can be used in combination with any available transgenic expression system to study the physiologic role of specific cell populations.

    Funded by: NIAID NIH HHS: R01 AI061174, R01 AI063444; NIDDK NIH HHS: R01 DK060979

    Nature medicine 2008;14;1;98-103

  • CD59 or C3 are not requred for angiotensin II-dependent hypertension or hypertrophy in mice.

    Coles B, Lewis R, Anning PB, Morton J, Baalasubramanian S, Morgan BP and O'Donnell VB

    Department of Medical Biochemistry & Immunology, School of Medicine, Cardiff University, Cardiff, UK.

    Complement is a major pro-inflammatory innate immune system whose serum activity correlates with systolic blood pressure in humans. To date, no studies using in vivo models have directly examined the role of individual complement components in regulating vessel function, hypertension and cardiac hypertrophy. Herein, in vivo responses to angiotensin (ang) II were characterized in mice deficient in CD59a or C3. CD59a(-/-) mice had slightly but significantly elevated systolic blood pressure (107.2 +/- 1.7 mmHg versus 113.8 +/- 1.31 mmHg, P < 0.01, for wild-type and CD59a(-/-), respectively). Aortic rings from CD59a(-/-) mice showed significantly less platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, with elevated deposition of membrane attack complex. However, acetylcholine- and sodium nitroprusside-dependent dilatation, plasma nitrate/nitrite and aortic cyclic guanosine monophosphate levels were unchanged from wild-type. Also, in vivo infusion with either ang II or noradrenaline caused similar hypertension and vascular hypertrophy to wild-type. Mice deficient in C3 had similar basal blood pressure to wild type and showed no differences in hypertension or hypertrophy responses to in vivo infusion with ang II. These data indicate that CD59a deficiency is associated with some vascular alterations that may represent early damage occurring as a result of increased complement attack. However, a direct role for CD59a or C3 in modulating development of ang II-dependent hypertension or hypertrophy in vivo is excluded and we suggest caution in development of complement intervention strategies for hypertension and heart failure.

    Funded by: Wellcome Trust

    Immunology 2007;121;4;518-25

  • Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo.

    Zhang X, Kimura Y, Fang C, Zhou L, Sfyroera G, Lambris JD, Wetsel RA, Miwa T and Song WC

    Institute for Translational Medicine and Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Toll-like receptors (TLRs) and complement are 2 components of innate immunity that are critical for first-line host defense and elicitation of adaptive immune responses. Many pathogen-associated molecular patterns activate both TLR and complement, but whether and how these 2 systems, when coactivated in vivo, interact with each other has not been well studied. We demonstrate here a widespread regulation of TLR signaling by complement in vivo. The TLR ligands lipopolysacharride (TLR4), zymosan (TLR2/6), and CpG oligonucleotide (TLR9) caused, in a complement-dependent manner, strikingly elevated plasma interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-1beta, and/or decreased plasma IL-12 levels in mice deficient in the membrane complement inhibitor decay-accelerating factor (DAF). A similar outcome was observed in wild-type mice cotreated with the TLR ligands and cobra venom factor, a potent complement activator. The regulatory effect of complement on TLR-induced cytokine production in vivo was mediated by the anaphylatoxin receptors C5aR and C3aR. Additionally, changes in lipopolysaccharide (LPS)-induced cytokine production in DAF-deficient mice correlated with increased mitogen-activated protein kinase and nuclear factor-kappaB activation in the spleen. These results reveal a strong interaction between complement and TLR signaling in vivo and suggest a novel mechanism by which complement promotes inflammation and modulates adaptive immunity.

    Funded by: CSR NIH HHS: RG 3671-A-1; NIAID NIH HHS: AI-44970, AI-49344, AI-63288, R01 AI044970, R01 AI049344, R01 AI063288, R21 AI049344; NIGMS NIH HHS: GM-069736, R24 GM069736

    Blood 2007;110;1;228-36

  • CD59a deficiency exacerbates influenza-induced lung inflammation through complement-dependent and -independent mechanisms.

    Longhi MP, Williams A, Wise M, Morgan BP and Gallimore A

    Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, UK. mlonghi@mail.rockefeller.edu

    Influenza-specific immune activity not only promotes virus clearance but also causes immunopathology, thereby underlining the importance of mounting a measured anti-viral immune response. Since complement bridges both the innate and adaptive immune systems and has been implicated in defence against influenza, the role of the complement regulator CD59a in modulating the response to influenza was explored. For this purpose, immune responses to influenza virus, strain E61-13-H17, in mice deficient in the complement regulator protein CD59a (Cd59a(-/-) mice) were compared to those in wild-type mice. The severity of lung inflammation was significantly enhanced in the lungs of Cd59a(-/-) mice with increased numbers of infiltrating neutrophils and CD4(+) T cells. When complement was inhibited using soluble complement receptor 1, the frequency of lung-infiltrating neutrophils in influenza-infected Cd59a(-/-) mice was much reduced whilst numbers of CD4(+) T cells remained unchanged. These results demonstrate that CD59a, previously defined as a complement regulator, modulates both the innate and adaptive immune response to influenza virus by both complement-dependent and -independent mechanisms.

    Funded by: Medical Research Council: G0500617, G117/488; Wellcome Trust: 068590, 073055

    European journal of immunology 2007;37;5;1266-74

  • CD59a deficient mice display reduced B cell activity and antibody production in response to T-dependent antigens.

    Sivasankar B, Donev RM, Longhi MP, Hughes TR, Davies R, Cole DS, Morgan BP and Marchbank KJ

    Department of Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Cardiff, United Kingdom.

    CD59a is the primary regulator of membrane attack complex in mice. Recently, we have shown that CD59a-deficient (Cd59a-/-) mice exhibit enhanced CD4+ T cell responses. Here, we explored the effects of CD59a on B cell function and antibody production. Contrary to our expectations, Cd59a-/- mice showed a decreased humoral immune response to a T cell dependent antigen, sheep red blood cells. We found that the decreased humoral immune response was associated with a reduction in plasma cell number in vivo and reduced ability to respond to stimuli during in vitro culture experiments. Using MLR studies in which purified wild type or Cd59a-/- CD4+ T cells were mixed with purified B cells from each source, we found that the reduced B cell activation was largely due to the absence of CD59a on CD4+ T cells. Furthermore, a CD59a fusion protein bound specifically to mouse B cells, and enhanced B cell proliferation in a MLR, demonstrating that B cells express an as yet unidentified ligand for CD59a that aids in B cell activation.

