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1.
Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process. 相似文献
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Purification and characterization of the adenosine A2-like binding site from human placental membrane 总被引:1,自引:0,他引:1
We have purified and characterized the adenosine A2-like binding site from human placental membranes. 5'-N-Ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binds to this site, with a Kd of 240 nM and a Bmax of 13.0 pmol/mg in human placental membranes. The adenosine A2-like binding site was purified after extraction from placental membranes with 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The purification included ammonium sulfate precipitation and concanavalin A, DEAE-Sephadex, and Sepharose 6B gel filtration chromatographies. The protein was purified 127-fold to homogeneity, with a final specific activity of 1.5-1.9 nmol/mg of protein and a 5.5-8.1% yield of binding activity from the membranes. The purified protein had similar binding properties and an identical potency order for displacement of [3H] NECA by adenosine analogs as the initial membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified protein revealed a single band at 98 kDa which coeluted with [3H]NECA binding activity during Sepharose 6B gel filtration chromatography. In 0.1% Triton X-100, the binding complex has a Stokes radius of 70 A, a sedimentation coefficient of 6.9 S, and a partial specific volume of 0.698 ml/g. The detergent-protein complex has a calculated molecular mass of 230 kDa. The estimated frictional ratio is 1.5. The native binding complex appears to consist of a dimer of identical subunits. The function of this ubiquitous protein remains unclear. 相似文献
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Mineo Shibasaka Sizuka Sasano Sigeko Utsugi Maki Katsuhara 《Plant signaling & behavior》2012,7(12):1648-1652
Water homeostasis is crucial to the growth and survival of plants. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. We characterized a novel PIP2 gene, HvPIP2;8 in barley (Hordeum vulgare). HvPIP2;8 shared 72–76% identity with other HvPIP2s and 74% identity with rice OsPIP2;8. The gene was expressed in all organs including the shoots, roots and pistil at a similar level. When HvPIP2;8 was transiently expressed in onion epidermal cells, it was localized to the plasma membrane. HvPIP2;8 showed transport activity for water in Xenopus oocytes, however its interaction with HvPIP1;2 was not observed. These results suggest that HvPIP2;8 plays a role in water homeostasis although further functional analysis is required in future. 相似文献
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Structural characterization of the fusion core in syncytin, envelope protein of human endogenous retrovirus family W 总被引:5,自引:0,他引:5
Gong R Peng X Kang S Feng H Huang J Zhang W Lin D Tien P Xiao G 《Biochemical and biophysical research communications》2005,331(4):1193-1200
Syncytin is a captive retroviral envelope protein, possibly involved in the formation of the placental syncytiotrophoblast layer generated by trophoblast cell fusion at the maternal-fetal interface. We found that syncytin and type I viral envelope proteins shared similar structural profiling, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR). We expressed the predicted regions of NHR (41aa) and CHR (34aa) in syncytin as a native single chain (named 2-helix protein) to characterize it. 2-Helix protein exists as a trimer and is highly alpha-helix, thermo-stable, and denatured by low pH. NHR and CHR could form a protease-resistant complex. The complex structure built by the molecular docking demonstrated that NHR and CHR associated in an antiparallel manner. Overall, the 2-helix protein could form a thermo-stable coiled coil trimer. The fusion core structure of syncytin was first demonstrated in endogenous retrovirus. These results support the explanation how syncytin mediates cytotrophoblast cell fusion involved in placental morphogenesis. 相似文献
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The function of the syncytiotrophoblast in maternal-fetal exchange is related to the properties of its microvillous (maternal-facing) and basal (fetal-facing) plasma membranes. We have previously reported the properties of the microvillous membrane (Smith, C.H., Nelson, D.M., King, B.F., Donohue, T.M., Ruzycki, S.M. and Kelley, L.K. (1977) Am. J. Obstet. Gynecol. 