Protruding disordered loop of gC1qR is specifically exposed and related to antiapoptotic property in germ cell lineage

We established a monoclonal antibody (MAb), 5G9, with the use of a fixed seminoma tissue from an archival paraffin-embedded specimen, as an immunogen. Without antigen retrieval, positive 5G9-immunohistochemical staining was confined mostly to primordial germ cells, spermatogonia and various germ cell tumors. 5G9 recognized a mitochondrial 32-kD protein with an isoelectric point of pH 4.2, identified as a multifunctional ubiquitous protein, receptor for globular head of C1q (gC1qR), whose epitope was mapped in a disordered loop connecting the β3 and the β4 strands. Reflecting the ubiquitous distribution of gC1qR, with antigen retrieval, 5G9 was found reactive to a wide range of normal and tumor tissues. Since several co-precipitated and phosphorylated bands were observed in various human cell lines but not in germ cell tumor cell lines by in vitro phosphorylation assay, we speculate that the epitope of gC1qR is specifically unmasked in the germ cell lineage. By reducing gC1qR by siRNA, a significant increase was observed in the number of apoptotic cells in ITO-II and TCam-2 cell lines, but to a lesser extent in the Colo201 colon cancer cell line, showing an antiapoptotic property of gC1qR in the germ cells. Since protein–protein interaction is partially preserved by fixation, archival paraffin-embedded specimens can be a valuable source of immunogens for generating monoclonal antibodies (MAbs) that recognize tissue-specific protein conformation.     

gC1qR expression in normal and pathologic human tissues: differential expression in tissues of epithelial and mesenchymal origin

The gC1qR (i.e., gC1q receptor, gC1q binding protein, p32, p33) is a multifunctional cellular protein that interacts with components of the complement, kinin, and coagulation cascades and select microbial pathogens. Enhanced gC1qR expression has been reported in adenocarcinomas arising in a variety of organs. The present study compared gC1qR expression in normal, inflammatory, dysplastic, and malignant tissue of epithelial and mesenchymal origin. gC1qR expression was visualized in tissue sections by immunohistochemistry using the 60.11 monoclonal antibody (i.e., IgG(1) mouse monoclonal antibody directed against gC1qR) and the UltraVision LP Detection System. Sections were counterstained with hematoxylin and examined by light microscopy. Strongest gC1qR expression was noted in epithelial tumors of breast, prostate, liver, lung, and colon, as well as in squamous and basal cell carcinoma of the skin. However, increased gC1qR staining was appreciated also in inflammatory and proliferative lesions of the same cell types, as well as in normal continuously dividing cells. In contrast, tumors of mesenchymal origin generally stained weakly, with the exception of osteoblasts, which stained in both benign and malignant tissues. The data suggest that increased gC1qR expression may be a marker of benign and pathologic cell proliferation, particularly in cells of epithelial origin, with potential diagnostic and therapeutic applications.     

Bioinformatic identification of genes encoding C1q-domain containing proteins in zebrafish

C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular (gC1q) domain similar with C1q was also found in many non-complement C1q-domain-containing (C1qDC) proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor (TNF) ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing (C1qDC) proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.     

Structure of the Third Intracellular Loop of the Vasopressin V2 Receptor and Conformational Changes upon Binding to gC1qR

The V2 vasopressin receptor is a G-protein-coupled receptor that regulates the renal antidiuretic response. Its third intracellular loop is involved in the coupling not only with the GαS protein but also with gC1qR, a potential chaperone of G-protein-coupled receptors. In this report, we describe the NMR solution structure of the V2 i3 loop under a cyclized form (i3_cyc) and characterize its interaction with gC1qR. i3_cyc formed a left-twisted α-helical hairpin structure. The building of a model of the entire V2 receptor including the i3_cyc NMR structure clarified the side-chain orientation of charged residues, in agreement with literature mutagenesis reports. In the model, the i3 loop formed a rigid helical column, protruding deep inside the cytoplasm, as does the i3 loop in the recently elucidated structure of squid rhodopsin. However, its higher packing angle resulted in a different structural motif at the intracellular interface, which may be important for the specific recognition of GαS. Moreover, we could estimate the apparent K_d of the i3_cyc/gC1qR complex by anisotropy fluorescence. Using a shorter and more soluble version of i3_cyc, which encompassed the putative site of gC1qR binding, we showed by NMR saturation transfer difference spectroscopy that the binding surface corresponded to the central arginine cluster. Binding to gC1qR induced the folding of the otherwise disordered short peptide into a spiral-like path formed by a succession of I and IV turns. Our simulations suggested that this folding would rigidify the arginine cluster in the entire i3 loop and would alter the conformation of the cytosolic extensions of TM V and TM VI helices. In agreement with this conformational rearrangement, we observed that binding of gC1qR to the full-length receptor modifies the intrinsic tryptophan fluorescence binding curves of V2 to an antagonist.     

Specific Interactions Between gC1qR and α1‐Adrenoceptor Subtypes

Abstract

The multi‐functional protein gC1qR has been reported to interact with an arginine‐rich motif in the C‐tail of hamster α_1B‐adrenoceptors (ARs), controlling their expression and subcellular localization. Since a similar motif is present in α_1D‐, but not α_1A‐ARs, we studied the specificity of this interaction. Human α₁‐ARs, tagged at their amino termini with Flag epitopes, were coexpressed in HEK293 cells with gC1qR containing a hemaglutinin (HA) tag at its carboxy terminus. Immunoprecipitation studies showed that Flag‐α_1B‐ or α_1D‐, but not α_1A‐ARs, caused coimmunoprecipitation of HA‐gC1qR, while immunoprecipitation of HA‐gC1qR caused coimmunoprecipitation of Flag‐α_1B‐ or α_1D‐, but not α_1A‐ARs, supporting specific interactions between subtypes. C‐terminal truncation of Flag‐α₁‐ARs prevented interaction with HA‐gC1qR, supporting previous conclusions about the role of the C‐terminal arginine‐rich motif. These studies suggest that gC1qR interacts specifically with α_1B‐ and α_1D‐, but not α_1A‐ARs, and this interaction depends on the presence of an intact C‐tail.     