    Funded by: Medical Research Council: G0700102; Wellcome Trust: 068541, 068590, 070355

    Molecular immunology 2007;44;11;2978-87

  • Decay-accelerating factor but not CD59 limits experimental immune-complex glomerulonephritis.

    Bao L, Haas M, Minto AW and Quigg RJ

    Section of Nephrology, The University of Chicago, Chicago, IL 60637, USA. lbao@medicine.bsd.uchicago.edu

    The complex balance between the pro-activating and regulatory influences of the complement system can affect the pathogenesis of immune complex-mediated glomerulonephritis (ICGN). Key complement regulatory proteins include decay accelerating factor (DAF) and CD59, which inhibit C3 activation and C5b-9 generation, respectively. Both are glycosylphosphatidylinositol-linked cell membrane proteins, which are widely distributed in humans and mice. Chronic serum sickness induced by daily immunization with horse spleen apoferritin over 6 weeks was used to induce ICGN in DAF-, CD59- and DAF/CD59-deficient mice, with wild-type littermate mice serving as controls. Both DAF and DAF/CD59-deficient mice had an increased incidence of GN relative to wild-type controls associated with significantly increased glomerular C3 deposition. Disease expression in CD59-deficient mice was no different than wild-type controls. DAF- and DAF/CD59-deficient mice also had increased monocyte chemoattractant protein-1 mRNA expression and glomerular infiltration with CD45(+) leukocytes. Our findings suggest that activation of C3 is strongly associated with experimental ICGN while downstream formation of C5b-9 is of lesser pathogenic importance in this model.

    Funded by: NIDDK NIH HHS: R01DK041873

    Laboratory investigation; a journal of technical methods and pathology 2007;87;4;357-64

  • CD59, a complement regulatory protein, controls choroidal neovascularization in a mouse model of wet-type age-related macular degeneration.

    Bora NS, Kaliappan S, Jha P, Xu Q, Sivasankar B, Harris CL, Morgan BP and Bora PS

    Department of Ophthalmology, Jones Eye Institute, Pat and Willard Walker Eye Research Center, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA. NBora@UAMS.edu

    We have shown that membrane attack complex (MAC) formation via the activation of the alternative pathway plays a central role in the laser-induced choroidal neovascularization (CNV). This study was undertaken to understand the role of a complement regulatory protein, CD59, which controls MAC assembly and function, in this model. CNV was induced by laser photocoagulation in C57BL/6 and Cd59a(-/-) mice using an argon laser. Animals from each group were sacrificed on day 1, 3, 5, and 7 postlaser. Retinal pigment epithelium-choroid-scleral tissue was examined to determine the incidence and size of CNV complex, and semiquantitative RT-PCR and Western blot analysis for CD59a was studied. Recombinant soluble mouse CD59a-IgG2a fusion (rsCD59a-Fc) protein was injected via i.p. or intravitreal routes 24 h before laser. Our results demonstrated that CD59a (both mRNA and protein) was down-regulated during laser-induced CNV. Cd59a(-/-) mice developed CNV complex early in the disease process. Increased MAC deposition was also observed in these Cd59a(-/-) mice. Administration of rsCD59a-Fc inhibited the development of CNV complex in the mouse model by blocking MAC formation and also inhibited expression of angiogenic growth factors. These data provide strong evidence that CD59a plays a crucial role in regulating complement activation and MAC formation essential for the release of growth factors that drive the development of laser-induced CNV in mice. Thus, our results suggest that the inhibition of complement by soluble CD59 may provide a novel therapeutic alternative to current treatment.

    Funded by: NCRR NIH HHS: 1S10 RR19395, 2P20 RR16460; NEI NIH HHS: EY014623, EY13335, R01 EY013335-05

    Journal of immunology (Baltimore, Md. : 1950) 2007;178;3;1783-90

  • WT1-mediated gene regulation in early urogenital ridge development.

    Klattig J, Sierig R, Kruspe D, Makki MS and Englert C

    Leibniz Institute for Age Research, Jena, Germany.

    The Wilms tumor protein WT1 is involved in the development of several organs, including the gonads. WT1 mutations in humans lead to syndromes associated with impaired sexual development and Wt1 knockout mice show regression of gonad anlagen. As a transcription factor, WT1 fulfills its function by regulating a set of target genes. With respect to gonad development only few in vivo WT1 targets, e.g. steroidogenic factor 1 (SF1) have been identified so far. To get a comprehensive view of WT1 targets in the gonad, we compared gene expression in urogenital ridges of wild-type and Wt1(-/-) embryos. We found almost 150 genes differentially expressed higher than factor three, using microarray analysis. To confirm these results we performed quantitative real-time RT-PCR for many genes and observed a high degree of concordance between microarray and real-time RT-PCR results. Employing in situ hybridization we found 'WT1 activated genes' to be expressed in gonads, mesonephroi and coelomic epithelium--those parts of the urogenital ridge with Wt1 expression. Interestingly, many of the differentially expressed genes are known to show sex-specific expression at later time-points. These results provide a basis for investigation of developmental pathways in the urogenital ridge downstream of WT1 and for identification of new candidate genes involved in early urogenital ridge development. For example we provide a first potential target of WT1 in the coelomic epithelium--Muc16, and a gene regulated by the WT1 target SF1--Gata4.

    Sexual development : genetics, molecular biology, evolution, endocrinology, embryology, and pathology of sex determination and differentiation 2007;1;4;238-54

  • Deficiency of decay accelerating factor and CD59 leads to crisis in experimental myasthenia.

    Kaminski HJ, Kusner LL, Richmonds C, Medof ME and Lin F

    Department of Neurology, Case Western Reserve University, Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH, USA. Henry.Kaminski@case.edu

    In myasthenia gravis (MG), neuromuscular transmission is disrupted due to the production of autoantibodies against acetylcholine receptors (AChR). In previous work, we showed that decay accelerating factor (DAF or CD55), an intrinsic cell surface complement regulator that disables C3/C5 amplification convertases, protects against receptor loss and muscle weakness. In this study, we examined whether, and if so, to what extent CD59, a downstream intrinsic cell surface regulator that prevents assembly of membrane attack complexes (MACs), contributes to this protection. Twenty-four hours after anti-AChR injection, we found that CD59a-/- mice did not significantly differ from WTs, all Daf1-/- CD59a-/- mice either died or required euthanasia. At 48h, Daf1-/- were significantly weaker than CD59a-/- and WT mice, and for these mice immunohistochemistry revealed marked C9 deposition at postsynaptic junctions, radioimmunoassays showed reductions in AChR levels, and electron microscopy demonstrated massive junctional damage. These data indicate that DAF serves as the initial shield that protects the neuromuscular junction whereas CD59 is a further barrier. They argue that complement inhibitor, particularly if targeted to the receptor, could then have therapeutic value in human MG.

