The P25 component of Galleria silk

The water-insoluble core of lepidopteran silk is composed of four major proteins, but only three genes have been identified. This study demonstrates that the 29- and 30-kDa components of Galleria mellonella silk are derived from a single gene designated P25. The gene is expressed exclusively in the posterior section of the silk glands as a 2-kb mRNA, which accumulates in the feeding larvae and declines at molting. The mRNA encodes a peptide of 24 864 Da that exhibits 51% identity with the putative product of the P25 gene of Bombyx. The conservation of several amino acid stretches, including the relative positions of all 8 cysteines in the mature polypeptide, implies that the P25 proteins play similar, and apparently significant roles in silk formation in the two species. A Galleria P25 cDNA yields a peptide of about 25 kDa when translated in vitro; the 29- and 30-kDa forms present in the silk are derived from this primary translation product by differential glycosylation. Received: 4 June 1997 / Accepted: 5 September 1997     

Low molecular weight silk proteins in Galleria mellonella

Galleria cocoon proteins have been extracted by different solubilizing agents. Nine protein bands were observed by gel electrophoresis, with molecular weights ranging from 18 to 420 kD. Three silk proteins of 24, 29 and 30 kD were extracted only in the presence of β-mercaptoethanol, suggesting that they are covalently linked by disulfide bonds to the large fibroin. They are likely to be the products of the highly abundant mRNA of the posterior silk gland cells. In vitro translation analysis of this mRNA yielded 24, 29 and 30 kD proteins. Thus, as in Bombyx, the Galleria silk is composed of several subunits, including fibroin and low molecular weight polypeptides. However, the genes coding for fibroin or low molecular weight silk proteins in Bombyx and Galleria do not show nucleotide base homology.     

CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori

Dietary carotenoids are absorbed in the intestine and delivered to various tissues by circulating lipoproteins; however, the mechanism underlying selective delivery of different carotenoid species to individual tissues remains elusive. The products of the Yellow cocoon (C) gene and the Flesh (F) gene of the silkworm Bombyx mori determine the selectivity for transport of lutein and β-carotene, respectively, to the silk gland. We previously showed that the C gene encodes Cameo2, a CD36 family member, which is thought to function as a transmembrane lipoprotein receptor. Here, we elucidated the molecular identity of the F gene product by positional cloning, as SCRB15, a paralog of Cameo2 with 26% amino acid identity. In the F mutant, SCRB15 mRNA structure was severely disrupted, due to a 1.4 kb genomic insertion in a coding exon. Transgenic expression of SCRB15 in the middle silk gland using the binary GAL4-UAS expression system enhanced selective β-carotene uptake by the middle silk gland, while transgenic expression of Cameo2 enhanced selective lutein uptake under the same GAL4 driver. Our findings indicate that divergence of genes in the CD36 family determines the selectivity of carotenoid species uptake by silk gland tissue and that CD36-homologous proteins can discriminate among carotenoid species.     

Microbial production of spider silk proteins

The remarkable properties of spider dragline silk and related protein polymers will find many applications if the materials can be produced economically. We have demonstrated the production of high molecular weight spider dragline silk analog proteins encoded by synthetic genes in several microbial systems, including Escherichia coli and Pichia pastoris. In E. coli, proteins of up to 1000 amino acids in length could be produced efficiently, but the yield and homogeneity of higher molecular weight silk proteins were found to be limited by truncated synthesis, probably as a result of ribosome termination errors. No such phenomenon was observed in the yeast P. pastoris, where higher molecular weight silk proteins could be produced without heterogeneity due to truncated synthesis. Spider dragline silk analog proteins could be secreted by P. pastoris when fused to both the signal sequence and N-terminal pro-sequence of the Saccharomyces cerevisiae alpha-mating factor gene.     

Intramolecular homologous recombination event occurred in the spider egg case silk gene CySp2 of wasp spider Argiope bruennichi

To gain further understanding of egg case silk proteins gene family, Zhao et al. (2006) isolated two full-length cDNAs for egg case silk proteins, cylindrical silk protein 1 (CySp1) and cylindrical silk protein 2 (CySp2), from the wasp spider, Argiope bruennichi. CySp2 was reported to contain no apparent Signal peptide sequences, and the CySp1-CySp2 complex, which would possess a signal peptide, would be transported across the endoplasmic reticulum and secreted to the Golgi. According to a report by Hayashi, genomic DNA sequencing is one approach that can be successfully utilized to retrieve 5′ ends of silk genes; using this method, we retrieved the 5’ end of CySp1. We found that CySp2 contained a typical signal peptide similar to that found in CySp1; thus, due to technical limitations, an artificial error had occurred in the CySp2 sequence reported by Zhao et al.     