Bioinformatic identification of genes encoding Clq-domaincontaining proteins in zebrafish

C1q is the first subcomponent of classical pathway in the complement system and a major link between innate and acquired immunities. The globular （gC1q） domain similar with C1q was also found in many non-complement C1q-domain-containing （C1qDC） proteins which have similar crystal structure to that of the multifunctional tumor necrosis factor （TNF） ligand family, and also have diverse functions. In this study, we identified a total of 52 independent gene sequences encoding C1q-domain-containing proteins through comprehensive searches of zebrafish genome, cDNA and EST databases. In comparison to 31 orthologous genes in human and different numbers in other species, a significant selective pressure was suggested during vertebrate evolution. Domain organization of C1q-domain-containing （C1qDC） proteins mainly includes a leading signal peptide, a collagen-like region of variable length, and a C-terminal C1q domain. There are 11 highly conserved residues within the C1q domain, among which 2 are invariant within the zebrafish gene set. A more extensive database searches also revealed homologous C1qDC proteins in other vertebrates, invertebrates and even bacterium, but no homologous sequences for encoding C1qDC proteins were found in many species that have a more recent evolutionary history with zebrafish. Therefore, further studies on C1q-domain-containing genes among different species will help us understand evolutionary mechanism of innate and acquired immunities.     

Excessive reactive oxygen species induces apoptosis in fibroblasts: role of mitochondrially accumulated hyaluronic acid binding protein 1 (HABP1/p32/gC1qR)

Constitutively expressed HABP1 in normal murine fibroblast cell line induces growth perturbation, morphological abnormalities along with initiation of apoptosis. Here, we demonstrate that though HABP1 accumulation started in mitochondria from 48 hr of growth, induction of apoptosis with the release of cytochrome c and apoptosome complex formation occurred only after 60 hr. This mitochondrial dysfunction was due to gradual increase in ROS generation in HABP1 overexpressing cells. Along with ROS generation, increased Ca 2+ influx in mitochondria leading to drop in membrane potential was evident. Interestingly, upon expression of HABP1, the respiratory chain complex I was shown to be significantly inhibited. Electronmicrograph confirmed defective mitochondrial ultrastructure. The reduction in oxidant generation and drop in apoptotic cell population accomplished by disruption of HABP1 expression, corroborating the fact that excess ROS generation in HABP1 overexpressing cells leading to apoptosis was due to mitochondrial HABP1 accumulation.     

C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus

Murine C3d were utilized to enhance immunogenicity of pseudorabies virus (PrV) gC DNA vaccination. Three copies of C3d and four copies of CR2-binding domain M28(4) were fused, respectively, to truncated gC gene encoding soluble glycoprotein C (sgC) in pcDNA3.1. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine. The antibody ELISA titer for sgC-C3d3 DNA was 49-fold more than that for sgC DNA, and the neutralizing antibody obtained 8-fold rise. Protection of mice from death after lethal PrV (316 LD50) challenge was augmented from 25% to 100%. Furthermore, C3d fusion increased Th2-biased immune response by inducing IL-4 production. The IL-4 level for sgC-C3d3 DNA immunization approached that for the inactivated vaccine. Compared to C3d, M28 enhanced sgC DNA immunogenicity to a lesser extent. In conclusion, we demonstrated that murine C3d fusion significantly enhanced gC DNA immunity by directing Th1-biased to a balanced and more effective Th1/Th2 response.     

p32 Is a Novel Target for Viral Protein ICP34.5 of Herpes Simplex Virus Type 1 and Facilitates Viral Nuclear Egress

As a large double-stranded DNA virus, herpes simplex virus type 1 (HSV-1) assembles capsids in the nucleus where the viral particles exit by budding through the inner nuclear membrane. Although a number of viral and host proteins are involved, the machinery of viral egress is not well understood. In a search for host interacting proteins of ICP34.5, which is a virulence factor of HSV-1, we identified a cellular protein, p32 (gC1qR/HABP1), by mass spectrophotometer analysis. When expressed, ICP34.5 associated with p32 in mammalian cells. Upon HSV-1 infection, p32 was recruited to the inner nuclear membrane by ICP34.5, which paralleled the phosphorylation and rearrangement of nuclear lamina. Knockdown of p32 in HSV-1-infected cells significantly reduced the production of cell-free viruses, suggesting that p32 is a mediator of HSV-1 nuclear egress. These observations suggest that the interaction between HSV-1 ICP34.5 and p32 leads to the disintegration of nuclear lamina and facilitates the nuclear egress of HSV-1 particles.     

猕猴B病毒gC蛋白特异性抗原表位的合成和表达

目的获得B病毒gC蛋白的特异性表位抗原。方法利用长片段基因合成的方法,合成B病毒C蛋白的特性抗原表位基因,将该基因连接到pMAL-5x载体,转化到BL21受体菌进行表达,并纯化表达产物。结果成功的获得了B病毒gC蛋白的特异性抗原蛋白,该蛋白以可溶的形式表达。结论利用原核表达系统,可以产生B病毒gC蛋白的可溶性抗原,可以作为B病毒的检测抗原。