    Funded by: NEI NIH HHS: EY11373, P30 EY11370, R01EY013238, R24EY014837

    Experimental neurology 2006;202;2;287-93

  • The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice.

    Morgan BP, Chamberlain-Banoub J, Neal JW, Song W, Mizuno M and Harris CL

    Department of Pathology, School of Medicine, Cardiff University, Cardiff, UK. morganbp@cardiff.ac.uk

    The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end-plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end-plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild-type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end-plates. Cd59a-deficient mice had very mild disease with some muscle inflammation and essentially undamaged end-plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end-plates. Inhibition of MAC assembly abrogated clinical disease in these double-deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti-C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.

    Funded by: Wellcome Trust: 068590, 068823

    Clinical and experimental immunology 2006;146;2;294-302

  • Complement activation contributes to both glomerular and tubulointerstitial damage in adriamycin nephropathy in mice.

    Turnberg D, Lewis M, Moss J, Xu Y, Botto M and Cook HT

    Rheumatology Section, Faculty of Medicine, Imperial College, Hammersmith Campus, London, United Kingdom.

    Adriamycin nephropathy is a model of focal segmental glomerulosclerosis, characterized by proteinuria and progressive glomerulosclerosis and tubulointerstitial damage. In this study, we examined the role of complement in the etiology of adriamycin nephropathy in mice. We used mice deficient in C1q, factor D, C3, and CD59, and compared them with strain-matched controls. C3 deposition occurred in the glomeruli of wild-type mice as early as 48 h following a single i.v. injection of adriamycin. C3-deficient mice developed significantly less proteinuria and less podocyte injury at day 3 postadriamycin than controls, suggesting that complement is important in mediating the early podocyte injury. At later time points, C3-deficient mice were protected from glomerulosclerosis, tubulointerstitial injury, and renal dysfunction. Factor D-deficient mice were also protected from renal disease, confirming the importance of alternative pathway activation in this model. In contrast, C1q-deficient mice developed similar disease to controls, indicating that the complement cascade was not activated via the classical pathway. CD59-deficient mice, which lack adequate control of C5b-9 formation, developed significantly worse histological and functional markers of renal disease than controls. Interestingly, although more C9 deposited in glomeruli of CD59-deficient mice than controls, in neither group was tubulointerstitial C9 staining apparent. We have demonstrated for the first time that alternative pathway activation of complement plays an important role in mediating the initial glomerular damage in this in vivo model of focal segmental glomerulosclerosis. Lack of CD59, which regulates the membrane attack complex, led to greater glomerular and tubulointerstitial injury.

    Funded by: Wellcome Trust: 071467

    Journal of immunology (Baltimore, Md. : 1950) 2006;177;6;4094-102

  • BGEM: an in situ hybridization database of gene expression in the embryonic and adult mouse nervous system.

    Magdaleno S, Jensen P, Brumwell CL, Seal A, Lehman K, Asbury A, Cheung T, Cornelius T, Batten DM, Eden C, Norland SM, Rice DS, Dosooye N, Shakya S, Mehta P and Curran T

    Department of Developmental Neurobiology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States.

    Funded by: NINDS NIH HHS: 5R37NS036558, N01-NS-0-2331, R37 NS036558

    PLoS biology 2006;4;4;e86

  • Cutting edge: murine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner.

    Longhi MP, Sivasankar B, Omidvar N, Morgan BP and Gallimore A

    Medical Biochemistry and Immunology, School of Medicine, Cardiff University, Wales, United Kingdom. longhimp@Cardiff.ac.uk

    CD59 blocks formation of the membrane attack complex of complement by inhibiting binding of C9 to the C5b-8 complex. To investigate a role for CD59 in promoting T cell responses, we compared T cell activation in CD59a-deficient (Cd59a-/-) and wild-type (WT) mice after in vitro stimulation and after infection with rVV. Virus-specific CD4+ T cell responses were significantly enhanced in Cd59a-/- mice compared with WT mice. Similarly, Cd59a-/- T cells responded more vigorously to in vitro stimulation with CD3-specific Abs compared with WT mice. This effect of CD59a on T cell proliferation was found to be complement-independent. Collectively, these results demonstrate that CD59a down-modulates CD4+ T cell activity in vitro and in vivo, thereby revealing another link between complement regulators and T cell activation.

    Funded by: Wellcome Trust: 073055

    Journal of immunology (Baltimore, Md. : 1950) 2005;175;11;7098-102

  • The role of complement in cryoglobulin-induced immune complex glomerulonephritis.

    Trendelenburg M, Fossati-Jimack L, Cortes-Hernandez J, Turnberg D, Lewis M, Izui S, Cook HT and Botto M

    Rheumatology Section, Imperial College, London, UK.

    Many forms of glomerulonephritis are triggered by Ab localization in the glomerulus, but the mechanisms by which this induces glomerular inflammation are not fully understood. In this study we investigated the role of complement in a mouse model of cryoglobulin-induced immune complex glomerulonephritis. Several complement-deficient mice on a C57BL/6 and BALB/c genetic background were used and compared with strain-matched, wild-type controls. Cryoglobulinemia was induced by i.p. injection of 6-19 hybridoma cells producing an IgG3 cryoglobulin with rheumatoid factor activity against IgG2a of allotype a present in BALB/c, but not C57BL/6, mice. Thus, the cryoprecipitate in C57BL/6 mice consisted of the IgG3 cryoglobulin only (type I cryoglobulinemia) compared with IgG3-IgG2a complexes in BALB/c (type II cryoglobulinemia). The survival of mice was not affected by complement deficiency. Glomerular influx of neutrophils was significantly less in C3-, factor B-, and C5-deficient mice compared with wild-type and C1q-deficient mice. It did not correlate with C3 deposition, but did correlate with the amount of C6 deposited. Deficiency of CD59a, the membrane inhibitor of the membrane attack complex, did not induce an increase in neutrophil infiltration, suggesting that the generation of C5a accounts for the effects observed. There was no apparent difference between cryoglobulinemia types I and II regarding the role of complement. Our results suggest that in this model of cryoglobulin-induced glomerulonephritis the neutrophil influx was mediated by C5 activation with the alternative pathway playing a prominent role in its cleavage. Thus, blocking C5 is a potential therapeutic strategy for preventing renal injury in cryoglobulinemia.

    Funded by: Wellcome Trust

    Journal of immunology (Baltimore, Md. : 1950) 2005;175;10;6909-14

  • On the integration of alcohol-related quantitative trait loci and gene expression analyses.

