Characterization of a glycosyl-phosphatidylinositol degrading activity in Dictyostelium discoideum membranes

Antigen 117 is a glycolipid-anchored cell surface protein implicated in cell-cell cohesion of Dictyostelium discoideum amoebae. Previous studies have demonstrated that during cell aggregation some of the protein is released from the cell surface. Here we report the characterization of the enzymatic activity involved in the 117 antigen release. The data indicate that the releasing enzyme is a phosphatidylinositol phospholipase C. The data also indicate that structural features of glycolipid anchors are conserved in a variety of organisms.     

Expression and function of the Trypanosoma brucei major surface protease (GP63) genes

The genome of the African trypanosome Trypanosoma brucei (Tb) contains at least three gene families (TbMSP-A, -B, and -C) encoding homologues of the abundant major surface protease (MSP, previously called GP63), which is found in all Leishmania species. TbMSP-B mRNA occurs in both procyclic and bloodstream trypanosomes, whereas TbMSP-A and -C mRNAs are detected only in bloodstream organisms. RNA interference (RNAi)-mediated gene silencing was used to investigate the function of TbMSP-B protein. RNAi directed against TbMSP-B but not TbMSP-A ablated the steady state TbMSP-B mRNA levels in both procyclic and bloodstream cells but had no effect on the kinetics of cultured trypanosome growth in either stage. Procyclic trypanosomes have been shown previously to have an uncharacterized cell surface metalloprotease activity that can release ectopically expressed surface proteins. To determine whether TbMSP-B is responsible for this release, transgenic variant surface glycoprotein 117 (VSG117) was expressed constitutively in T. brucei procyclic TbMSP-RNAi cell lines, and the amount of surface VSG117 was determined using a surface biotinylation assay. Ablation of TbMSP-B but not TbMSP-A mRNA resulted in a marked decrease in VSG release with a concomitant increase in steady state cell-associated VSG117, indicating that TbMSP-B mediates the surface protease activity of procyclic trypanosomes. This finding is consistent with previous pharmacological studies showing that peptidomimetic collagenase inhibitors block release of transgenic VSG from procyclic trypanosomes and are toxic for bloodstream but not procyclic organisms.     

Mutations of the asparagine117 residue of a receptor activity-modifying protein 1-dependent human calcitonin gene-related peptide receptor result in selective loss of function

The initially orphan human calcitonin (CT) receptor-like receptor (hCRLR) interacts with novel accessory receptor activity-modifying protein 1 (RAMP1) to reveal a functional CT gene-related peptide (CGRP) receptor. In mammalian cells, RAMP1 is required for mature N-glycosylation of the hCRLR predicted to occur at Asn(60), Asn(112), and/or Asn(117) in the amino-terminal extracellular domain. Here we have shown that the substitution of Asn(117) with Ala, Gln, Thr, or Pro abolished CGRP-evoked cAMP formation which was left unchanged when the Asn(117) was replaced with Asp. Moreover, the hCRLR and the Asn(117) mutants exhibited comparable N-glycosylation and cell surface expression, and the association with RAMP1 was only slightly impaired. In contrast, the hCRLR Asn(60,112) to Thr double mutant exhibited defective RAMP1-dependent N-glycosylation, and impaired cell surface expression and CGRP receptor function. Unlike Asn(60) and Asn(112), Asn(117) is normally not N-glycosylated, but essential for CGRP binding to the hCRLR-RAMP1 complex.     

CD137 induces proliferation of murine hematopoietic progenitor cells and differentiation to macrophages

CD137 is a member of the TNFR family, and reverse signaling through the CD137 ligand, which is expressed as a cell surface transmembrane protein, costimulates or activates APCs. CD137 and CD137 ligand are expressed on small subsets of bone marrow cells. Activation of bone marrow cells through CD137 ligand induces proliferation, colony formation and an increase in cell numbers. Compared with total bone marrow cells, the small subpopulation of progenitor cells that express no lineage markers but express CD117 cells (or Lin(-), CD117(+) cells) responds with the same activities to CD137 ligand signaling, but at a significantly enhanced rate. Concomitantly to proliferation, the cells differentiate to CFU granulocyte-macrophage and CFU macrophage, and then to monocytes and macrophages but not to granulocytes or dendritic cells. Hematopoietic progenitor cells differentiated in the presence of CD137 protein display enhanced phagocytic activity, secrete high levels of IL-10 but little IL-12 in response to LPS, and are incapable of stimulating T cell proliferation. These data demonstrate that reverse CD137 ligand signaling takes place in hematopoietic progenitor cells, in which it induces proliferation, an increase in cell numbers, colony formation, and differentiation toward monocytes and macrophages.     