An Exported Rhodanese-Like Protein Is Induced during Growth of Acidithiobacillus ferrooxidans in Metal Sulfides and Different Sulfur Compounds

By proteomic analysis we found a 21-kDa protein (P21) from Acidithiobacillus ferrooxidans ATCC 19859 whose synthesis was greatly increased by growth of the bacteria in pyrite, thiosulfate, elemental sulfur, CuS, and ZnS and was almost completely repressed by growth in ferrous iron. After we determined the N-terminal amino acid sequence of P21, we used the available preliminary genomic sequence of A. ferrooxidans ATCC 23270 to isolate the DNA region containing the p21 gene. The nucleotide sequence of this DNA fragment contained a putative open reading frame (ORF) coding for a 23-kDa protein. This difference in size was due to the presence of a putative signal peptide in the ORF coding for P21. When p21 was cloned and overexpressed in Escherichia coli, the signal peptide was removed, resulting in a mature protein with a molecular mass of 21 kDa and a calculated isoelectric point of 9.18. P21 exhibited 27% identity and 42% similarity to the Deinococcus radiodurans thiosulfate-sulfur transferase (rhodanese; EC 2.8.1.1) and similar values in relation to other rhodaneses, conserving structural domains and an active site with a cysteine, both characteristic of this family of proteins. However, the purified recombinant P21 protein did not show rhodanese activity. Unlike cytoplasmic rhodaneses, P21 was located in the periphery of A. ferrooxidans cells, as determined by immunocytochemical analysis, and was regulated depending on the oxidizable substrate. The genomic context around gene p21 contained other ORFs corresponding to proteins such as thioredoxins and sulfate-thiosulfate binding proteins, clearly suggesting the involvement of P21 in inorganic sulfur metabolism in A. ferrooxidans.     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.     

Characterization of mleR, a positive regulator of malolactic fermentation and part of the acid tolerance response in Streptococcus mutans

Background

The periodontal pathogen Porphyromonas gingivalis is an obligate anaerobe that requires heme for growth. To understand its heme acquisition mechanism, we focused on a hemin-binding protein (HBP35 protein), possessing one thioredoxin-like motif and a conserved C-terminal domain, which are proposed to be involved in redox regulation and cell surface attachment, respectively.

Results

We observed that the hbp35 gene was transcribed as a 1.1-kb mRNA with subsequent translation resulting in three proteins with molecular masses of 40, 29 and 27 kDa in the cytoplasm, and one modified form of the 40-kDa protein on the cell surface. A recombinant 40-kDa HBP35 exhibited thioredoxin activity in vitro and mutation of the two putative active site cysteine residues abolished this activity. Both recombinant 40- and 27-kDa proteins had the ability to bind hemin, and growth of an hbp35 deletion mutant was substantially retarded under hemin-depleted conditions compared with growth of the wild type under the same conditions.

Conclusion

P. gingivalis HBP35 exhibits thioredoxin and hemin-binding activities and is essential for growth in hemin-depleted conditions suggesting that the protein plays a significant role in hemin acquisition.     

Macromolecules Involved in the Amoeba-Bacteria Symbiosis1

ABSTRACT. In the Amoeba-bacteria symbiosis, rod-shaped Gram-negative bacterial endosymbionts reside within symbiosomes in the host cytoplasm, and the host and symbionts are mutually dependent for survival. Three proteins and one group of lipopolysaccharides (LPS) synthesized by the bacterial endosymbionts and two proteins derived from the host cells have been found to be involved in the host-symbiont interactions, although their respective roles are not yet fully known. The symbiont-derived molecules included proteins with molecular weights of 29 kDa, 67 kDa and 96 kDa and LPS. The 29-kDa protein was most abundant in the host cytoplasm, while the 96-kDa protein and LPS were found mostly on the symbiosome membranes. The 67-kDa protein was a GroEL analog and stayed within the symbionts. The host-derived 43-kDa protein, actin, was selectively accumulated by the symbionts, while the 220/225-kDa protein, spectrin, was attached to the symbiosome membranes. The symbiont genes coding for the 29-kDa and 67-kDa proteins were cloned and sequenced. The 29-kDa protein gene was unique with no relation to any known DNA sequences but has a leucine zipper-like motif, suggesting a possible DNA-binding function. The DNA sequence of the 67-kDa protein gene showed a 70% identity with heat-shock-protein genes of Escherichia coli and Coxiella burnetii.     

Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets

Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing. To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) [alpha-32P]GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes. The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound [alpha-32P]GTP prominently but was only poorly detected with the GTP gamma S-binding assay. The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu). A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids. In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets. One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein. Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) J. Biol. Chem. 264, 16378-16382). These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds [alpha-32P]GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.     

