Differences In Amino Acid Sequence Research Articles

Chaperones are necessary for the expression of catalytically active potato apyrases in prokaryotic cells.

NTPDases (nucleoside triphosphate diphosphohydrolases) (also called in plants apyrases) hydrolyze nucleoside 5′-tri- and/or diphosphate bonds producing nucleosides di or monophosphate and inorganic phosphate. For years, studies have been carried out to use both plant and animal enzymes for medicine. Therefore, there is a need to develop an efficient method for the quick production of large amounts of homogeneous proteins with high catalytic activity. Expression of proteins in prokaryotic cells is the most common way for the protein production. The aim of our study was to develop a method of expression of potato apyrase (StAPY4, 5, and 6) genes in bacterial cells under conditions that allowed the production of catalytically active form of these enzymes. Apyrase 4 and 6 were overexpressed in BL21-CodonPlus (DE3) bacteria strain but they were accumulated in inclusion bodies, regardless of the culture conditions and induction method. Co-expression of potato apyrases with molecular chaperones allowed the expression of catalytically active apyrase 5. However, its high nucleotidase activity could be toxic for bacteria and is therefore synthesized in small amounts in cells. Our studies show that each protein requires other conditions for maturation and even small differences in amino acid sequence can essentially affect protein folding regardless of presence of chaperones.

A subtle alternative splicing event gives rise to a widely expressed human RNase k isoform.

Subtle alternative splicing leads to the formation of RNA variants lacking or including a small number of nucleotides. To date, the impact of subtle alternative splicing phenomena on protein biosynthesis has been studied in frame-preserving incidents. On the contrary, mRNA isoforms derived from frame-shifting events were poorly studied and generally characterized as non-coding. This work provides evidence for a frame-shifting subtle alternative splicing event which results in the production of a novel protein isoform. We applied a combined molecular approach for the cloning and expression analysis of a human RNase κ transcript (RNase κ-02) which lacks four consecutive bases compared to the previously isolated RNase κ isoform. RNase κ-02 mRNA is expressed in all human cell lines tested end encodes the synthesis of a 134-amino-acid protein by utilizing an alternative initiation codon. The expression of RNase κ-02 in the cytoplasm of human cells was verified by Western blot and immunofluorescence analysis using a specific polyclonal antibody developed on the basis of the amino-acid sequence difference between the two protein isoforms. The results presented here show that subtle changes during mRNA splicing can lead to the expression of significantly altered protein isoforms.

Reprolysin metalloproteases from Ixodes persulcatus, Rhipicephalus sanguineus and Rhipicephalus microplus ticks

Metalloproteases (MPs) have been considered essential for blood feeding and other physiological functions in several hematophagous animals, including ticks. We report the characterization of MP sequences of three important ticks from Asia, Africa and America: Ixodes persulcatus (Ip-MPs), Rhipicephalus sanguineus (Rs-MPs) and R. microplus (BrRm-MPs). Amino acid sequence identity between R. microplus and R. sanguineus MPs ranged from 76 to 100 %, and identities among I. persulcatus, I. ricinus and I. scapularis MP sequences ranged from 88 to 97 %. This high sequence identity and typical functional motifs show that all sequences are MPs. The presence of a zinc binding site, a Met-turn and cysteine rich domain at the C-terminal region indicates that these proteins belong to the reproplysin family of MPs. Differences in amino acid sequences of BrRm-MP1, BrRm-MP2, BrRm-MP4 and BrRm-MP5 (from Porto Alegre strain ticks) were 6, 2, 7 and 5 %, respectively, when compared with sequences deposited in GenBank for the same genes from other R. microplus isolates. Analyses of MPs predicted that they have various highly antigenic regions. Semi-quantitative RT-PCR analysis revealed the presence of transcripts in salivary glands of partially and fully fed female ticks. None of these transcripts were observed in males (except BrRm-MP4) and eggs. These enzymes may be functional components required during tick feeding to manipulate host defenses and support tick hematophagy.

