A novel chicken membrane-associated complement regulatory protein: molecular cloning and functional characterization

A cDNA encoding a membrane-associated complement (C) regulatory protein was identified here for the first time in an oviparous vertebrate, chicken. This protein, named Cremp, possessed five short consensus repeats (SCRs) and one SCR-like domain followed by a transmembrane domain and a cytoplasmic tail. SCR1/SCR2 of Cremp were 43.6% identical with SCR2/SCR3 of human decay-accelerating factor (CD55), and SCR3/SCR4 were 45.3% identical with those of human membrane cofactor protein (CD46). Cremp is likely to be an ancestral hybrid protein of human decay-accelerating factor and membrane cofactor protein rather than a homolog of rodent C receptor 1-related protein y, which structurally resembles human CR1 (CD35). Chinese hamster ovary cells transfected with Cremp were efficiently protected from chicken C but not from human or rabbit C in both classical and alternative pathways. Thus, chicken Cremp is a membrane C regulator for cell protection against homologous C. Cremp mRNA was seen as a doublet comprised of a faint band of 2.2 kb and a thick band of 3.0 kb on RNA blotting analysis. An Ab against chicken Cremp recognized a single band of 46.8 kDa on immunoblotting. mRNA and protein of Cremp were ubiquitously expressed in all chicken organs tested. Minute amounts of dimer were present in some tissues. Surface expression of Cremp was confirmed by flow cytometry and immunofluorescence analysis. These results suggested that even in nonmammals a C regulatory membrane protein with ubiquitous tissue distribution should be a prerequisite for protection of host cells from homologous C attack.     

Regulator of complement activation (RCA) gene cluster in <Emphasis Type="Italic">Xenopus tropicalis</Emphasis>

Genome and expressed sequence tag information of Xenopus tropicalis suggested that short-consensus repeat (SCR)-containing proteins are encoded by three genes that are mapped within a 300-kb downstream of PFKFB2, which is a marker gene for the regulator of complement activation (RCA) loci in human and chicken. Based on this observation, we cloned the three cDNAs of these proteins using 3′- or 5′-RACE technique. Since their primary structures and locations of the proximity to the PFKFB2 locus, we named them amphibian RCA protein (ARC) 1, 2, and 3. Expression in human HEK293 or CHO cells suggested that ARC1 is a soluble protein of Mr ∼67 kDa, ARC2 is a membrane protein with Mr 44 kDa, and ARC3 a secretary protein with a putative transmembrane region. They were N-glycosylated during maturation. In human and chicken RCA clusters, the order in which genes for soluble, GPI-anchored, and membrane forms of SCR proteins are arranged is from the distant to proximity to the PFKFB2 gene. However, the amphibian ARC1, 2, and 3 resembled one another and did not reflect the same order found in human and chicken RCA genes. This may be due to self-duplication of ARCs to form a family, and it evolved after the amphibia separated from the ancestor of the amniotes, which possessed soluble, GPI-anchored, and membrane forms of SCR protein members. Taken together, frog possesses a RCA locus, but the constitution of the ARC proteins differs from that of the amniotes with a unique self-resemblance.     

Regulator of complement activation (RCA) group 2 gene cluster in zebrafish: identification, expression, and evolution

The activation of the complement system is tightly regulated by a group of plasma and cell membrane-associated proteins for host cell protection. In humans, these regulatory protein genes are clustered in a region named the regulator of complement activation (RCA) gene locus and can be categorized into two groups. The group 1 gene cluster has been reported in zebrafish, but information regarding the RCA locus remains scarce in fish. Here we identified two closely linked RCA group 2 genes in zebrafish, ZRC1 and ZRC2, which had all the features characteristic of known RCA group 2 genes. Both ZRC1 and ZRC2 were closely linked to the PFKFB1 gene and located 17 Mkb downstream of the PFKFB2 gene; in contrast, RCA group 2 genes are closely linked to PFKFB2 in frogs, chickens, and humans. However, both the direction of the RCA group 2 genes relative to PFKFB2 and the order of the RCA group 2 gene-encoded proteins in zebrafish were comparable to those in frogs, chickens, and humans. ZRC1 and ZRC2 shared 71.1% identity to each other, implicating that they might have originated by gene duplication after the split of the fish/mammalian common ancestor. Moreover, ZRC1 and ZRC2 encoded a membrane-associated protein and a soluble protein, respectively, and displayed different expression patterns, suggesting that functional divergence has already occurred. This is the first report showing the presence of the RCA group 2 cluster as well as the membrane-associated complement regulatory protein in zebrafish, providing a better understanding of the origin and evolution of RCA proteins.     

