Sea urchin TgBMP2/4 gene encoding a bone morphogenetic protein closely related to vertebrate BMP2 and BMP4 with maximal expression at the later stages of embryonic development.

We have cloned a gene fragment (named TgBMP2/4) that encodes a protein homologous to vertebrate bone morphogenetic protein (BMP) 2 and BMP4 in the sea urchin Tripneustes gratilla. This peptide sequence contains 204 amino acids with 7 conserved cysteine residues at the C-terminus of the coding region and a cluster of basic amino acids that may serve as a signal for proteolytic cleavage. Sequence comparison and phylogenetic analyses reveal that TgBMP2/4 is closely related to vertebrate BMP2 and BMP4 as well as to amphioxus BMP2/4, with similarity levels ranging from 90% to 94% at the mature C-terminal domain. Northern blot analyses show that a 6.3-kb TgBMP2/4 mRNA appears first at the mesenchyme blastula stage and increases to a maximal level at the gastrula and pluteus stages. This expression pattern is different from that of a BMP2/4-related gene previously found in sea urchin.     

Isolation, molecular cloning and antimicrobial activity of novel defensins from common chickweed (Stellaria media L.) seeds

Two novel highly homologous defensins, Sm-AMP-D1 and Sm-AMP-D2, were isolated from seeds of common chickweed Stellaria media L. (family Cariophyllaceae). They show sequence homology to defensins of the Brassicaceae plants and display strong inhibitory activity against phytopathogenic fungi and oomycetes in the micromolar range (IC₅₀ ≤ 1 μM). The cDNA sequences coding for Sm-AMP-D1 and Sm-AMP-D2 were obtained. They code for highly homologous precursor proteins, consisting of a signal peptide of 32 amino acid residues and the mature peptide domain of 50 amino acid residues. The Sm-AMP-D1 and Sm-AMP-D2 precursors differ by two amino acids: one in the signal peptide region, and the other, in the mature peptide domain. Two Sm-D1-encoding genes were identified in S. media genome by PCR amplification from the genomic DNA using Sm-D1-specific primers. They contain a single 599-bp intron in the signal peptide domain and differ from each other by nucleotide substitutions in the intron and 3′-untranslated regions, while the coding sequences are well conserved. One of the genes matched perfectly the sm-D1 cDNA sequence. The sm-D genes show promise for engineering pathogen resistance in crops and expand our knowledge on weed genomics.     

Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants.     

The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signaling activities

Nodal proteins are secreted signaling factors of the transforming growth factor beta (TGFbeta) family with essential roles in embryonic development in vertebrates. Mutations affecting the Nodal factors have severe consequences in mammals and fish. Furthermore, increased Nodal levels have been associated with melanoma tumor progression. Like other TGFbeta-related proteins, Nodal factors consist of a pro-domain and a mature domain. The pro-domain of mouse Nodal protein stabilizes its precursor. However, the mechanisms by which the pro-domains exert their activities are unknown. Here, we characterize the zebrafish Nodal-related factor Cyclops (Cyc) and find unexpected functions for the pro-domain in regulating Cyc activity. We identified a lysosome-targeting region in the Cyc pro-domain that destabilizes the precursor and restricts Cyc activity, revealing the molecular basis for the short-range signaling activities of Cyc. We show that both the pro- and mature-domains of Cyc regulate its stability. We also characterize a mutation in the pro-domain of human NODAL (hNODAL) that underlies congenital heterotaxia. Heterologous expression of mutant hNODAL increases expression of Nodal-response genes. Our studies reveal unexpected roles for the pro-domain of the Nodal factors and provide a possible mechanism for familial heterotaxia.     

Secretion of Active-Form Streptoverticillium mobaraense Transglutaminase by Corynebacterium glutamicum: Processing of the Pro-Transglutaminase by a Cosecreted Subtilisin-Like Protease from Streptomyces albogriseolus

The transglutaminase secreted by Streptoverticillium mobaraense is a useful enzyme in the food industry. A fragment of transglutaminase was secreted by Corynebacterium glutamicum when it was coupled on a plasmid to the promoter and signal peptide of a cell surface protein from C. glutamicum. We analyzed the signal peptide and the pro-domain of the transglutaminase gene and found that the signal peptide consists of 31 amino acid residues and the pro-domain consists of 45 residues. When the pro-domain of the transglutaminase was used, the pro-transglutaminase was secreted efficiently by C. glutamicum but had no enzymatic activity. However, when the plasmid carrying the S. mobaraense transglutaminase also encoded SAM-P45, a subtilisin-like serine protease derived from Streptomyces albogriseolus, the peptide bond to the C side of 41-Ser of the pro-transglutaminase was hydrolyzed, and the pro-transglutaminase was converted to an active form. Our findings suggest that C. glutamicum has potential as a host for industrial-scale protein production.     

Repressive BMP2 gene regulatory elements near the BMP2 promoter

The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.     