Elements Modulating the Prion Species Barrier and Its Passage Consequences

The specific characteristics of Transmissible Spongiform Encephalopathy (TSE) strains may be altered during passage across a species barrier. In this study we investigated the biochemical and biological characteristics of Bovine Spongiform Encephalopathy (BSE) after transmission in both natural host species (cattle, sheep, pigs and mice) and in transgenic mice overexpressing the corresponding cellular prion protein (PrPC) in comparison with other non-BSE related prions from the same species. After these passages, most features of the BSE agent remained unchanged. BSE-derived agents only showed slight modifications in the biochemical properties of the accumulated PrPSc, which were demonstrated to be reversible upon re-inoculation into transgenic mice expressing bovine-PrPC. Transmission experiments in transgenic mice expressing bovine, porcine or human-PrP revealed that all BSE-derived agents were transmitted with no or a weak transmission barrier. In contrast, a high species barrier was observed for the non-BSE related prions that harboured an identical PrP amino acid sequence, supporting the theory that the prion transmission barrier is modulated by strain properties (presumably conformation-dependent) rather than by PrP amino acid sequence differences between host and donor.As identical results were observed with prions propagated either in natural hosts or in transgenic mouse models, we postulate that the species barrier and its passage consequences are uniquely governed by the host PrPC sequence and not influenced by other host genetic factors. The results presented herein reinforce the idea that the BSE agent is highly promiscuous, infecting other species, maintaining its properties in the new species, and even increasing its capabilities to jump to other species including humans. These data are essential for the development of an accurate risk assessment for BSE.

The Key to the Extraordinary Thermal Stability of P. furiosus Holo-Rubredoxin: Iron Binding-Guided Packing of a Core Aromatic Cluster Responsible for High Kinetic Stability of the Native Structure

Pyrococcus furiosus rubredoxin (PfRd), a small, monomeric, 53 residues-long, iron-containing, electron-transfer protein of known structure is sometimes referred to as being the most structurally-stable protein known to man. Here, using a combination of mutational and spectroscopic (CD, fluorescence, and NMR) studies of differently made holo- and apo-forms of PfRd, we demonstrate that it is not the presence of iron, or even the folding of the PfRd chain into a compact well-folded structure that causes holo-PfRd to display its extraordinary thermal stability, but rather the correct iron binding-guided packing of certain residues (specifically, Trp3, Phe29, Trp36, and also Tyr10) within a tight aromatic cluster of six residues in PfRd's hydrophobic core. Binding of the iron atom appears to play a remarkable role in determining subtle details of residue packing, forcing the chain to form a hyper-thermally stable native structure which is kinetically stable enough to survive (subsequent) removal of iron. On the other hand, failure to bind iron causes the same chain to adopt an equally well-folded native-like structure which, however, has a differently-packed aromatic cluster in its core, causing it to be only as stable as any other ordinary mesophile-derived rubredoxin. Our studies demonstrate, perhaps for the very first time ever that hyperthermal stability in proteins can owe to subtle differences in residue packing vis a vis mesostable proteins, without there being any underlying differences in either amino acid sequence, or bound ligand status.

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (Pi) residues linked by "high-energy" phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyP-driven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities.

Wild and attenuated vaccine RS-12 strains of mumps virus exhibit differences in amino acid sequences of their proteins.

Attenuated mumps virus (MuV) RS-12 strain-based vaccine is one of several effective vaccines available in the prevention of mumps. Since previous studies have unveiled only about one-third of the attenuated vaccine RS-12 strain genome sequence, the rest of sequence and molecular basis for attenuation remained unsolved. Therefore, in this study, the full-length genome sequences of wild and attenuated RS-12 strains were determined and compared. The comparison revealed nucleotide substitutions at 9 positions leading to amino acid substitutions at 6 positions in P, V, I, M, and L proteins, while the remaining substitutions were silent. This result indicates that the observed mutations in P, V, I, M, and L proteins of MuV might be responsible for the attenuation of the RS-12 vaccine strain.