128, 190–196), and now describe the purification and partial characterization of the basal plasma membrane. Sonication and incubation with EDTA were used to isolate selectively the basal cell membrane. These steps were followed by a more conventional purification by centrifugation. The trophoblast was disrupted and its microvillous membrane and cytoplasmic contents were removed by sonication. The exposed basal cell membrane was selectively released from the underlying basal lamina by sonication in the presence of EDTA and further purified by discontinuous Ficoll gradient centrifugation. The material at the 4–10% Ficoll interface consisted of smooth membrane vesicles with internal microfilaments. It was 45-fold enriched in dihydroalprenolol binding activity and 11-fold enriched in ouabain binding activity. Other enzymatic analyses, including alkaline phosphatase, cytochrome-c oxidase, cytochrome-c reductase and galactosyl transferase indicated low contamination by other organelles. This procedure yields a preparation of relatively high purity which should be suitable for investigation of transport and other functions of the basal surface membrane of trophoblast. In principle, the purification procedures used may be applicable to other transporting epithelia. 相似文献
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Patarroyo MA Plaza DF Ocampo M Curtidor H Forero M Rodriguez LE Patarroyo ME 《Biochemical and biophysical research communications》2008,372(4):935-940
Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine. 相似文献
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R Bützow M L Huhtala H Bohn I Virtanen M Sepp?l? 《Biochemical and biophysical research communications》1988,150(1):483-490
This report describes the purification of placental protein 5, PP5, from the human placenta by two affinity chromatography steps, the first with Heparin-Sepharose and the second with Sepharose-linked monoclonal anti-PP5 antibody. The final purification is achieved by reversed-phase high performance liquid chromatography. In SDS-polyacrylamide gel electrophoresis under reducing or nonreducing conditions, PP5 purified in this study migrates as one major band at 36 kD. The previously purified PP5 is more heterogeneous: under nonreducing conditions it migrates at 30 kD and, after reduction, it gives three bands at 16.8 kD, 18.3 kD, and 19.0 kD. In Western blot analysis, both purified proteins react with polyclonal and monoclonal anti-PP5 antibodies. Three N-terminal amino acid sequences are obtained for the previously purified PP5, whereas the N-terminal of PP5 purified in this study is blocked. These results suggest that PP5 previously purified in the absence of protease inhibitors, does not represent the native form of PP5. Computer comparison of the obtained amino acid sequences revealed no significant homology to known protein sequences. 相似文献
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《Biochimica et Biophysica Acta - Proteins and Proteomics》2020,1868(8):140436
Surfactant Protein D (SP-D) is a collectin protein that participates in the innate immune defense of the lungs. SP-D mediates the clearance of invading microorganisms by opsonization, aggregation or direct killing, which are lately removed by macrophages.SP-D is found as a mixture of trimers, hexamers, dodecamers and higher order oligomers, “fuzzy balls”. However, it is unknown whether there are differences between these oligomeric forms in functions, activity or potency.In the present work, we have obtained fractions enriched in trimers, hexamers and fuzzy balls of full-length recombinant human (rh) SP-D by size exclusion chromatography, in a sufficient amount to perform functional assays. We have evaluated the differences in protein lectin-dependent activity relative to aggregation and binding to E. coli, one of the ligands of SP-D in vivo. Fuzzy balls are the most active oligomeric form in terms of binding and aggregation of bacteria, achieving 2-fold binding higher than hexamers and 50% bacteria aggregation at very short times. Hexamers, recently described as a defined oligomeric form of the protein, have never been isolated or tested in terms of protein activity. rhSP-D hexamers efficiently bind and aggregate bacteria, achieving 50–60% aggregation at final time point and high protein concentrations. Nevertheless, trimers are not able to aggregate bacteria, although they bind to them. Therefore, SP-D potency, in functions that relay on the C-lectin activity of the protein, is proportional to the oligomeric state of the protein. 相似文献
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The bacterial genus Brucella consists of a group of facultative intracellular pathogens which produces abortion and infertility in animals and a chronic debilitating febrile illness in humans. BMFP is a basic protein of Brucella abortus that belongs to a highly conserved group of homologue proteins of unknown structure and function in proteobacteria (COG2960). In this study, we report the structural and biochemical characterization of this protein. We found that BMFP has two structural domains: a carboxyl-terminal coiled-coil domain through which the protein self-associates as a trimer and a natively disordered amino-terminal domain which has propensity to adopt an amphipathic alpha-helical structure. This natively unfolded domain undergoes a structural rearrangement from unfolded to alpha-helix in the presence of high ionic strength, acidic pH, detergents, and phospholipid vesicles. Moreover, we demonstrated that the interaction of BMFP with phospholipid vesicles promotes in vitro membrane fusion. These results contribute to the elucidation of the structural and functional properties of this protein and its homologues present in most proteobacteria. 相似文献