    Hitzemann R, Reed C, Malmanger B, Lawler M, Hitzemann B, Cunningham B, McWeeney S, Belknap J, Harrington C, Buck K, Phillips T and Crabbe J

    Department of Behavioral Neuroscience and Portland Alcohol Research Center, Oregon Health & Science University, Portland, Oregon 97201, USA. hitzeman@ohsu.edu

    Background: Quantitative trait loci (QTLs) have been detected for a wide variety of ethanol-related phenotypes, including acute and chronic ethanol withdrawal, acute locomotor activation, and ethanol preference. This study was undertaken to determine whether the process of moving from QTL to quantitative trait gene (QTG) could be accelerated by the integration of functional genomics (gene expression) into the analysis strategy.

    Methods: Six ethanol-related QTLs, all detected in C57BL/6J and DBA/2J intercrosses were entered into the analysis. Each of the QTLs had been confirmed in independent genetic models at least once; the cumulative probabilities for QTL existence ranged from 10 to 10. Brain gene expression data for the C57BL/6 and DBA/2 strains (n = 6 per strain) and an F2 intercross sample (n = 56) derived from these strains were obtained by using the Affymetrix U74Av2 and 430A arrays; additional data with the U74Av2 array were available for the extended amygdala, dorsomedial striatum, and hippocampus. Low-level analysis was performed by using multiple methods to determine the likelihood that a transcript was truly differentially expressed. For the 430A array data, the F2 sample was used to determine which of the differentially expressed transcripts within the QTL intervals were cis-regulated and, thus, strong candidates for QTGs.

    Results: Within the 6 QTL intervals, 39 transcripts (430A array) were identified as being highly likely to be differentially expressed between the C57BL/6 and DBA/2 strains at a false discovery rate of 0.01 or better. Twenty-eight of these transcripts showed significant (logarithm of odds > or =3.6) to highly significant (logarithm of odds >7) cis-regulation. The process correctly detected Mpdz (chromosome 4) as a candidate QTG for acute withdrawal.

    Conclusions: Although improvements are needed in the expression databases, the integration of QTL and gene expression analyses seems to have potential as a high-throughput strategy for moving from QTL to QTG.

    Funded by: NIAAA NIH HHS: AA 06243, AA 10760, AA 11043, AA 11114, AA 11384, P60 AA010760; NIDA NIH HHS: DA 05228

    Alcoholism, clinical and experimental research 2004;28;10;1437-48

  • CD59a is the primary regulator of membrane attack complex assembly in the mouse.

    Baalasubramanian S, Harris CL, Donev RM, Mizuno M, Omidvar N, Song WC and Morgan BP

    Complement Biology Group, Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Cardiff, United Kingdom.

    Gene-deleted mice have provided a potent tool in efforts to understand the roles of complement and complement-regulating proteins in vivo. In particular, mice deficient in the membrane regulators complement receptor 1-related gene/protein y, decay-accelerating factor, or CD59 have demonstrated homeostatic relevance and backcrossing between the strains has revealed cooperativity in regulation. In mouse, genes encoding decay-accelerating factor and CD59 have been duplicated and show differential expression in tissues, complicating interpretation and extrapolation of findings to man. The first described form of CD59, CD59a, is broadly distributed and deletion of the cd59a gene causes a mild hemolytic phenotype with increased susceptibility in complement-mediated disease models. The distribution of the second form, CD59b, was originally described as testis specific, but later by some as widespread. Deletion of the cd59b gene caused a severe hemolytic and thrombotic phenotype. To apply data from these mouse models to man it is essential to know the relative distribution and functional roles of these two forms of CD59. We have generated new specific reagents and used them in sensitive quantitative analyses to comprehensively characterize expression of mRNA and protein and functional roles of CD59a and CD59b in wild-type (wt) and CD59a-negative mice. cd59b mRNA was detected only in testis and, at very low levels, in bone marrow. CD59b protein was present on mature spermatozoa and precursors and, in trace amounts, erythrocytes. Erythrocyte CD59b did not inhibit complement lysis except when CD59a was absent or blocked. These data confirm that CD59a is the primary regulator of complement membrane attack in mouse.

    Journal of immunology (Baltimore, Md. : 1950) 2004;173;6;3684-92

  • CD59a deficiency exacerbates ischemia-reperfusion injury in mice.

    Turnberg D, Botto M, Lewis M, Zhou W, Sacks SH, Morgan BP, Walport MJ and Cook HT

    Rheumatology Section, Eric Bywaters Centre, London, United Kingdom.

    The terminal complement components C5a and the membrane attack complex are involved in the pathogenesis of ischemia-reperfusion injury in many organs. CD59 is the major regulator of membrane attack complex formation. Mice deficient in the Cd59a gene (mCd59a-/-) were used to investigate the role of CD59 in renal ischemia-reperfusion injury. Unilateral ischemia-reperfusion injury was induced by clamping the left renal pedicle for 30 minutes under general anesthetic. Mice were studied at 72 hours and 2 weeks after ischemia-reperfusion injury. mCd59a-/- mice developed significantly greater tubular injury (P = 0.01), tubulointerstitial apoptosis (P = 0.02), and neutrophil influx (P = 0.04) than controls at 72 hours after ischemia-reperfusion. Two weeks after ischemia-reperfusion, mCd59a-/- mice exhibited more severe tubular damage predominantly in a corticomedullary distribution than controls (P = 0.02). Quantification of interstitial leukocytes revealed significantly greater numbers of infiltrating lymphocytes (but not macrophages) in mCd59a-/- mice than controls (P = 0.04) at 2 weeks. At both time points, significantly more C9 (as a marker of membrane attack complex) deposition occurred in a peritubular distribution in mCd59a-/- mice than controls. In conclusion, these results demonstrate that the lack of CD59a, by allowing unregulated membrane attack complex deposition, exacerbates both the tubular injury and the interstitial leukocyte infiltrate after ischemia-reperfusion injury in mice.

    Funded by: Medical Research Council: G0000771

    The American journal of pathology 2004;165;3;825-32

  • Deletion of the gene encoding CD59a in mice increases disease severity in a murine model of rheumatoid arthritis.

    Williams AS, Mizuno M, Richards PJ, Holt DS and Morgan BP

    University of Wales College of Medicine, Cardiff, UK.

    Objective: To investigate the roles of CD59a in the protection of joint tissue in the context of murine antigen-induced arthritis (AIA).