The comparison of peroxynitrite action on bovine, porcine and human fibrinogens

Abstract Subjects of bovine and porcine flocks are sometimes susceptible to death before time of slaughter, and some of those deaths may be due to cardiovascular problems connected with stress. The role of oxidative stress in farm animals is yet unexplored. Human fibrinogen seems to be highly susceptible to nitration. Peroxynitrite produced from superoxide and nitric oxide initiates noticeable changes in the structure of human fibrinogen molecule. The objective of this work is to compare the in vitro interactions of peroxynitrite with human fibrinogen and with fibrinogen from mammals of great economic importance, namely cows and pigs. Using western blots and ELISA we show that porcine fibrinogen is susceptible to tyrosine nitration induced by peroxynitrite whereas, bovine fibrinogen is more resistant. Moreover, porcine fibrinogen polymerization is susceptible to peroxynitrite action, whereas bovine fibrinogen is the least susceptible to inhibition of polymerization caused by peroxynitrite. These observed differences may result from differences in amino acid sequence of fibrinogen chains, mostly including tyrosine content and location in the Aα chain. Protection against toxic effects of peroxynitrite activity in the circulatory system seems to be important in avoiding cardiovascular diseases and may prevent production loss in pig breeding herds.

Interface dynamics explain assembly dependency of influenza neuraminidase catalytic activity

Influenza virus neuraminidase (iNA) is a homotetrameric surface protein of the influenza virus and an established target for antiviral drugs. In contrast to neuraminidases (NAs) of other biological systems (non-iNAs), enzymatic activity of iNA is only observed in a quaternary assembly and iNA needs the tetramerization to mediate enzymatic activity. Obviously, differences on a molecular level between iNA and non-iNAs are responsible for this intriguing observation. Comparison between protein structures and multiple sequence alignment allow the identification of differences in amino acid composition in crucial regions of the enzyme, such as next to the conserved D151 and the 150-loop. These differences in amino acid sequence and protein tetramerization are likely to alter the dynamics of the system. Therefore, we performed molecular dynamics simulations to investigate differences in the molecular flexibility of monomers, dimers, and tetramers of iNAs of subtype N1 (avian 2004, pandemic 1918 and pandemic 2009 iNA) and as comparison the non-iNA monomer from Clostridium perfringens. We show that conformational transitions of iNA are crucially influenced by its assembly state. The protein–protein interface induces a complex hydrogen-bonding network between the 110-helix and the 150-loop, which consequently stabilizes the structural arrangement of the binding site. Therefore, we claim that these altered dynamics are responsible for the dependence of iNA’s catalytic activity on the tetrameric assembly. Only the tetramerization-induced balance between stabilization and altered local flexibility in the binding site provides the appropriate arrangement of key residues for iNA’s catalytic activity.

B-Cell Epitopes Within The A2 and C2 Domains Of Coagulant Factor VIII Refined By Orthologous Protein Antigenicity