Interaction of voltage-gated sodium channel Nav1.6 (SCN8A) with microtubule-associated protein Map1b

The mechanism by which voltage-gated sodium channels are trafficked to the surface of neurons is not well understood. Our previous work implicated the cytoplasmic N terminus of the sodium channel Na(v)1.6 in this process. We report that the N terminus plus the first transmembrane segment (residues 1-153) is sufficient to direct a reporter to the cell surface. To identify proteins that interact with the 117-residue N-terminal domain, we carried out a yeast two-hybrid screen of a mouse brain cDNA library. Three clones containing overlapping portions of the light chain of microtubule-associated protein Map1b (Mtap1b) were recovered from the screen. Interaction between endogenous Na(v)1.6 channels and Map1b in mouse brain was confirmed by co-immunoprecipitation. Map1b did not interact with the N terminus of the related channel Na(v)1.1. Alanine-scanning mutagenesis of the Na(v)1.6 N terminus demonstrated that residues 77-80 (VAVP) contribute to interaction with Map1b. Co-expression of Na(v)1.6 with Map1b in neuronal cell line ND7/23 resulted in a 50% increase in current density, demonstrating a functional role for this interaction. Mutation of the Map1b binding site of Na(v)1.6 prevented generation of sodium current in transfected cells. The data indicate that Map1b facilitates trafficking of Na(v)1.6 to the neuronal cell surface.     

Role of Survivin Phosphorylation by Aurora B in Mitosis

The chromosomal protein passenger complex, a key mitotic regulator, consists of at least four proteins, INCENP, Aurora B, Survivin and Borealin. Survivin, in contrast to the other members of the chromosomal protein passenger complex (CPC), is mobile at metaphase. This protein is also phosphorylated by Aurora B at Threonine 117. In this work we have studied the role of the phosphorylation of Survivin in mitotosis by using non phosphorylable T117A and phosphomimic T117E silent resistant Survivin mutants, inducible cell lines expressing these mutants and a combination of siRNA, time-lapse microscopy and FRAP analysis. Time lapse microscopy and FRAP analysis show that Survivin T117A mutant is very stably associated with centromeres and its expression induces a prometaphasic arrest in endogenous survivin depleted cells. In addition, Survivin T117A was unable to rescue the phenotypes of the endogenous survivin depleted cells. Expressed in these cells, the phosphomimic Survivin T117E mutant exhibits a very weak interaction with the centromeres and behaves as a dominant negative mutant inducing severe mitotic defects. Our data suggest that the Aurora B generated phosphorylation/dephosphorylation cycle of Survivin is required for proper proceeding of mitosis.     

Re-expression of 117 antigen, a cell surface glycoprotein of aggregating cells, during terminal differentiation of Dictyostelium discoideum prespore cells

117 antigen is a glycoprotein expressed on the surface of D. discoideum cells at aggregation. It then disappears and is later re-expressed on the surface of a subpopulation of cells at culmination, the terminal differentiation stage (Sadeghi et al. 1987). A cDNA clone was used to show that the appearance of cell surface 117 antigen accurately reflects the expression of the 117 gene as measured by mRNA levels. It was also shown that during multicellular development there is a reciprocal relationship between the levels of 117 mRNA and the mRNA which codes for prespore surface glycoprotein, PsA. Dual parameter flow cytometry was used to demonstrate that the 117 antigen is found on the surface of maturing prespore cells after the PsA glycoprotein disappears, but that it is not found on mature spores. Using three monoclonal antibodies which identify respectively 117 antigen, PsA, and MUD3 antigen (a spore coat glycoprotein--probably Sp96), two new stages of final spore maturation were defined. These results indicate that there is a recapitulation of at least one aggregative cell surface glycoprotein in the prespore subpopulation of cells as they rise up the stalk during final spore development. This raises the possibility that culmination, which involves complex three dimensional morphogenetic movements not unlike those observed during animal embryogenesis, involves components of the two-dimensional pattern seen during aggregation.     