Secretion of foreign proteins mediated by chicken lysozyme gene regulatory sequences.

Exploitation of the insulating properties of the complete chicken lysozyme gene domain may facilitate the production of transgenic chicken bioreactors with the capacity to deposit valuable proteins in the egg white. Chimeric genes consisting of the chicken lysozyme gene regulatory sequences and sequences encoding foreign proteins could be inserted randomly into the chicken genome and retain appropriate expression levels. The research reported here established that chicken lysozyme gene regulatory sequences can be used to direct the production and secretion of green fluorescent protein (used as a reporter protein) in transiently transfected chicken blastodermal cells. Attempts to verify these findings in transgenic hens are currently in progress. To provide a rapid means of generating constructs encoding other foreign proteins under the control of lysozyme gene regulatory sequences that can facilitate the secretion of heterologous proteins in vivo, a generic lysozyme gene regulatory scaffold was created using a poxvirus-mediated gene targeting system.     

CSMD1 is a novel multiple domain complement-regulatory protein highly expressed in the central nervous system and epithelial tissues

In this study, we describe the identification and in vitro functional activity of a novel multiple domain complement regulatory protein discovered based on its homology to short consensus repeat (SCR)-containing proteins of the regulators of complement activation (RCA) gene family. The rat cDNA encodes a predicted 388-kDa protein consisting of 14 N-terminal CUB domains that are separated from each other by a SCR followed by 15 tandem SCR domains, a transmembrane domain, and a short cytoplasmic tail. This protein is the homolog of the human protein of unknown function called the CUB and sushi multiple domains 1 (CSMD1) protein. A cloning strategy that incorporates the two C-terminal CUB-SCR domains and 12 of the tandem SCR repeats was used to produce a soluble rat CSMD1 protein. This protein blocked classical complement pathway activation in a comparable fashion with rat Crry but did not block alternative pathway activation. Analysis of CSMD1 mRNA expression by in situ hybridization and immunolabeling of neurons indicates that the primary sites of synthesis are the developing CNS and epithelial tissues. Of particular significance is the enrichment of CSMD1 in the nerve growth cone, the amoeboid-leading edge of the growing neuron. These results suggest that CSMD1 may be an important regulator of complement activation and inflammation in the developing CNS, and that it may also play a role in the context of growth cone function.     

Complement-dependent enhancement of CD8+ T cell immunity to lymphocytic choriomeningitis virus infection in decay-accelerating factor-deficient mice

Decay-accelerating factor (DAF, CD55) is a GPI-anchored membrane protein that regulates complement activation on autologous cells. In addition to protecting host tissues from complement attack, DAF has been shown to inhibit CD4+ T cell immunity in the setting of model Ag immunization. However, whether DAF regulates natural T cell immune response during pathogenic infection is not known. We describe in this study a striking regulatory effect of DAF on the CD8+ T cell response to lymphocytic choriomeningitis virus (LCMV) infection. Compared with wild-type mice, DAF knockout (Daf-1(-/-)) mice had markedly increased expansion in the spleen of total and viral Ag-specific CD8+ T cells after acute or chronic LCMV infection. Splenocytes from LCMV-infected Daf-1(-/-) mice also displayed significantly higher killing activity than cells from wild-type mice toward viral Ag-loaded target cells, and Daf-1(-/-) mice cleared LCMV more efficiently. Importantly, deletion of the complement protein C3 or the receptor for the anaphylatoxin C5a (C5aR) from Daf-1(-/-) mice reversed the enhanced CD8+ T cell immunity phenotype. These results demonstrate that DAF is an important regulator of CD8+ T cell immunity in viral infection and that it fulfills this role by acting as a complement inhibitor to prevent virus-triggered complement activation and C5aR signaling. This mode of action of DAF contrasts with that of CD59 in viral infection and suggests that GPI-anchored membrane complement inhibitors can regulate T cell immunity to viral infection via either a complement-dependent or -independent mechanism.     