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Purification and characterization of human placental ferredoxin 总被引:1,自引:0,他引:1
A ferredoxin-type iron-sulfur protein was isolated from human placenta mitochondria. The properties of the purified protein were very similar to those of adrenal ferredoxin (adrenodoxin), and immunological cross-reactivity with polyclonal antibodies to bovine adrenodoxin was observed. The N-terminal amino acid sequence and the visible absorption spectrum were identical to bovine adrenodoxin. The molecular mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 13,500), however, is slightly smaller than that of adrenodoxin, and the C-terminal sequence is different. Human placental ferredoxin can substitute for bovine adrenodoxin in reactions reconstituted with bovine adrenal enzymes which catalyze the side chain cleavage of cholesterol to pregnenolone and the 11 beta-hydroxylation of deoxycorticosterone to corticosterone. 相似文献
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Peroxins are proteins involved in peroxisome biogenesis and are encoded by PEX genes. The human PEX2 gene encodes a 35-kDa peroxisomal integral membrane protein which is a member of the zinc finger protein family. Mutations in the PEX2 gene are the primary defect in a subset of patients with Zellweger syndrome and related peroxisome biogenesis disorders. The role of zinc finger proteins in peroxisome assembly and function is poorly understood. Here we report the cloning and characterisation of the human PEX2 structural gene. PEX2 was assigned to human chromosome 8q13-q21 and its murine homologue to mouse chromosome 3. The gene is approximately 17.5 kb in length, and contains four exons. The entire coding sequence is included in one exon, exon 4. The 5'-flanking region has features of housekeeping genes (GC enrichment, two Sp1 sites) and tissue-specific, inducible genes (two CCAAT boxes). In more than 1.5 kb of 5'-flanking sequences we did not identify consensus peroxisomal proliferator responsive elements (PPRE). 相似文献
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Angeletti S Sanchez JM Chamley LW Genti-Raimondi S Perillo MA 《Biochimica et biophysica acta》2012,1818(3):425-433
StarD7 is a surface active protein, structurally related with the START lipid transport family. So, the present work was aimed at elucidating a potential mechanism of action for StarD7 that could be related to its interaction with a lipid-membrane interface. We applied an assay based on the fluorescence de-quenching of BD-HPC-labeled DMPC-DMPS 4:1 mol/mol SUVs (donor liposomes) induced by the dilution with non-labeled DMPC-DMPS 4:1 mol/mol LUVs (acceptor liposomes). Recombinant StarD7 accelerated the dilution of BD-HPC in a concentration-dependent manner. This result could have been explained by either a bilayer fusion or monomeric transport of the labeled lipid between donor and acceptor liposomes. Further experiments (fluorescence energy transfer between DPH-HPC/BD-HPC, liposome size distribution analysis by dynamic light scattering, and the multinuclear giant cell formation induced by recombinant StarD7) strongly indicated that bilayer fusion was the mechanism responsible for the StarD7-induced lipid dilution. The efficiency of lipid dilution was dependent on StarD7 electrostatic interactions with the lipid-water interface, as shown by the pH- and salt-induced modulation. Moreover, this process was favored by phosphatidylethanolamine which is known to stabilize non-lamellar phases considered as intermediary in the fusion process. Altogether these findings allow postulate StarD7 as a fusogenic protein. 相似文献
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S Q Liu A Bhatnagar B Das S K Srivastava 《Archives of biochemistry and biophysics》1989,275(1):112-121
Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species. 相似文献
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Identification and characterization of membrane cofactor protein of human spermatozoa. 总被引:6,自引:0,他引:6
F Cervoni T J Oglesby E M Adams C Milesifluet M Nickells P Fenichel J P Atkinson B L Hsi 《Journal of immunology (Baltimore, Md. : 1950)》1992,148(5):1431-1437