Complete nucleotide sequence of complementary DNA coding for a variant surface glycoprotein from Trypanosoma brucei

The complete nucleotide sequence of complementary DNA coding for a variant surface glycoprotein (VSG 117) of Trypanosoma brucei has been determined and compared with amino acid sequence data for the mature protein. This has revealed several interesting and novel features about the synthesis and processing of VSG 117: (1) the primary translation product of the VSG 117 gene includes hydrophobic extensions at both the NH₂ and COOH termini that are not found on mature VSG 117; (2) the glycosylated residue at the mature COOH terminus is aspartate, a residue that is not known to be glycosylated in any other system; (3) the nucleotide sequence shows an unusual dinucleotide frequency and codon usage for the gene.     

Butanol-extractable and detergent-solubilized cell surface components from murine large cell lymphoma cells associated with adhesion to organ microvessel endothelial cells.

Metastatic variant cell lines of the murine RAW117 large cell lymphoma were used to study the cell surface components involved in syngeneic tumor cell/microvessel endothelial cell interactions. Poorly liver-metastatic parental RAW117-P cell line adhered to murine hepatic sinusoidal endothelial cell monolayers at significantly lower rates than the liver-selected, highly liver-metastatic RAW117-H10 line and both cell lines were poorly adherent to lung microvessel and bovine aorta endothelial cells. Viable, 2% 1-butanol-treated RAW117-H10 tumor cells formed fewer liver tumor nodules in experimental metastasis assays than untreated H10 cells and were significantly less adherent to murine hepatic sinusoidal endothelial cell monolayers. When 2% 1-butanol extracts of metabolically labeled or CHAPS detergent lysates of cell surface-labeled tumor cells were analyzed for their ability to bind to fixed microvessel endothelial cell monolayers, quantitative differences were found in the extractable tumor cell surface components that bound to the different organ-derived microvessel endothelial cells. Cell surface components (1-butanol extractable), of Mr approximately 85,000-90,000 and approximately 37,000-40,000 bound to hepatic sinusoidal endothelial cell monolayers to a greater extent than to murine lung microvessel endothelial or bovine aortic endothelial cell monolayers, whereas tumor cell surface components of Mr approximately 45,000, approximately 33,000, and approximately 25,000 bound similarly to endothelial cells regardless of origin. The results suggest but do not prove that tumor cell/endothelial cell adhesion involves multiple tumor cell surface components, some of which commonly bind to various endothelial cells and others for which binding may be dictated by the tissue origin and type of endothelial cell. Particular RAW117 butanol-extractable cell membrane components were associated with tumor cell-endothelial cell adhesion, and these components could be responsible, in part, for the preferential adhesion of RAW117 cells to liver sinusoidal endothelial cells and metastasis to liver.     

水稻及其他禾本科植物体内硅结合 蛋白的免疫印迹检测

为了检测硅结合蛋白在水稻和禾本科植物体内的分布,根据硅结合蛋白 (silica-binding protein 117, SBP117) 的氨基酸保守片段和天然抗原表位,合成了 SBP117 的两个肽段做抗原,与匙孔血蓝蛋白载体偶联后免疫兔子,获得了抗 SBP117 的专一性抗体. 蛋白质印迹和免疫印迹检测表明, SBP117 的抗体不仅能识别水稻硅结合蛋白,而且与其他累积硅的禾本科植物中提取的硅结合蛋白有交叉反应,但它不识别不累积硅的双子叶植物番茄叶中的蛋白质以及牛血清蛋白,说明与 SBP117 同源的硅结合蛋白广泛存在于禾本科植物之中. 组织印迹法定位显示, SBP117 主要分布在水稻根茎叶的外表皮中,在根和叶的维管组织中也有分布,这与前人报道的硅在水稻体内的分布是一致的,说明此蛋白质可能参与到诱导和控制硅在植物体内的沉积.     

