Amyloid-forming peptides from beta2-microglobulin-Insights into the mechanism of fibril formation in vitro

Beta(2)-Microglobulin (beta(2)m) is one of over 20 proteins known to be involved in human amyloid disease. Peptides equivalent to each of the seven beta-strands of the native protein, together with an eighth peptide (corresponding to the most stable region in the amyloid precursor conformation formed at pH 3.6, that includes residues in the native strand E plus the eight succeeding residues (named peptide E')), were synthesised and their ability to form fibrils investigated. Surprisingly, only two sequences, both of which encompass the region that forms strand E in native beta(2)m, are capable of forming amyloid-like fibrils in vitro. These peptides correspond to residues 59-71 (peptide E) and 59-79 (peptide E') of intact beta(2)m. The peptides form fibrils under the acidic conditions shown previously to promote amyloid formation from the intact protein (pH <5 at low and high ionic strength), and also associate to form fibrils at neutral pH. Fibrils formed from these two peptides enhance fibrillogenesis of the intact protein. No correlation was found between secondary structure propensity, peptide length, pI or hydrophobicity and the ability of the peptides to associate into amyloid-like fibrils. However, the presence of a relatively high content of aromatic side-chains correlates with the ability of the peptides to form amyloid fibrils. On the basis of these results we propose that residues 59-71 may be important in the self-association of partially folded beta(2)m into amyloid fibrils and discuss the relevance of these results for the assembly mechanism of the intact protein in vitro.     

Molecular cloning and expression of cDNAS encoding rat aldosterone synthase: variants of cytochrome P-450(11 beta)

Two distinct forms of cDNA encoding rat aldosterone synthase were cloned from an adrenal capsular tissue cDNA library. The deduced amino acid sequences showed that one of the enzymes (P-450(11 beta),aldo-1) had a long extension peptide composed of 34 amino acid residues while the other (P-450(11 beta),aldo-2) had an extension peptide identical to that of rat P-450(11 beta). Glu at the 320th position of P-450(11 beta),aldo-1 was replaced with Lys in P-450(11 beta),aldo-2. The amino acid sequence of the aldosterone synthase was highly homologous (81%) to rat P-450(11 beta). Constructed expression vector containing the cDNA for extension peptide of P-450(11 beta) and the mature protein of P-450(11 beta),aldo-1 was transfected into COS-7 cells. The cells converted 11-deoxycorticosterone into corticosterone, 18-hydroxycorticosterone, and aldosterone.     

Core and heterogeneity of beta2-microglobulin amyloid fibrils as revealed by H/D exchange

Dialysis-related amyloidosis, which occurs in the patients receiving a long-term hemodialysis with high frequency, accompanies the deposition of amyloid fibrils composed of beta(2)-microglobulin (beta2-m). In vitro, beta2-m forms two kinds of fibrous structures at acidic pH. One is a rigid "mature fibril", and the other is a flexible thin filament often called an "immature fibril". In addition, a 22-residue peptide (K3 peptide) corresponding to Ser20 to Lys41 of intact beta2-m forms rigid amyloid-like fibrils similar to mature fibrils. We compared the core of these three fibrils at single-residue resolution using a recently developed hydrogen/deuterium (H/D) exchange method with the dissolution of fibrils by dimethylsulfoxide (DMSO). The exchange time-course of these fibrils showed large deviations from a single exponential curve showing that, because of the supramolecular structures, the same residue exists in different environments from molecule to molecule, even in a single fibril. The exchange profiles revealed that the core of the immature fibril is restricted to a narrow region compared to that of the mature fibril. In contrast, all residues were protected from exchange in the K3 fibril, indicating that a whole region of the peptide is engaged in the beta-sheet network. These results suggest the mechanism of amyloid fibril formation, in which the core beta-sheet formed by a minimal sequence propagates to form a rigid and extensive beta-sheet network.     

Molecular cloning of interleukin 1beta from rainbow trout Oncorhynchus mykiss reveals no evidence of an ice cut site.