    Methods: AIA was triggered in CD59a-deficient (CD59a(-/-)) mice and in CD59a-sufficient (CD59a(+/+)) controls; the course and severity of disease were compared between groups. The effects on arthritis of restoring CD59 to the joint in CD59a(-/-) mice by use of a membrane-targeted recombinant CD59 were also explored.

    Results: Disease, as assessed clinically by measurement of joint swelling on day 1 (P < 0.0001), day 2 (P < 0.01), and day 7 (P < 0.02) and histologically from indicators of joint damage on day 21 (P < 0.02), was significantly enhanced in CD59a(-/-) mice compared with CD59a(+/+) wild-type controls. Membrane attack complex (MAC) deposition in the arthritic joints of CD59a(-/-) mice was also increased compared with that in the joints of CD59a(+/+) controls. Restitution of CD59 activity in joints of CD59a(-/-) mice was attempted with soluble recombinant rat CD59 (sCD59) or with a novel membrane-targeted rat CD59 derivative (sCD59-APT542). Strong immunohistochemical staining of the synovial membrane and subsynovial tissue was apparent in sCD59-APT542-injected joints, but not in joints injected with untargeted sCD59. Intraarticular administration of sCD59-APT542 markedly ameliorated disease severity in CD59a(-/-) mice, knee swelling was significantly reduced over the time course of the disease, and joint damage, assessed histologically, was significantly milder on day 21 (P < 0.05).

    Conclusion: These data firmly implicate the MAC of complement as a major effector of joint damage in the murine AIA model of rheumatoid arthritis (RA), and they provide a rationale for the inhibition of MAC assembly as a therapeutic strategy for RA.

    Arthritis and rheumatism 2004;50;9;3035-44

  • Genomic analysis of mouse retinal development.

    Blackshaw S, Harpavat S, Trimarchi J, Cai L, Huang H, Kuo WP, Weber G, Lee K, Fraioli RE, Cho SH, Yung R, Asch E, Ohno-Machado L, Wong WH and Cepko CL

    Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts, USA.

    The vertebrate retina is comprised of seven major cell types that are generated in overlapping but well-defined intervals. To identify genes that might regulate retinal development, gene expression in the developing retina was profiled at multiple time points using serial analysis of gene expression (SAGE). The expression patterns of 1,051 genes that showed developmentally dynamic expression by SAGE were investigated using in situ hybridization. A molecular atlas of gene expression in the developing and mature retina was thereby constructed, along with a taxonomic classification of developmental gene expression patterns. Genes were identified that label both temporal and spatial subsets of mitotic progenitor cells. For each developing and mature major retinal cell type, genes selectively expressed in that cell type were identified. The gene expression profiles of retinal Müller glia and mitotic progenitor cells were found to be highly similar, suggesting that Müller glia might serve to produce multiple retinal cell types under the right conditions. In addition, multiple transcripts that were evolutionarily conserved that did not appear to encode open reading frames of more than 100 amino acids in length ("noncoding RNAs") were found to be dynamically and specifically expressed in developing and mature retinal cell types. Finally, many photoreceptor-enriched genes that mapped to chromosomal intervals containing retinal disease genes were identified. These data serve as a starting point for functional investigations of the roles of these genes in retinal development and physiology.

    Funded by: NCI NIH HHS: P20 CA096470, P20 CA96470; NEI NIH HHS: EY08064, R01 EY008064

    PLoS biology 2004;2;9;E247

  • Critical protection from renal ischemia reperfusion injury by CD55 and CD59.

    Yamada K, Miwa T, Liu J, Nangaku M and Song WC

    Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    Renal ischemia-reperfusion injury (IRI) is a feature of ischemic acute renal failure and it impacts both short- and long-term graft survival after kidney transplantation. Complement activation has been implicated in renal IRI, but its mechanism of action is uncertain and the determinants of complement activation during IRI remain poorly understood. We engineered mice deficient in two membrane complement regulatory proteins, CD55 and CD59, and used them to investigate the role of these endogenous complement inhibitors in renal IRI. CD55-deficient (CD55(-/-)), but not CD59-deficient (CD59(-/-)), mice exhibited increased renal IRI as indicated by significantly elevated blood urea nitrogen levels, histological scores, and neutrophil infiltration. Remarkably, although CD59 deficiency alone was inconsequential, CD55/CD59 double deficiency greatly exacerbated IRI. Severe IRI in CD55(-/-)CD59(-/-) mice was accompanied by endothelial deposition of C3 and the membrane attack complex (MAC) and medullary capillary thrombosis. Complement depletion in CD55(-/-)CD59(-/-) mice with cobra venom factor prevented these effects. Thus, CD55 and CD59 act synergistically to inhibit complement-mediated renal IRI, and abrogation of their function leads to MAC-induced microvascular injury and dysfunction that may exacerbate the initial ischemic assault. Our findings suggest a rationale for anti-complement therapies aimed at preventing microvascular injury during ischemia reperfusion, and the CD55(-/-)CD59(-/-) mouse provides a useful animal model in this regard.

    Funded by: NIAID NIH HHS: AI-44970, AI-49344

    Journal of immunology (Baltimore, Md. : 1950) 2004;172;6;3869-75

  • Respective roles of decay-accelerating factor and CD59 in circumventing glomerular injury in acute nephrotoxic serum nephritis.

    Lin F, Salant DJ, Meyerson H, Emancipator S, Morgan BP and Medof ME

    Institute of Pathology, Case Western Reserve University, Cleveland, OH 44106, USA.

    Decay-accelerating factor (DAF or CD55) and CD59 are regulators that protect self cells from C3b deposition and C5b-9 assembly on their surfaces. Their relative roles in protecting glomeruli in immune-mediated renal diseases in vivo are unknown. We induced nephrotoxic serum (NTS) nephritis in Daf1(-/-), CD59a(-/-), Daf1(-/-)CD59a(-/-), and wild-type (WT) mice by administering NTS IgG. After 18 h, we assessed proteinuria, and performed histological, immunohistochemical, and electron microscopic analyses of kidneys. Twenty-four mice in each group were studied. Baseline albuminuria in the Daf1(-/-), CD59a(-/-), and Daf1(-/-)CD59a(-/-) mice was 82, 83, and 139 as compared with 92 microg/mg creatinine in the WT controls (p > 0.1). After NTS, albuminuria in CD59a(-/-) and WT mice (186 +/- 154 and 183 +/- 137 microg/mg creatinine, p > 0.1) was similar. In contrast, Daf1(-/-) mice developed severe albuminuria (378 +/- 520, p < 0.05) that was further exacerbated in Daf1(-/-)CD59a(-/-) mice (577 +/- 785 micro g/mg creatinine, p < 0.05). Glomerular histology showed essentially no infiltrating leukocytes in any group. In contrast, electron microscopy revealed prominent podocyte foot process effacement in Daf1(-/-) mice with more widespread and severe damage in the double knockouts compared with only mild focal changes in CD59a(-/-) or WT mice. In all animals, deposition of administered (sheep) NTS Ig was equivalent. This contrasted with marked deposition of both C3 and C9 in Daf1(-/-)CD59a(-/-) and Daf1(-/-) mice, which was evident as early as 2 h post-NTS injection. The results support the proposition that in autoantibody-mediated nephritis, DAF serves as the primary barrier to classical pathway-mediated injury, while CD59 limits consequent C5b-9-mediated cell damage.