Role of the C-terminal pro-domain of aqualysin I in the maturation and translocation of the precursor across the cytoplasmic membrane in Escherichia coli

Aqualysin I, which is a subtilisin-type, extracellular protease secreted by Thermus aquaticus YT-1, is synthesized as a unique precursor bearing pro-domains at both N- and C-terminus of the mature protease domain as well as an N-terminal signal peptide. To investigate the function of the C-terminal pro-domain in maturation and export pathway of the precursor in E. coli cells, aqualysin I variants were constructed in which deletion mutants of the C-terminal pro-domain lacking its own signal peptide were inserted into pIN-III-ompA3. When E. coli harboring wild type and mutant plasmids were induced by 0.2 mM IPTG, active aqualysin I was produced by heat treatment at 65 °C. Aqualysin I precursors with deletions of more than 5 amino acid residues at the C-terminal end of pro-domain were much more rapidly processed than that of wild type, indicating that the C-terminal pro-domain functions as a inhibitor for processing of aqualysin I precursor. With the wild type, most of aqualysin I was present in membrane fraction (probably the outer membrane), whereas for the truncated mutants, it remained in the cytoplasm, indicating that for deletion mutants, their precursors expressed in cells were not translocated across the cytoplasmic membrane, despite the existence of an N-terminal signal peptide.     

Signal Peptide-rheostat Dynamics Delay Secretory Preprotein Folding

Sec secretory proteins are distinguished from cytoplasmic ones by N-terminal signal peptides with multiple roles during post-translational translocation. They contribute to preprotein targeting to the translocase by slowing down folding, binding receptors and triggering secretion. While signal peptides get cleaved after translocation, mature domains traffic further and/or fold into functional states. How signal peptides delay folding temporarily, to keep mature domains translocation-competent, remains unclear. We previously reported that the foldon landscape of the periplasmic prolyl-peptidyl isomerase is altered by its signal peptide and mature domain features. Here, we reveal that the dynamics of signal peptides and mature domains crosstalk. This involves the signal peptide’s hydrophobic helical core, the short unstructured connector to the mature domain and the flexible rheostat at the mature domain N-terminus. Through this cis mechanism the signal peptide delays the formation of early initial foldons thus altering their hierarchy and delaying mature domain folding. We propose that sequence elements outside a protein’s native core exploit their structural dynamics to influence the folding landscape.     

The bovine chromogranin A gene: structural basis for hormone regulation and generation of biologically active peptides.

The structure of the gene encoding bovine chromogranin-A has been determined by characterization of two isolated genomic clones. Chromogranin-A is encoded by eight exons, which organize the coding region into several distinct structural and functional domains. Exons 1-5 represent the highly conserved signal peptide and N-terminal domain, which are separated into regions corresponding to the signal peptide, N-terminal sequence, disulfide-bonded loop, and remainder of the conserved N-terminal domain. Exon 6 represents the variable domain and encodes a region that is identical to the novel chromogranin-A-derived peptide chromostatin. Exon 7 encodes the biologically active peptide pancreastatin as well as most of the conserved C-terminal domain, with the remainder found on exon 8. The mRNA sequence obtained from the gene contains five nucleotide differences from the consensus sequence of four reported bovine chromogranin-A cDNA clones. Two of the differences in the gene result in two amino acid changes in the region encoded by exon 6. The structural organization of the chromogranin-A gene resembles that of the chromogranin-B gene in the exons corresponding to the signal peptide, N-terminal sequence, disulfide loop, and C-terminal sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolution of extracellular Dpp modulators in insects: The roles of tolloid and twisted-gastrulation in dorsoventral patterning of the Tribolium embryo

The formation of the BMP gradient which patterns the DV axis in flies and vertebrates requires several extracellular modulators like the inhibitory protein Sog/Chordin, the metalloprotease Tolloid (Tld), which cleaves Sog/Chordin, and the CR domain protein Twisted gastrulation (Tsg). While flies and vertebrates have only one sog/chordin gene they possess several paralogues of tld and tsg. A simpler and probably ancestral situation is observed in the short-germ beetle Tribolium castaneum (Tc), which possesses only one tld and one tsg gene. Here we show that in T. castaneum tld is required for early BMP signalling except in the head region and Tc-tld function is, as expected, dependent on Tc-sog. In contrast, Tc-tsg is required for all aspects of early BMP signalling and acts in a Tc-sog-independent manner. For comparison with Drosophila melanogaster we constructed fly embryos lacking all early Tsg activity (tsg;;srw double mutants) and show that they still establish a BMP signalling gradient. Thus, our results suggest that the role of Tsg proteins for BMP gradient formation has changed during insect evolution.     

甘薯贮藏蛋白(sporam in)A基因编码区克隆及序列同源性分析

采用 PCR技术 ,从我国广泛栽培甘薯品种南薯 88基因组中扩增和克隆到甘薯贮藏蛋白 A基因编码区段 ,并测定了其全部核苷酸序列 .该编码区长 65 7bp,编码一个长 2 1 9个氨基酸残基的蛋白质 ,其中信号肽长 37个氨基酸残基 ,成熟蛋白质长 1 82个氨基酸残基 ,其分子量为 2 0 k D.将该片段的核苷酸序列与已登录在 Gen Bank中的另外 6个甘薯贮藏蛋白 A基因编码区序列进行比较 ,发现其同源性高达 90 % ,说明甘薯贮藏蛋白 A基因编码区序列具有高度保守性 .虽然 7个基因编码区的核苷酸总变异为 1 0 % ,但在每两个基因之间的比较则表明其核苷酸的变异范围小于 7% .     

Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis

Two different hybrid genes were constructed which fuse the Bacillus amyloliquefaciens alkaline protease gene (apr[BamP]) promoter and signal peptide coding region to a synthetic bpr gene coding for the mature bovine pancreatic RNase A. The first gene fusion (apr-bpr1) contained the apr[BamP] signal peptide coding region fused to mature bpr through a linker coded 3-amino acid region and retained the signal processing site ala-ala of the alkaline protease. The second fusion (apr-bpr2) joined the end of the apr[BamP] signal peptide coding sequence to the mature bpr resulting in a hybrid signal processing site ala-lys. B. subtilis strains harboring these gene fusions secreted bovine pancreatic RNase A into the growth medium. Cleavage at the hybrid signal processing site ala-lys resulted in the secretion of bovine pancreatic RNase A from B. subtilis which had an N-terminal amino acid sequence that was identical to the native RNase A. Bovine pancreatic RNase A contains four disulfide bonds and the proper formation of these bonds is required for activity. RNase activity could be detected in the culture supernatants of strains carrying the apr-bpr gene fusions, which suggests that the proper disulfide bonds have formed spontaneously.     

Secretion of Human Interleukin-2 in Biologically Active Form by Bacillus brevis Directly into Cultute Medium

We constructed an efficient system for synthesis and secretion of human interleukin-2 (IL-2) by Bacillus brevis. The secretion vector we constructed had strong promoters and contained the region coding for the signal peptide of the gene for B. brevis 47 cell-wall protein, followed directly by the gene encoding mature IL-2. Modification of the signal peptide and use of a protease-deficient mutant of B. brevis HPD31 increased productivity. When the signal peptide was more basic near its amino terminal and more hydrophobic in the middle region, IL-2 production increased 20 fold. Production by the mutant harboring the secretion vector was four fold that of the parent harboring the same plasmid. The yield of IL-2 increased further to 0.12 g/liter, when cultural conditions were made optimal, such by the addition of Tween 40 to the medium. The IL-2 produced by B. brevis had the same biological activity as authentic IL-2. Biologically active human IL-2 was produced efficiently and secreted directly into the medium by B. brevis.     

Molecular characterization,balancing selection,and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene

The major histocompatibility complex (MHC) class I genes play a pivotal role in the adaptive immune response among vertebrates. Accordingly, in numerous mammals the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated. To date, however, little is known about these genes in tree shrews, despite the increasingly popularity of its usage as an animal model. To address this deficiency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that the full-length cDNA sequence of the tree shrew MHC class I is 1074 bp in length. The deduced peptide is composed of 357 amino acids containing a leader peptide, an α1 and α2 domain, an α3 domain, a transmembrane domain and a cytoplasmic domain. Among these peptides, the cysteines, CD8⁺ interaction and N-glycosylation sites are all well conserved. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identified to be 3180 bp in length, containing 8 exons and 7 introns. In 21 MHC class I sequences, we conducted an extensive study of nucleotide substitutions. The results indicated that in the peptide binding region (PBR) the rate of non-synonymous substitutions (dN) to synonymous substitutions (dS) was greater than 1, suggesting balancing selection at the PBR. These findings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research.     

Cloning and sequencing of a molluscan endo-beta-1,4-glucanase gene from the blue mussel, Mytilus edulis.

Using polymerase chain reaction, cloning and sequencing techniques, a complementary DNA encoding a low molecular mass cellulase (endo-1,4-beta-D-glucanase, EC 3.2.1.4) has been identified in the digestive gland of the marine mussel, Mytilus edulis. It contains a 5' untranslated region, a 633-nucleotide ORF encoding a 211 amino-acid protein, including a 17 amino-acid signal peptide and a complete 3' untranslated region. At the C-terminal end of the purified mature protein, a 13 amino-acid peptide is lacking in comparison to the protein sequence deduced from the ORF. This peptide is probably removed as a consequence of post-translational amidation of the C-terminal glutamine. The endoglucanase genes have been isolated and sequenced from both Swedish and French mussels. The coding parts of these two sequences are identical. Both genes contain two introns, the positions of which are conserved. However the length of the introns are different due to base substitutions, insertions or deletions showing the existence of interspecies length polymorphism. The percentage of similarity for the introns of the two gene sequences is 96.9%. This is the first time a molluscan cellulase is characterized at DNA level. Amino acid sequence-based classification has revealed that the enzyme belongs to the glycosyl hydrolase family 45 [B. Henrissat (Centre de Recherches sur les Macromolecules Végétales, CNRS, Joseph Fourier Université, Grenoble, France), personal communication]. There is no cellulose binding domain associated with the sequence.