Characterization of the chicken histone H1 gene complement. Generation of a complete set of vertebrate H1 protein sequences

Sequence analysis of four chicken H1 histone genes described here completes the characterization of the full complement of six H1 genes in the chicken genome. Each of the six genes codes for a different H1 protein sequence, and these range in size from 217 to 224 amino acids. The proteins are distinct in sequence from the H1-related chicken H5 protein and appear to be analogous to the standard somatic mammalian H1 subtypes. The protein sequence data deduced from the genes represent the first complete set of vertebrate H1 protein sequences. Comparison of the chicken H1 gene noncoding sequences with each other and with H1 gene sequences from other organisms reveals conservation of an H1 gene-specific element, a G-rich element, and histone gene-specific 3' elements. Additional sequences are conserved between H1 genes of the chicken and other vertebrates. Comparisons also reveal variation in promoter and 3' elements between chicken genes that could play a role in the differential expression of H1 gene protein products.     

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses.     

虹鳟补体活化调节因子CD46的分子特征及功能

为进一步研究硬骨鱼类中补体活化调节因子的分子特征和功能,研究克隆了虹鳟(Oncorhynchus mykiss)的CD46基因,对其分子特征进行了系统分析,结果显示:虹鳟CD46基因由10个外显子和9个内含子组成,cDNA序列全长2812 bp,编码317个氨基酸,蛋白序列由1个信号肽、4个SCR结构域、1个跨膜区和1个胞内区组成,预测分子量为33.9 kD。基因组共线性分析显示,虹鳟CD46基因位于16号染色体,其基因座在脊椎动物中具有保守的共线性。组织和白细胞亚群表达分析显示,虹鳟CD46基因在各种组织和白细胞亚群中均有表达。为了进一步阐明虹鳟CD46的免疫功能,研究原核表达纯化了标签蛋白GST和融合蛋白GST-CD46。溶血活性实验表明,与GST相比, GST-CD46能够显著抑制虹鳟血清对兔红细胞的溶血活性,且呈现剂量依赖效应,表明虹鳟CD46是补体活化的调节因子。此外,研究用HEK293T细胞过表达了GFP和GFP-CD46。细胞损伤实验显示,与GFP相比, GFP-CD46能够显著抑制虹鳟血清对HEK293T细胞的损伤,进一步表明虹鳟CD46是补体活化的调节因子,能够保护细...     

miwi,a murine homolog of piwi,encodes a cytoplasmic protein essential for spermatogenesis

The piwi family genes are crucial for stem cell self-renewal, RNA silencing, and translational regulation in diverse organisms. However, their function in mammals remains unexplored. Here we report the cloning of a murine piwi gene (miwi) essential for spermatogenesis. miwi encodes a cytoplasmic protein specifically expressed in spermatocytes and spermatids. miwi(null) mice display spermatogenic arrest at the beginning of the round spermatid stage, resembling the phenotype of CREM, a master regulator of spermiogenesis. Furthermore, mRNAs of ACT (activator of CREM in testis) and CREM target genes are downregulated in miwi(null) testes. Whereas MIWI and CREM do not regulate each other's expression, MIWI complexes with mRNAs of ACT and CREM target genes. Hence, MIWI may control spermiogenesis by regulating the stability of these mRNAs.     

Cell surface expression of the C3b/C4b receptor (CR1) protects Chinese hamster ovary cells from lysis by human complement.