Proteomic evaluation of gymnosperm pollination drop proteins indicates highly conserved and complex biological functions

The pollination droplet is a highly conservative pollination mechanism that is observed in all major gymnosperm taxa. Proteomics analysis of the pollination drops was carried out on four gymnosperm species: Juniperus communis (common juniper), Juniperus oxycedrus (prickly juniper), Chamaecyparis lawsoniana (Port Orford cedar), and Welwitschia mirabilis. Pollination drop proteins were purified by SDS-PAGE, and the most abundant proteins were analyzed by mass spectrometry and sequenced. Based on BLAST searching of combined amino acid sequences, the following proteins were identified in the following species: an 83-kDa subtilisin-like proteinase, a 62-kDa glycosyl hydrolase, a 47.5-kDa glucan 1,3-β-glucosidase precursor, a 30-kDa chitinase, and a 25-kDa thaumatin-like protein were identified in J. communis; a 30-kDa chitinase, a 25-kDa thaumatin-like protein, and a 32.5-kDa glucanase-like protein were identified in J. oxycedrus; an 83-kDa subtilisin-like proteinase, a 62-kDa β-d-glucan exohydrolase, a 47.5-kDa glucan 1,3-β-glucosidase, and two 25-kDa thaumatin-like proteins were identified in C. lawsoniana, and a 25-kDa chitinase was identified in W. mirabilis. Based on protein identifications, there is strong evidence that the pollination drop functions in both pathogen defense and pollen development. The discovery of similarities in terms of peptide sequence and protein identifications indicates that ovular secretions are functionally conservative, and that they are essential to reproductive success.     

Truncation analysis of an alkaline cellulase from an alkalophilic Bacillus species

A series of truncated gene products from an alkaline cellulase from an alkalophilic Bacillus sp. No. 1139 has been prepared. The variously sized proteins were products of in vitro insertional mutagenesis constructs made by gene inserts containing translational terminators. One product, a 46-kDa protein, which had about half the M_r of the original cellulase, had a similar enzyme activity and pH optimum to the original 92-kDa protein. In contrast, a slightly smaller product protein (43 kDa) did not show cellulase activity.     

Construction and Characterization of a Helicobacter pylori clpB Mutant and Role of the Gene in the Stress Response

Antiserum raised against whole Helicobacter pylori cells identified a novel 94-kDa antigen. The nucleotide sequence of the gene encoding the 94-kDa antigen was determined, and analysis of the deduced amino acid sequence revealed structural features typical of the ClpB ATPase family of stress response proteins. An isogenic H. pylori clpB mutant showed increased sensitivity to high-temperature stress, indicating that the clpB gene product functions as a stress response protein in H. pylori.     

Profiling of Viral Proteins Expressed from the Genomic RNA of Japanese Encephalitis Virus Using a Panel of 15 Region-Specific Polyclonal Rabbit Antisera: Implications for Viral Gene Expression

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, is closely related to West Nile (WN), yellow fever (YF), and dengue (DEN) viruses. Its plus-strand genomic RNA carries a single open reading frame encoding a polyprotein that is cleaved into three structural (C, prM/M, and E) and at least seven nonstructural (NS1/NS1'', NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins, based on previous work with WNV, YFV, and DENV. Here, we aimed to profile experimentally all the viral proteins found in JEV-infected cells. We generated a collection of 15 JEV-specific polyclonal antisera covering all parts of the viral protein-coding regions, by immunizing rabbits with 14 bacterially expressed glutathione-S-transferase fusion proteins (for all nine viral proteins except NS2B) or with a chemically synthesized oligopeptide (for NS2B). In total lysates of JEV-infected BHK-21 cells, immunoblotting with these antisera revealed: (i) three mature structural proteins (~12-kDa C, ~8-kDa M, and ~53-kDa E), a precursor of M (~24-kDa prM) and three other M-related proteins (~10-14 kDa); (ii) the predicted ~45-kDa NS1 and its frameshift product, ~58-kDa NS1'', with no evidence of the predicted ~25-kDa NS2A; (iii) the predicted but hardly detectable ~14-kDa NS2B and an unexpected but predominant ~12-kDa NS2B-related protein; (iv) the predicted ~69-kDa NS3 plus two major cleavage products (~34-kDa NS3^N-term and ~35-kDa NS3^C-term), together with at least nine minor proteins of ~16-52 kDa; (v) the predicted ~14-kDa NS4A; (vi) two NS4B-related proteins (~27-kDa NS4B and ~25-kDa NS4B''); and (vii) the predicted ~103-kDa NS5 plus at least three other NS5-related proteins (~15 kDa, ~27 kDa, and ~90 kDa). Combining these data with confocal microscopic imaging of the proteins’ intracellular localization, our study is the first to provide a solid foundation for the study of JEV gene expression, which is crucial for elucidating the regulatory mechanisms of JEV genome replication and pathobiology.