    Funded by: NEI NIH HHS: EY11373; NIAID NIH HHS: AI23598; NIDDK NIH HHS: DK30932, DK62945, K01 DK062945, K01 DK062945-02

    Journal of immunology (Baltimore, Md. : 1950) 2004;172;4;2636-42

  • Deficiency of the complement regulator CD59a enhances disease severity, demyelination and axonal injury in murine acute experimental allergic encephalomyelitis.

    Mead RJ, Neal JW, Griffiths MR, Linington C, Botto M, Lassmann H and Morgan BP

    Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Heath Park, Cardiff, UK.

    There is a growing body of evidence implicating complement and, in particular, the terminal pathway (membrane attack complex; MAC) in inducing demyelination in multiple sclerosis and experimental allergic encephalomyelitis. In this paper, we examined the disease course and pathological changes in mice deficient in the major regulator of MAC assembly, CD59a, during the course of acute experimental allergic encephalomyelitis induced by immunisation with recombinant myelin oligodendrocyte glycoprotein. Disease incidence and severity were significantly increased in CD59a-deficient mice. The extent of inflammation, demyelination and axonal injury were assessed in spinal cord cross-sections from CD59a-deficient and control mice, and all these parameters were enhanced in the absence of CD59a. Areas of myelin loss and axonal damage in CD59a-deficient mice were associated with deposits of MAC, firmly implicating MAC as a cause of the observed injury. These findings are relevant to some types of human demyelination, where abundant deposits of MAC are found in association with pathology.

    Funded by: Multiple Sclerosis Society: 560

    Laboratory investigation; a journal of technical methods and pathology 2004;84;1;21-8

  • Wnk1 kinase deficiency lowers blood pressure in mice: a gene-trap screen to identify potential targets for therapeutic intervention.

    Zambrowicz BP, Abuin A, Ramirez-Solis R, Richter LJ, Piggott J, BeltrandelRio H, Buxton EC, Edwards J, Finch RA, Friddle CJ, Gupta A, Hansen G, Hu Y, Huang W, Jaing C, Key BW, Kipp P, Kohlhauff B, Ma ZQ, Markesich D, Payne R, Potter DG, Qian N, Shaw J, Schrick J, Shi ZZ, Sparks MJ, Van Sligtenhorst I, Vogel P, Walke W, Xu N, Zhu Q, Person C and Sands AT

    Lexicon Genetics, 8800 Technology Forest Place, The Woodlands, TX 77381, USA. brian@lexgen.com

    The availability of both the mouse and human genome sequences allows for the systematic discovery of human gene function through the use of the mouse as a model system. To accelerate the genetic determination of gene function, we have developed a sequence-tagged gene-trap library of >270,000 mouse embryonic stem cell clones representing mutations in approximately 60% of mammalian genes. Through the generation and phenotypic analysis of knockout mice from this resource, we are undertaking a functional screen to identify genes regulating physiological parameters such as blood pressure. As part of this screen, mice deficient for the Wnk1 kinase gene were generated and analyzed. Genetic studies in humans have shown that large intronic deletions in WNK1 lead to its overexpression and are responsible for pseudohypoaldosteronism type II, an autosomal dominant disorder characterized by hypertension, increased renal salt reabsorption, and impaired K+ and H+ excretion. Consistent with the human genetic studies, Wnk1 heterozygous mice displayed a significant decrease in blood pressure. Mice homozygous for the Wnk1 mutation died during embryonic development before day 13 of gestation. These results demonstrate that Wnk1 is a regulator of blood pressure critical for development and illustrate the utility of a functional screen driven by a sequence-based mutagenesis approach.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;24;14109-14

  • The regulation of the complement system: insights from genetically-engineered mice.

    Turnberg D and Botto M

    Rheumatology Section, Division of Medicine, Faculty of Medicine, Imperial College, Hammersmith Campus, Du Cane Road, London W12 0NN, UK.

    The complement system is very tightly regulated by fluid-phase and membrane-bound factors that prevent injury to self-tissues. The study of genetically engineered animals with targeted deletion or gain of function mutations has highlighted the important role that many of the complement inhibitors play in vivo. The advantages and disadvantages of this type of approach are discussed and the insights gained from the investigation of these animals are reviewed.

    Molecular immunology 2003;40;2-4;145-53

  • A large-scale, gene-driven mutagenesis approach for the functional analysis of the mouse genome.

    Hansen J, Floss T, Van Sloun P, Füchtbauer EM, Vauti F, Arnold HH, Schnütgen F, Wurst W, von Melchner H and Ruiz P

    Institute of Developmental Genetics, GSF-National Research Center for Environment and Health, D-85764 Neuherberg, Germany.

    A major challenge of the postgenomic era is the functional characterization of every single gene within the mammalian genome. In an effort to address this challenge, we assembled a collection of mutations in mouse embryonic stem (ES) cells, which is the largest publicly accessible collection of such mutations to date. Using four different gene-trap vectors, we generated 5,142 sequences adjacent to the gene-trap integration sites (gene-trap sequence tags; http://genetrap.de) from >11,000 ES cell clones. Although most of the gene-trap vector insertions occurred randomly throughout the genome, we found both vector-independent and vector-specific integration "hot spots." Because >50% of the hot spots were vector-specific, we conclude that the most effective way to saturate the mouse genome with gene-trap insertions is by using a combination of gene-trap vectors. When a random sample of gene-trap integrations was passaged to the germ line, 59% (17 of 29) produced an observable phenotype in transgenic mice, a frequency similar to that achieved by conventional gene targeting. Thus, gene trapping allows a large-scale and cost-effective production of ES cell clones with mutations distributed throughout the genome, a resource likely to accelerate genome annotation and the in vivo modeling of human disease.

    Proceedings of the National Academy of Sciences of the United States of America 2003;100;17;9918-22

  • BayGenomics: a resource of insertional mutations in mouse embryonic stem cells.