The C3b/C4b receptor, also known as complement receptor type 1 (CR1, CD35), is a single chain glycoprotein consisting of 30 repeating homologous protein domains known as short consensus repeats (SCR) followed by transmembrane and cytoplasmic domains. A series of recombinant proteins derived from CR1 has been prepared and assessed for the capacity to inhibit complement lysis of the host Chinese hamster ovary (CHO) cells. The full-length recombinant CR1 inhibited human complement-mediated CHO cell lysis, and the efficiency of inhibition was directly proportional to the number of receptors/cell. The SCR 15-18 of CR1, but not SCR 15-16, inhibited complement lysis of the host CHO cell, bound monomeric C3b (Kd,app = 6.5 x 10(-7) M), and dimeric C3b (Kd = 1.8 x 10(-8) M), and served as a cofactor in the proteolysis of C3b by factor I, confirming and extending the observations of Fearon and colleagues (Kalli, K. R., Hsu, P., Bartow, T. J., Ahearn, J. M., Matsumoto, A. K., Klickstein, L. B., and Fearon, D. T. (1991) J. Exp. Med. 174, 1451-1460). The SCR 1-4 of CR1, but not SCR 1-2, also inhibited complement lysis of the host CHO cell, indicating that more than two SCR are necessary and that four SCR are sufficient for optimal C4b binding to CR1. Thus, the structural requirements for C4b binding are analogous to those for C3b binding, namely, four SCR of CR1 form the binding sites for each of these proteins. CR1 has long been recognized to regulate extrinsic complement activation, that is, to bind to and promote the degradation of fluid phase C3b and of C3b attached to immune complex. These results demonstrate that CR1 is also an intrinsic regulator of complement activation in that, under appropriate conditions, CR1 inhibits complement-mediated lysis of the cell on which it is expressed.     

The host immune regulator factor H interacts via two contact sites with the PspC protein of Streptococcus pneumoniae and mediates adhesion to host epithelial cells

Pneumococcal surface protein C (PspC) of Streptococcus pneumoniae is a key virulence factor that mediates adhesion to host cells and immune evasion of the host complement. PspC binds the host immune and complement regulator factor H, which is composed of 20 short consensus repeats (SCR). This interaction contributes to pneumococcal virulence. In this study, we identified within the factor H protein two separate PspC binding regions, which were localized to SCR8-11 and SCR19-20, by using recombinant factor H deletion constructs for Western blotting assays and surface plasmon resonance studies. A detailed analysis of binding epitopes in these SCR by peptide spot arrays identified several linear binding regions within the sequences of SCR8-11 and SCR19-20. In addition, the factor H binding site was mapped within the pneumococcal PspC protein to a 121-aa-long stretch positioned in the N terminus (residues 38-158). Factor H attached to the surface of pneumococci via PspC significantly enhanced pneumococcal adherence to host epithelial and endothelial cells. This adhesion was specific and was blocked with a truncated N-terminal factor H-binding fragment of PspC. In conclusion, the acquisition of factor H by pneumococci via PspC occurs via two contact sites located in SCR8-11 and SCR19-20, and factor H attached to the surface of the pneumococcus promotes adhesion to both host epithelial and endothelial cells.     

Selection of secretory protein-encoding genes by fusion with PHO5 in Saccharomyces cerevisiae.

Secretory protein-encoding genes of Saccharomyces cerevisiae have been cloned by a novel procedure that is based on the functional selection of their fusions with acid phosphatase (APase) at the DNA level. DNA fragments that functionally replace the promoter and signal sequence-encoding regions of the PHO5 gene (encoding APase) have been obtained by positive selection from a pool of cloned random DNA fragments. Five unique DNA sequences containing the promoter, and encoding signal sequences have been isolated. We have also isolated the complete gene, SSP120, encoding one of these S. cerevisiae secretory proteins, SSP120. Gene disruption studies have shown that the SSP120 gene is not essential for viability and growth. The SSP120 amino acid (aa) sequence has 13.5% identity with the middle 88-250 aa residues of the chicken glycosylation site-binding protein. However, SSP120 disruption did not affect protein glycosylation in yeast. The present study provides an alternative approach for the isolation of genes encoding secretory proteins, in contrast to classical genetic approaches that require isolation of functionally defective mutations followed by gene isolation by functional complementation. The present procedure should contribute to our understanding of protein sorting by permitting the cloning of genes encoding proteins targeted to different organelles in the secretory pathway.