The complete coding sequence of rainbow trout IL-1beta has been obtained. The gene contains a short 5' UTR (97 bp), a 780 bp open reading frame and a 466 bp 3' UTR, which includes a polyadenylation signal, 7 ATTTA motifs and an 18 bp poly A tail. The predicted amino acid sequence (260 amino acids) contains 3 potential glycosylation sites, with a predicted molecular weight of 29 kDa, and shows between 49 and 56% amino acid similarity to mammalian IL-1betas and 57% similarity to carp IL-1beta. Greatest homology was apparent within the secondary structure of the gene, with few of the amino acids known to bind to the IL-1 receptor being conserved. No ICE cut site was apparent but multiple alignment with mammalian sequences allowed a putative mature peptide of 166 amino acids to be identified, in which Ala(95)would be the amino terminus. Northern blot analysis showed that whilst no IL-1beta expression was detectable in head kidney leukocytes immediately after isolation, expression could be induced by stimulation with LPS for 4 h in culture. Similarly, with isolated head kidney macrophages expression was significantly increased following stimulation with LPS.     

Human Zn-α2-glycoprotein cDNA cloning and expression analysis in benign and malignant breast tissues

Two cDNA clones coding Zn-α₂-glycoprotein (Zn-α₂-gp) have been isolated from a human breast library and their nucleotide sequences determined. The deduced amino acid sequence contains the coding information for a hydrophobic signal peptide and the 278 residues of the mature proteine. Comparison of this sequence with that from the protein puriried from plasma reveals four differences: two amino acid changes (Gln-67 and Glu-222) and insertion of two residues (Ile-75 and Phe-76). Northern-blot analysis showed that the Zn-α₂-gp gene is expressed in liver and normal breast, but not in placenta, ovary and thyroid. A comparative analysis in mammary tissues from women with different diseases revealed enhanced expression of Zn-α₂-gp gene in benign breast lesions and a variable expression level in breast cancers.     

Molecular characterization of a stigma-specific gene encoding an arabinogalactan-protein (AGP) from Nicotiana alata

Arabinogalactan-proteins (AGPs) were isolated from the pistils of Nicotiana alata , deglycosylated, and the protein backbones fractionated by reversed-phase HPLC as previously reported. A major fraction, RT35 was isolated and peptide sequences were obtained after protease digestion. A gene-specific degenerate oligonucleotide was designed according to the amino acid sequences and a 380 bp PCR fragment was amplified in vitro from pistil RNA. The PCR fragment was used to screen a pistil cDNA library and a 762 bp cDNA clone (AGP Na 3) was isolated and sequenced. The AGP Na 3 cDNA encodes a 169 amino acid protein which consists of three domains: an N-terminal secretion signal, a Pro-rich domain and a C-terminal Cys-rich domain. The mature protein has 145 amino acid residues (16.7 kDa) and a predicted pl of 7.5. Northern blot analyses showed that the AGP Na 3 gene was only expressed in the pistils of N. alata and of closely related Nicotiana species but not in other plants or suspension-cultured cells. Further Northern blot analysis and in situ hybridization showed that within the pistil, it was primarily expressed in the stigmatic tissues of mature flowers.     

中华蜜蜂蜂毒磷脂酶A_2基因在大肠杆菌中的表达(英文)

将中华蜜蜂蜂毒磷脂酶A2(AcPLA2)蛋白成熟肽编码区基因(405 bp)克隆至表达载体pETBlue-1,在大肠杆菌Tuner(DE3)plac I中诱导表达,经SDS-PAGE电泳检测,表达产物分子量为15kD,约占细菌总蛋白的4.6％;用意大利蜜蜂蜂毒磷脂酶A2(AmPLA2)纯品制备的兔源多克隆抗体为一抗作Western blot,表达产物显示类似于天然纯AmPLA2的特异性印迹,证实AcPLA2基因已在大肠杆菌中得到表达。     

Two different types of hepcidins from the Japanese flounder Paralichthys olivaceus