    Stryke D, Kawamoto M, Huang CC, Johns SJ, King LA, Harper CA, Meng EC, Lee RE, Yee A, L'Italien L, Chuang PT, Young SG, Skarnes WC, Babbitt PC and Ferrin TE

    Department of Pharmaceutical Chemistry, University of California San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.

    The BayGenomics gene-trap resource (http://baygenomics.ucsf.edu) provides researchers with access to thousands of mouse embryonic stem (ES) cell lines harboring characterized insertional mutations in both known and novel genes. Each cell line contains an insertional mutation in a specific gene. The identity of the gene that has been interrupted can be determined from a DNA sequence tag. Approximately 75% of our cell lines contain insertional mutations in known mouse genes or genes that share strong sequence similarities with genes that have been identified in other organisms. These cell lines readily transmit the mutation to the germline of mice and many mutant lines of mice have already been generated from this resource. BayGenomics provides facile access to our entire database, including sequence tags for each mutant ES cell line, through the World Wide Web. Investigators can browse our resource, search for specific entries, download any portion of our database and BLAST sequences of interest against our entire set of cell line sequence tags. They can then obtain the mutant ES cell line for the purpose of generating knockout mice.

    Funded by: NCRR NIH HHS: P41 RR001081, P41 RR01081; NHLBI NIH HHS: U01 HL066621, U01 HL66621

    Nucleic acids research 2003;31;1;278-81

  • Crry, but not CD59 and DAF, is indispensable for murine erythrocyte protection in vivo from spontaneous complement attack.

    Miwa T, Zhou L, Hilliard B, Molina H and Song WC

    Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.

    Decay-accelerating factor (DAF) and CD59 are 2 glycosylphosphatidylinositol-anchored membrane proteins that inhibit complement activation at the C3 and C5b-9 step, respectively. CD59 is considered critical for protecting erythrocytes from spontaneous complement attack, as deficiency of CD59 or CD59/DAF, but not of DAF alone, on human erythrocytes renders them sensitive to complement lysis in paroxysmal nocturnal hemoglobinuria syndrome. To evaluate the relative roles of CD59 and DAF in vivo, we have generated and studied a CD59 knockout and a CD59/DAF double-knockout mouse. CD59-deficient and CD59/DAF-double-deficient mouse erythrocytes were highly sensitive to antibody-induced complement lysis in vitro, yet neither CD59 knockout nor CD59/DAF double-knockout mouse developed spontaneous hemolytic anemia. Consistent with the latter observation, erythrocytes from the 2 strains of mutant mice were shown to have a normal lifespan in vivo. In contrast, mouse erythrocytes deficient in complement receptor 1 (CR1)-related gene y (Crry), a membrane C3 inhibitor with DAF and membrane cofactor protein activities, were rapidly eliminated from the circulation by a complement-dependent mechanism. Compared with DAF-deficient erythrocytes, Crry-deficient erythrocytes incurred higher levels of spontaneous C3 deposition in vivo. These findings demonstrate that CD59 and DAF are not indispensable on murine erythrocytes. Rather, effective C3 regulation on the cell surface, provided by Crry rather than DAF, is necessary for mouse erythrocytes to resist spontaneous complement attack. Our results raise the possibility that proper control of C3 activation may also be critical on human erythrocytes, where CR1 but not DAF could be the principal regulator of spontaneous C3 activation.

    Funded by: NIAID NIH HHS: AI 44970

    Blood 2002;99;10;3707-16

  • Genomic structure, functional comparison, and tissue distribution of mouse Cd59a and Cd59b.

    Qin X, Miwa T, Aktas H, Gao M, Lee C, Qian YM, Morton CC, Shahsafaei A, Song WC and Halperin JA

    Harvard Medical School, Laboratory for Membrane Transport, 240 Longwood Ave., C1-607, Boston, Massachusetts 02115, USA.

    CD59 is a crucial complement regulatory protein that inhibits the terminal step of the complement activation cascade by interfering with the binding of C9 to C5b-8, thus preventing the formation of the membrane attack complex (MAC). We recently reported that the mouse genome contains two Cd59 genes, while the human and rat genomes each contain only one Cd59 gene (Qian et al. 2000). Here, we describe the genomic structure, comparative activity, and tissue distribution of these two mouse genes, designated Cd59a and Cd59b. The mouse Cd59 genes encompass a total of 45.6 kb with each gene having four exons. Cd59a spans 19 kb, and Cd59b spans 15 kb, with approximately 11.6 kb of genomic DNA separating the two genes. The overall sequence similarity between Cd59a and Cd59b is approximately 60%. The sequence similarity between exon 2, exon 3, and exon 4 and the respective flanking regions between the two genes is over 85%, but exon 1 and its flanking regions are totally different. Comparative studies of the activity of both genes as inhibitors of MAC formation revealed that Cd59b has a specific activity that is six times higher than that of Cd59a. Using polyclonal antibodies specific to either Cd59a or Cd59b, we showed that Cd59a and Cd59b are both widely expressed in the kidneys, brain, lungs, spleen, and testis, as well as in the blood vessels of most mouse tissues. Interestingly, testicular Cd59a appeared to be expressed exclusively in spermatids, whereas Cd59b was expressed in more mature sperm cells. These results suggest that even though Cd59a and Cd59b are expressed in multiple tissues, they may play some different roles, particularly in reproduction.

    Funded by: NIDDK NIH HHS: 5 R01 DK52855-02

    Mammalian genome : official journal of the International Mammalian Genome Society 2001;12;8;582-9

  • Targeted deletion of the CD59 gene causes spontaneous intravascular hemolysis and hemoglobinuria.

    Holt DS, Botto M, Bygrave AE, Hanna SM, Walport MJ and Morgan BP

    Complement Biology Group, Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.

    The glycolipid-anchored glycoprotein CD59 inhibits assembly of the lytic membrane attack complex of complement by incorporation into the forming complex. Absence of CD59 and other glycolipid-anchored molecules on circulating cells in the human hemolytic disorder paroxysmal nocturnal hemoglobinuria is associated with intravascular hemolysis and thrombosis. To examine the role of CD59 in protecting host tissues in health and disease, CD59-deficient (CD59(-/-)) mice were produced by gene targeting in embryonic stem cells. Absence of CD59 was confirmed by staining cells and tissues with specific antibody. Despite the complete absence of CD59, mice were healthy and fertile. Erythrocytes in vitro displayed increased susceptibility to complement and were positive in an acidified serum lysis test. Despite this, CD59(-/-) mice were not anemic but had elevated reticulocyte counts, indicating accelerated erythrocyte turnover. Fresh plasma and urine from CD59(-/-) mice contained increased amounts of hemoglobin when compared with littermate controls, providing further evidence for spontaneous intravascular hemolysis. Intravascular hemolysis was increased following administration of cobra venom factor to trigger complement activation. CD59(-/-) mice will provide a tool for characterizing the importance of CD59 in protection of self tissues from membrane attack complex damage in health and during diseases in which complement is activated.