RGC-32 increases p34CDC2 kinase activity and entry of aortic smooth muscle cells into S-phase.

Proliferation of aortic smooth muscle cells contributes to atherogenesis and neointima formation. Sublytic activation of complement, particularly C5b-9, induces cell cycle progression in aortic smooth muscle cells. RGC-32 is a novel protein that may promote cell cycle progression in response to complement activation. We cloned human RGC-32 cDNA from a human fetal brain cDNA library. The human RGC-32 cDNA encodes a 117-amino acid protein with 92% similarity to the rat and mouse protein. Human RGC-32 maps to chromosome 13 and is expressed in most tissues. Sublytic complement activation enhanced RGC-32 mRNA expression in human aortic smooth muscle cells and induced nuclear translocation of the protein. RGC-32 was physically associated with cyclin-dependent kinase p34CDC2 and increased the kinase activity in vivo and in vitro. In addition, RGC-32 was phosphorylated by p34CDC2-cyclin B1 in vitro. Mutation of RGC-32 protein at Thr-91 prevented the p34CDC2-mediated phosphorylation and resulted in loss of p34CDC2 kinase enhancing activity. Overexpression of RGC-32 induced quiescent aortic smooth muscle cells to enter S-phase. These data indicate that cell cycle activation by C5b-9 may involve p34CDC2 activity through RGC-32. RGC-32 appears to be a cell cycle regulatory factor that mediates cell proliferation, both as an activator and substrate of p34CDC2.     

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.     

Crystal structure of Pseudomonas aeruginosa apo-azurin at 1.85 A resolution.

The 3D structure of apo-azurin from Pseudomonas aeruginosa has been determined at 1.85 A resolution. The crystal structure is composed of two different molecular forms of apo-azurin arranged as hetero-dimers in the tetramer of the asymmetric unit. Form 1 closely resembles the holo-protein lacking copper. Form 2 shows differences in the metal binding site region induced by the incorporation of a solvent molecule into this site. The positions of the copper ligands His46 and His117 are shifted by 0.6 A and 1.6 A. The His117 side chain adopts a position at the surface of the protein, thereby facilitating access to the copper site. The presence of two different molecular forms of apo-azurin in the crystal lattice may reflect an equilibrium between the two forms in solution. 1H-NMR spectra of apo-azurin recorded as a function of pH show that at high pH the line broadening of His35, His46 and His117 resonances is consistent with an interconversion between forms 1 and 2. At low pH, no broadening is observed. This may indicate that here the interconversion is fast on the NMR timescale.     

Novel surface expression of reticulocalbin 1 on bone endothelial cells and human prostate cancer cells is regulated by TNF-alpha

An unbiased cDNA expression phage library derived from bone-marrow endothelial cells was used to identify novel surface adhesion molecules that might participate in metastasis. Herein we report that reticulocalbin 1 (RCN1) is a cell surface-associated protein on both endothelial (EC) and prostate cancer (PCa) cell lines. RCN1 is an H/KDEL protein with six EF-hand, calcium-binding motifs, found in the endoplasmic reticulum. Our data indicate that RCN1 also is expressed on the cell surface of several endothelial cell lines, including human dermal microvascular endothelial cells (HDMVECs), bone marrow endothelial cells (BMEC), and transformed human bone marrow endothelial cells (TrHBMEC). While RCN1 protein levels were highest in lysates from HDMVEC, this difference was not statistically significant compared BMEC and TrHBMEC. Given preferential adhesion of PCa to bone-marrow EC, these data suggest that RCN1 is unlikely to account for the preferential metastasis of PCa to bone. In addition, there was not a statistically significant difference in total RCN1 protein expression among the PCa cell lines. RCN1 also was expressed on the surface of several PCa cell lines, including those of the LNCaP human PCa progression model and the highly metastatic PC-3 cell line. Interestingly, RCN1 expression on the cell surface was upregulated by tumor necrosis factor alpha treatment of bone-marrow endothelial cells. Taken together, we show cell surface localization of RCN1 that has not been described previously for either PCa or BMEC and that the surface expression on BMEC is regulated by pro-inflammatory TNF-alpha.     