The cysteine-rich peptide hepcidin is known to be an antimicrobial peptide and iron transport regulator that has been found in both fish and mammals. Recently, we found two different types (designated Hep-JF1 and Hep-JF2) of hepcidin cDNA in the Japanese flounder, Paralichthys olivaceus, by expressed sequence tag analysis. The identity of amino acid sequences between Hep-JF1 and Hep-JF2 was 51%. The Hep-JF1 and Hep-JF2 genes both consist of three exons and two introns, and both exist as single copies in the genome. The predicted mature regions of Hep-JF1 and Hep-JF2 have six and eight Cys residues, respectively. The first Cys residue of Hep-JF1 was deleted and the second was replaced with Gly. The number and positions of Cys residues in Hep-JF2 are the same as they are in human Hep. Hep-JF1 is specifically expressed in liver while the expression of Hep-JF2 was detected from gill, liver, heart, kidney, peripheral blood leucocytes, spleen and stomach. Gene expression of Hep-JF1 in liver decreased during experimental iron (iron-dextran) overload. Expression of Hep-JF1 in liver was decreased by injecting fish with iron-dextran and increased by injecting lipopolysaccharide. Iron overload did not significantly affect expression of Hep-JF2 in liver but it did increase expression of Hep-JF2 in kidney. Lipopolysaccharide injection increased expression of Hep-JF2 in both liver and kidney. In liver, some cells expressed both Hep-JF1 and Hep-JF2 while some other cells expressed just one of them. Synthesized Hep-JF2 peptide showed antimicrobial activity, while synthesized Hep-JF1 peptide did not against several bacteria including fish-pathogenic bacteria used in this study.     

Multiple β-defensin isoforms identified in early developmental stages of the teleost Paralichthys olivaceus

The β-defensin-like gene and its cloned isoforms (fBDI-1 to -5) were identified in an expressed sequence tag (EST) library from the early developmental stages of the olive flounder, Paralichthys olivaceus. The fBDI cDNA clones show identical amino acid sequences in 24 residues of the signal peptide and 38 residues of the mature peptide; however, the propiece region varies in sequence and length, from 5 to 15 amino acid residues. The predicted molecular weight of the mature peptide is 3.83 kDa, and its predicted isoelectric point is 4.1, showing anionic properties. The genomic organisation of the isoforms was analysed using bacterial artificial chromosome (BAC) DNA containing the fBDI gene. Southern blotting and sequence analyses of fBDI BAC DNA confirmed that the fBDI isoforms cluster at the same locus and exhibit the conserved gene organisation reported for other fish defensin genes. The fBDI mRNA was expressed constitutively in early developmental stages after hatching, and pathogen challenge induced fBDI expression in the head kidney of juvenile fish. We also produced a recombinant fBDI peptide (smfBD) using the expression plasmid pET32 and examined its bioactivity toward Escherichia coli.     

Structure of interacting segments in the growing amyloid fibril of beta2-microglobulin probed with IR spectroscopy

Inter-segmental interaction at the growing tip of the amyloid fibril of beta2-microglobulin (beta2m) was investigated using IR microscopy. Cross-seeded fibril formation was implemented, in which the amyloid fibril of the #21-31 fragment of beta2m (fA[#21-31]) was generated on the beta2m amyloid fibril (fA[beta2m]) as a seed. Differences between the IR spectra of the cross-seeded fibril and those of the seed were attributed to the contribution from the tip, whose structure is discussed. The results indicated that 6.5 +/- 1.0 out of 11 residues of the fA[#21-31] tip on fA[beta2m] are contained in a beta-sheet at pH 2.5, which was smaller than the corresponding value (7.5 +/- 1.1 residues) of the spontaneous fA[#21-31] at pH 2.5. The tip was suggested to have a planar structure, indicating the planarity of the interacting segment. The N-terminal region of fA[#21-31] in the fibril is more exposed to the solvent than that in the tip, and vice versa for the C-terminal region. This is consistent with the different protonation levels of these regions, and the direction of peptide in the fibrils is determined from these results.     