    Blood 2001;98;2;442-9

  • Identification and functional characterization of a new gene encoding the mouse terminal complement inhibitor CD59.

    Qian YM, Qin X, Miwa T, Sun X, Halperin JA and Song WC

    Center for Experimental Therapeutics and Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.

    CD59 is a 18- to 20-kDa, GPI-anchored membrane protein that functions as a key regulator of the terminal step of the complement activation cascade. It restricts binding of C9 to the C5b-8 complex, thereby preventing the formation of the membrane attack complex (C5b-9 of complement). A single human CD59 gene has been identified, and corresponding genetic homologues from rat, mouse, and pig have been characterized in previous studies. In this study, we report the discovery and functional characterization of a separate cd59 gene in the mouse (referred to as cd59b, the previously characterized mouse cd59 gene as cd59a). Mouse cd59b is 85% and 63% identical to cd59a at the nucleotide and amino acid level, respectively. In cDNA transfection experiments with Chinese hamster ovary cells, peptide-tagged cd59b was detected on the cell surface by flow cytometry and was shown to be susceptible to phosphatidylinositol-specific phospholipase C cleavage. Chinese hamster ovary cells expressing cd59b were significantly more resistant than control cells to human and mouse complement-mediated lysis. These results suggest that cd59b encodes a GPI-anchored protein that is functionally active as a membrane attack complex inhibitor. Northern blot analysis revealed that cd59b is expressed selectively in the mouse testis. In contrast, the major transcript of cd59a was shown to be expressed at high levels in the heart, kidney, liver, and lung, but only minimally in the testis. These results revealed the existence of two distinct cd59 genes in the mouse that are differentially regulated and that may have nonoverlapping physiological functions in vivo.

    Journal of immunology (Baltimore, Md. : 1950) 2000;165;5;2528-34

  • Genomic structure and chromosome location of the gene encoding mouse CD59.

    Holt DS, Powell MB, Rushmere NK and Morgan BP

    Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, UK.

    The gene encoding the mouse analogue of the human complement regulator CD59 was cloned using a combination of long range PCR and genomic library screening. Sequence obtained showed that its genomic structure closely resembled that of the human CD59 gene, comprising 4 exons, each separated by a long intron region. The sizes of introns and exons were comparable to those of the human gene with the exception of the third intron which is 2.5 kb in the mouse compared to 7 kb in the human gene. All exon/intron boundaries conformed to the GT-AG rules for splicing. Radiation hybrid mapping localised mouse Cd59 between D2Mit333 and D2Mit127 on chromosome 2, a region homologous with human chromosome 11p13 where the human CD59 gene is localised. These data have permitted the construction of a gene targeting vector for the generation of transgenic mice deficient in CD59.

    Cytogenetics and cell genetics 2000;89;3-4;264-7

  • The reticulocalbin gene maps to the WAGR region in human and to the Small eye Harwell deletion in mouse.

    Kent J, Lee M, Schedl A, Boyle S, Fantes J, Powell M, Rushmere N, Abbott C, van Heyningen V and Bickmore WA

    MRC Human Genetics Unit, Western General Hospital, Edinburgh, United Kingdom.

    We describe the localization of the gene encoding reticulocalbin, a Ca2+-binding protein of the endoplasmic reticulum, on human chromosome 11p13 midway between the WT1 and the PAX6 genes and show that it is hemizygously deleted in WAGR individuals. The mouse reticulocalbin gene is also shown to map to the region of conserved synteny on mouse chromosome 2 and to be deleted in the Small eye Harwell (SeyH) mutation. Loss of the reticulocalbin gene could contribute to the early lethality of SeyH and SeyDey homozygotes.

    Funded by: Medical Research Council: MC_U127527199; Wellcome Trust

    Genomics 1997;42;2;260-7

  • Molecular cloning, chromosomal localization, expression, and functional characterization of the mouse analogue of human CD59.

    Powell MB, Marchbank KJ, Rushmere NK, van den Berg CW and Morgan BP

    Department of Medical Biochemistry, University of Wales College of Medicine, Cardiff, United Kingdom.

    We have previously described the isolation and cloning of the rat analogue of the human complement inhibitor CD59 (hCD59). Using the rat CD59 (rCD59) coding region as probe, we have isolated positive cDNAs from a mouse kidney cDNA library. Sequence analysis of these clones indicated that they contained an open reading frame encoding a 124 amino acid protein. Comparisons with the known sequences of hCD59 and rCD59 suggested that the clones contained a full-length cDNA encoding the mouse analogue of CD59 (mCD59). The cDNA encoded a 81-bp 5'-flanking region, a 23 amino acid NH2-signal peptide, a 101 amino acid coding region including putative N-glycosylation sites and a glycosyl phosphatidylinositol (GPI) anchoring signal, and approximately 0.8 kb 3'-untranslated flanking region. Reverse transcriptase PCR revealed the presence of mCD59 mRNA in all mouse tissues examined. The gene for mCD59 was mapped by fluorescence in situ hybridization to the E2-E4 region of mouse chromosome 2, a region that includes areas syntenous with the location of the human CD59 gene on chromosome 11p13. Expression of mCD59 in a CD59-negative human cell line conferred protection against lysis by complement from rodent, human, and several other species, confirming that mCD59 was the functional analogue of hCD59 and that function was not species restricted. The expressed protein was glycosyl phosphatidylinositol anchored as demonstrated by its partial removal from U937 cells on treatment with phosphatidylinositol-specific phospholipase C. Abs raised against the expressed protein demonstrated the presence of mCD59 on all mouse blood cell types and on several mouse cell lines and neutralized function of mCD59 on mouse E and expressed on U937. Western blot analysis revealed that both expressed and endogenous mCD59 had a molecular mass of 22 to 24 kDa.

    Funded by: Wellcome Trust

    Journal of immunology (Baltimore, Md. : 1950) 1997;158;4;1692-702

Gene lists (2)

Gene List Source Species Name Description Gene count
L00000001 G2C Mus musculus Mouse PSD Mouse PSD adapted from Collins et al (2006) 1080
L00000008 G2C Mus musculus Mouse PSP Mouse PSP adapted from Collins et al (2006) 1121
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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