Characterization of EPPIN's Semenogelin I Binding Site: A Contraceptive Drug Target

Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.     

HeLa细胞中INI1通过与E2A作用下调CD117

目的:已经证明,CD117基因转录启始位点的上游-480bp包括4个E-box和一个GC-box。试验要确定INI1是否通过与E-box结合蛋白的相互作用来调控CD117。方法与结果:实验证明INI1过表达下调 CD117的mRNA水平和荧光素酶的活性,这个酶通过CD117启动子来启动。通过染色质共免疫沉淀反应实验,发现在HeLa细胞未损伤的染色质里面,INI1可以特定的结合CD117的启动子。绘制INI1在CD117启动子区域的结合位点图表明, E-boxes而不是GC-box对于下调CD117启动子的活性是必须的,这一点在Co-IP(共免疫沉淀)实验中被进一步的证实,在这个试验中INI1与E-box结合蛋白E2A在体内相互作用。结论:总的来说,INI1参与调节CD117的表达和细胞增殖。     

Gene expression and tumor cell escape from host effector mechanisms in murine large cell lymphoma

Using in vivo selection methods, we obtained metastatic sublines of the murine RAW117 large cell lymphoma that form multiple liver metastases. The highly metastatic subline RAW117-H10 has a low number of gp70 molecules expressed at the cell surface and low cytostatic sensitivity to activated syngeneic macrophages. This subline was infected with endogenous RNA tumor virus isolated from a high virus-expressing RAW117-P subline of low metastatic potential. After superinfection the H10 subline gradually increased its expression of cell surface gp70 and showed enhanced sensitivity to macrophage-mediated cytostasis, suggesting that gp70 might be involved in host macrophage-mediated surveillance. Culture of RAW117-P and H10 cells in media conditioned by activated macrophages indicated that parental cells are severely growth inhibited in a dose dependent fashion while H10 cells showed almost no effect. Examination of differentially expressed genes in the highly metastatic RAW117-H10 cells by analysis of RNA blots indicated that a mitochondrial gene was expressed at a level that was approximately 10 times higher in H10 cells than in parental cells. This gene was identified as ND5, which codes for a subunit of NADH dehydrogenase (complex I of the mitochondrial electron transport chain); this complex is the target for an activated macrophage-released cytostatic factor. Among other possibilities, the results are consistent with the suggestion that highly metastatic RAW117 cells may escape macrophage surveillance by decreasing the synthesis of specific cell-surface receptors for cytostatic molecules and increasing the synthesis of specific cellular targets for such molecules.     

The PilC adhesin of the Neisseria type IV pilus-binding specificities and new insights into the nature of the host cell receptor

Type IV pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate the first contact to human mucosal epithelial cells, an interaction which is also critical for the interaction with vascular endothelial cells. The PilC proteins have been characterized as the principal pilus-associated adhesin. Here we show that PilC2 exhibits a defined cell and tissue tropism, as it binds to human epithelial and endothelial cell lines, but not to human T cells or fibroblasts. Piliated gonococci and PilC2 exhibit similar patterns of binding to human epithelial and endothelial cells, supporting the function of PilC as the key pilus adhesin. Although CD46 has previously been suggested to be a pilus receptor, several observations indicate that neisserial type IV pili and the pilus adhesin PilC2 interact with epithelial cells in a CD46 independent manner. Biochemical approaches were used to characterize the nature of host cell factors mediating binding of piliated gonococci and PilC2 protein. Our data indicate that the putative host cell receptor for gonococcal pili and the PilC2 pilus adhesin is a surface protein. Glycostructures were found to not be involved in binding. Moreover, we observed the uptake of purified PilC2 protein together with its receptor via receptor-mediated endocytosis and subsequent receptor re-exposure on the cell surface. Our data support the existence of a specific pilus receptor and provide intriguing information on the nature of the receptor.