Structural basis of the differential stability and receptor specificity of H-2Db in complex with murine versus human beta2-microglobulin

beta(2)-Microglobulin (beta(2)m) is non-covalently linked to the major histocompatibility complex (MHC) class I heavy chain and interacts with CD8 and Ly49 receptors. Murine MHC class I heavy chains can bind human beta(2)m (hbeta(2)m) and peptide, and such hybrid molecules are often used in structural and functional studies. The replacement of mouse beta(2)m (mbeta(2)m) with hbeta(2)m has several functional consequences for MHC class I complex stability and specificity, but the structural basis for this is presently unknown. To investigate the impact of species-specific beta(2)m subunits on MHC class I conformation, we provide a crystallographic comparison of H-2D(b) in complex with LCMV-derived gp33 peptide and either hbeta(2)m or mbeta(2)m. The conformation of the gp33 peptide is not affected by the beta(2)m species. Comparison of the interface between beta(2)m and the alpha(1)alpha(2) domains of the heavy chain in these two crystal structures reveals a marked increase in both polarity and number of hydrogen bonds between hbeta(2)m and the alpha(1)alpha(2) domains of H-2D(b). We propose that the positioning of two hydrogen bond rich regions at the hbeta(2)m/alpha(1)alpha(2) interface plays a central role in the increased overall stability and peptide exchange capacity in the H-2D(b)/hbeta(2)m complex. These two regions act as bridges, holding and stabilizing the underside of the alpha(1) and alpha(2) helices, enabling a prolonged peptide-receptive conformation of the peptide binding cleft. Furthermore, analysis of H-2D(b) in complex with either mbeta(2)m or hbeta(2)m provides a structural explanation for the differential binding of H-2D(b)/hbeta(2)m to both Ly49A and Ly49C. Our comparative structural study emphasizes the importance of beta(2)m residues at positions 3, 6 and 29 for binding to Ly49A and suggests that sterical hindrance by residue K6 on hbeta(2)m impairs the recognition of Ly49C by H-2D(b)/gp33/hbeta(2)m. Finally, comparison of the two H-2D(b) crystal structures implies that the beta(2)m species may affect the strength of TCR recognition by affecting CD8 binding.     

Molecular cloning, differential expression and 3D structural analysis of the MHC class-II beta chain from sea bass (Dicentrarchus labrax L.)

The major histocompatibility complex class I and II molecules (MHC-I and MHC-II) play a pivotal role in vertebrate immune response to antigenic peptides. In this paper we report the cloning and sequencing of the MHC class II beta chain from sea bass (Dicentrarchus labrax L.). The six obtained cDNA sequences (designated as Dila-DAB) code for 250 amino acids, with a predicted 21 amino acid signal peptide and contain a 28bp 5'-UTR and a 478bp 3'-UTR. A multiple alignment of the predicted translation of the Dila-DAB sequences was assembled together with other fish and mammalian sequences and it showed the conservation of most amino acid residues characteristic of the MHC class II beta chain structure. The highest basal Dila-DAB expression was found in gills, followed by gut and thymus, lower mRNA levels were found in spleen, peripheral blood leucocytes (PBL) and liver. Stimulation of head kidney leukocytes with LPS for 4h showed very little difference in the Dila-DAB expression, but after 24h the Dila-DAB level decreased to a large extent and the difference was statistically significant. Stimulation of head kidney leukocytes with different concentrations of rIL-1beta (ranging from 0 to 100ng/ml) resulted in a dose-dependent reduction of the Dila-DAB expression. Moreover, two 3D Dila-DAB*0101 homology models were obtained based on crystallographic mouse MHC-II structures complexed with D10 T-cell antigen receptor or human CD4; features and differences between the models were evaluated and discussed. Taken together these results are of interest as MHC-II structure and function, molecular polymorphism and differential gene expression are in correlation with disease resistance to virus and bacteria in teleost fish.     

Plant low-molecular-weight phospholipase A2s (PLA2s) are structurally related to the animal secretory PLA2s and are present as a family of isoforms in rice (Oryza sativa)

Recently, we purified to homogeneity and characterized a low-molecular-weight calcium-dependent phospholipase A₂ (PLA₂) from developing elm seed endosperm. This represented the first purified and characterized PLA₂ from a plant tissue. The full sequences of two distinct but homologous rice (Oryza sativa) cDNAs are given here. These encode mature proteins of 119 amino acids (PLA₂-I, preceded by a 19 amino acid signal peptide) and 128 amino acids (PLA₂-II, preceded by a 25 amino acid signal peptide), and were derived from four expressed sequence tag (EST) clones. Both proteins were homologous to the N-terminal amino acid sequence of the elm PLA₂. They contained twelve conserved cysteine residues and sequences that are likely to represent the Ca²⁺-binding loop and active-site motif, which are characteristic of animal secretory PLA₂s. A soluble PLA₂ activity was purified 145 000-fold from green rice shoots. This had the same biochemical characteristics as the elm and animal secretory PLA₂s. The purified rice PLA₂ consisted of two proteins, with a molecular weight of 12 440 and 12 920, that had identical N-terminal amino acid sequences. This sequence was different from but homologous to the PLA₂-I and PLA₂-II sequences. Taken together, the results suggest that at least three different low-molecular-weight PLA₂s are expressed in green rice shoots. Southern blot analysis suggested that multiple copies of such genes are likely to occur in the rice and in other plant genomes.     