Membrane cofactor protein (MCP) regulates C activation by serving as a cofactor for the cleavage of C3b and C4b by the serine protease factor I. An MCP-like molecule on the inner acrosomal membrane of human spermatozoa has been characterized. Three mAb and a rabbit polyclonal antibody against MCP recognized the sperm protein. On SDS-PAGE, it migrated as a single band with a molecular mass of 38,000 and 44,000 Da under nonreducing or reducing conditions, respectively. The molecular mass was 10,000 to 20,000 Da less than the two forms of MCP expressed on others cells. The electrophoretic pattern, by one- and two-dimensional gel analysis, and the isoelectric point profile (4.5 to 5.0) of the sperm protein were similar among multiple individuals. In contrast to MCP of other cells, digestion with endoglycosidases did not alter either the m.w. or the pI of the protein, suggesting that it is a poorly or nonglycosylated form of MCP. The solubilized sperm protein bound C3 with broken thioester bond to Sepharose and possessed cofactor activity for factor I-mediated cleavage of C3 with the broken bond. A mAb that blocks the regulatory function of MCP inhibited the cofactor activity of the sperm lysate. Thus, the sperm protein is an antigenic and functional homologue of MCP but has the distinct structural features of a lower m.w. and an apparent lack of glycosylation. MCP may play an essential role in the survival of the acrosome-reacted spermatozoa by modulating C activation in the female genital tract. 相似文献
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Human placental anticoagulant protein: isolation and characterization 总被引:10,自引:0,他引:10
An anticoagulant protein was purified from the soluble fraction of human placenta by ammonium sulfate precipitation and column chromatography on DEAE-Sepharose, Sephadex G-75, and Mono S (Pharmacia). The yield of the purified protein was approximately 20 mg from one placenta. The purified protein gave a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 36,500. This protein prolonged the clotting time of normal plasma when clotting was induced either by brain thromboplastin or by kaolin in the presence of cephalin and Ca2+. It also prolonged the factor Xa induced clotting time of platelet-rich plasma but did not affect thrombin-induced conversion of fibrinogen to fibrin. The purified placental protein completely inhibited the prothrombin activation by reconstituted prothrombinase, a complex of factor Xa-factor Va-phospholipid-Ca2+. The placenta inhibitor had no effect on prothrombin activation when phospholipid was omitted from the above reaction. Also, it neither inhibited the amidolytic activity of factor Xa, nor did it bind to factor Xa. The placenta inhibitor, however, did bind specifically to phospholipid vesicles (20% phosphatidylserine and 80% phosphatidylcholine) in the presence of calcium ions. These results indicate that the placental anticoagulant protein (PAP) inhibits coagulation by binding to phospholipid vesicles. The amino acid sequences of three cyanogen bromide fragments of PAP aligned with those of two distinct regions of lipocortin I and II with a high degree of homology, showing that PAP is a member of the lipocortin family. 相似文献
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Kölzer M Ferlinz K Bartelsen O Hoops SL Lang F Sandhoff K 《Biological chemistry》2004,385(12):1193-1195
Degradation of membrane-bound sphingomyelin to phosphorylcholine and ceramide is catalyzed by the water-soluble lysosomal acid sphingomyelinase (A-SMase). The presence of sphingolipid activator proteins (Saps: saposins A-D; GM2 activator) is not essential to mediate this reaction at the water-lipid interface in vivo . A hypothesis based on amino acid sequence alignments suggests that the enzyme possesses an N-terminal saposin-homologous domain, which may facilitate the enzymatic reaction at the interface. We mutated one homologous and three conserved amino acid residues of this domain and studied the activity of the variant enzymes using different sphingomyelin degradation assays. A variant with an exchange of a conserved amino acid residue, Pro153Ala, still exhibited enzyme activity of approximately 52% of normal in a detergent-containing micellar assay, but only 13% of normal in a detergent-free liposomal assay system, which suggests that the Sap-homologous domain fulfills membrane-disturbing functions. Addition of saposin C to the liposomal assay mixtures increased the Pro153Ala variant sphingomyelinase activity to 46% of normal, indicating that the variant saposin-like domain can be substituted by the presence of the sphingolipid activator protein. On the other hand, the addition of saposin C did not result in complete restoration of the variant activity. Thus, the Sap-like domain may also have another role, e.g., to stabilize the fold of acid sphingomyelinase, which cannot be compensated by the presence of saposin C or a detergent. Such an essential second function of the saposin-like domain as an integral part of acid sphingomyelinase is confirmed by our observation that the Lys118Glu, Cys120Ser and Cys131Ser variants were almost completely devoid of activity in the detergent-containing micellar assay system as well as in the liposomal assay system in the presence of saposin C. 相似文献