烟实夜蛾信息素结合蛋白3 cDNA的克隆、序列分析与原核表达

利用RT-PCR技术从烟实夜蛾Helicoverpa assulta (Hass) 雄虫触角中扩增得到了信息素结合蛋白3(Hass PBP3)。克隆和测序结果表明,该基因核苷酸序列全长495 bp,编码164个氨基酸残基,预测分子量18.5 kD。并预测N-末端疏水区包含由22个氨基酸组成的信号肽。因此,成熟蛋白应包括142个氨基酸,预测分子量为16.1 kD,等电点为5.44。经氨基酸序列同源性分析发现,此序列与已知昆虫PBP3有较高的同源性,而且具有气味结合蛋白的典型特征。将该基因重组到表达载体pGEX-4T-2中进行原核表达。经IPTG诱导、SDS-PAGE分析和Western印迹检测,结果表明烟实夜蛾PBP3基因能在大肠杆菌BL21中表达,电泳检测到一条大约42 kD的外源蛋白,与预测的融合蛋白分子量相符。     

Complementary deoxyribonucleic acid cloning of a messenger ribonucleic acid encoding transforming growth factor beta 4 from chicken embryo chondrocytes

Using a human transforming growth factor beta 1 (TGF beta) cDNA probe, we have detected an RNA species migrating at about 1.7 kilobases in cultured primary chicken embryo chondrocytes that is distinct from chicken TGF beta 1. The cloning and sequencing of cDNAs corresponding to this chondrocyte RNA demonstrate that it represents a new member of the TGF beta family, which we have named TGF beta 4. Unlike previously described TGF beta which are 390 to 414 amino acids long, the predicted precursor protein of TGF beta 4 is only 304 amino acids and does not appear to contain a signal peptide. Also unique to this new TGF beta is an insertion of two amino acids near the N-terminus of the processed peptide which would result in a 114 amino acid mature protein after cleavage from the precursor at a tetrabasic arg-arg-arg-arg site. The nine cysteine residues characteristic of all TGF beta are conserved. TGF beta 4 shows 82%, 64%, and 71% identity with the amino acid sequences of processed TGF beta 1, 2, and 3, respectively.     

Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA.

Human beta 2-glycoprotein (beta 2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature beta 2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb, respectively, hybridizing specifically with the beta 2gpI cDNA. Upon isoelectric focusing, recombinant beta 2gpI obtained from expression of beta 2gpI cDNA in baby hamster kidney cells showed the same pattern of bands as beta 2gpI isolated from plasma, and at least 5 polypeptides were visible.     

EXPRESSION OF A BEE-VENOM PHOSPHOLIPASE A2 FROM APIS CERANA CERANA IN ESCHERICHIA COLI

Abstract  The venomous phospholipase A₂ (AcPLA₂) coding reading region of the Chinese honeybee ( Apis cerana cerana ), which is composed of 405 bp encoding a mature glycosylated peptide with 134 amino residues was transformed into the expression vector pETblue-1. Then the recombinant vector was introduced into Escherichia coli Tuner (DE3) plac I for expression. Analysis result of SDS-PAGE showed that the expression products had a protein band of about 15kD. Detection of western blot using ant-European honeybee ( Apis mellifera ) phospholipase A₂ (AmPLA₂) polyclonal serum as the first antibody showed that the expression products appeared a special blot same as the native AmPLA₂.The result demonstrated that the AcPLA₂ peptide had been expressed in E. coli and the AcPLA₂ has the similar antigenicity as the AmPLA₂.