Approximately 20-30% of severe hemophilia A patients develop inhibitors (anti-fVIII antibodies) to human fVIII (hfVIII) with the primary targets being the A2 and C2 domains. Studies of A2 domain-specific murine monoclonal antibodies (MAbs) targeting hfVIII identified 8 distinct groups of inhibitory MAbs displaying varying inhibitor titers and kinetics, with groups A, D, and E possessing the greatest inhibitory activity. Furthermore within the C2 domain, we previously characterized 5 distinct groups, whereby group BC antibodies were the most common and displayed the highest specific inhibitory activity. At present, a recombinant porcine fVIII (pfVIII) product (OBI-1, Baxter International) is being investigated within a pivotal clinical trial for persons with acquired hemophilia A. The rational for a recombinant pfVIII product stems from the limited cross reactivity of pfVIII, and potentially other fVIII orthologs, with antibodies developed against hfVIII and the prior clinical success of plasma-derived pfVIII. We have described the biochemical characterization of both pfVIII and, more recently, ovine fVIII (ofVIII) and now investigate their antigenic properties to define/refine key inhibitory epitopes and determine the amino acid differences that confer reduced antigenicity. Using an ELISA-based assay, 15 A2 and 12 C2 MAbs spanning all inhibitory groups were screened against B-domain deleted (BDD) pfVIII and ofVIII. Only 4 A2 domain targeting MAbs representing groups B, C, DE, and E were found to bind pfVIII and all displayed reduced cross-reactivity (Figure 1). Additionally, 3 MAbs within groups B, C, or E also bound ofVIII. No group A or D MAbs screened bound either ortholog. Of the C2 domain targeting MAbs tested, 3 MAbs (I109, D102, and ESH-8) from groups AB, B and C, respectively, bound pfVIII to lesser degrees. Nine of the 12 C2 targeting MAbs bound ofVIII, albeit at reduced signal strength, suggesting divergence in C2 epitopes between pfVIII and hfVIII as well as between pfVIII and ofVIII. Using these data and previous literature defining the specific epitopes of these MAbs, we suggest that species-specific amino acid sequence differences may be responsible for the differential binding. MAb413, a well characterized A2 group A inhibitor, was found not to bind either ortholog. Alanine scanning mutagenesis within the 484 – 508 epitope demonstrated that single point mutations at R484, Y487, R489, or P492 could each inhibit binding of MAb413 to below 10%. In addition, P485 or S488 mutations each reduced binding to approximately 40% (Lubin et al J Biol Chem 272:48). Both pfVIII and ofVIII share substitutions R484S, Y487H, R489G, and pfVIII contains unique substitutions P485A, S488P, and P492L. Since all but two A2 group A MAbs have been mapped to residues 484-508, and because all group A MAbs compete with each other for their epitope, we hypothesize that these substitutions are responsible for diminished binding of group A MAbs. Group D inhibitory antibodies have been mapped to residues 604-740. Within this region, there are 12 shared amino acid substitutions and 7 amino acids altered in either ortholog but not both. As it was demonstrated that the orthologs display differential cross reactivity, this suggests disparate amino acids alter inhibitor-epitope recognition. Walter et al. (J Biol Chem 2013 288:14) used small angle x-ray scattering (SAXS) to isolate the epitope of MAb 3E6, a C2 MAb group A inhibitor, to exposed loops R2209-S2216 and L2178-D2187. Our data show that this MAb is not cross reactive and both orthologs contain S2216T and S2182N substitutions. Finally, ESH-8, a well-studied C2 group C MAb, binds ofVIII and pfVIII equivalently at 29% of the hfVIII level. The epitope for ESH-8 contains amino acids 2248-2285 and within this region there is only one conserved substitution, F2275L. Therefore, we predict that substitution of this residue could restore or further diminish ESH-8 binding. These data provide evidence that the lack of A2 group A and D epitopes, as well as the C2 domain group BC epitopes within these orthologs may be responsible for the residual procoagulant activity in the context of inhibitor plasma. Furthermore, these data also suggest that 1) B-cell epitopes can be defined or refined through this approach, 2) less antigenic residues can be identified and 3) the information obtained can be utilized to rationally design recombinant fVIII products with reduced antigenicity. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Shell morphology and genetic characteristics of Corbula (Potamocorbula) spp. (Bivalvia: Corbulidae) from the waters adjacent to Japan, Korea and San Francisco Bay

Corbula (Potamocorbula) spp. are natively distributed in the waters adjacent to Japan and Korea. These populations are distinguished by 6 morphological characteristics. How- ever, they have high geographic variation, and it is quite difficult to distinguish among them. In the present study, we investigated the geographic distribution of the morphological and genetic characteristics of Corbula (Potamocorbula) spp. collected from the waters adjacent to Japan, Korea and San Francisco Bay, USA. Shell morphology was inferred through canonical discrimi- nant analysis (CDA) of 8 shell characteristics, and genetic characteristics were analysed using the DNA sequences of mitochondrial cytochrome c oxidase subunit 1 (CO1) and nuclear internal tran- scribed spacer 2 (ITS2) regions. The results of CDA showed that the samples from Ganghwa-do in western Korea could be clearly separated from those of all the other localities. The molecular phy- logenetic tree and haplotype network that were constructed from the CO1 and ITS2 regions, respectively, did not show clear groupings among the collected samples, except for 1 group com- posed of samples from Ganghwa-do. In addition, no differences in CO1 amino acid sequences could be found among any of the samples examined, including the Ganghwa-do samples. These facts suggest that Corbula (Potamocorbula) spp. distributed in the waters adjacent to Japan and Korea were separated into 2 groups. However, these 2 groups were genetically closely related, and it seems appropriate to treat them as 2 distinct species. The classification of the subgenus Potamocorbula should be re-examined at the species level.

Recombinant Human Prion Protein Inhibits Prion Propagation in vitro

Prion diseases are associated with the conformational conversion of the cellular prion protein (PrPC) into the pathological scrapie isoform (PrPSc) in the brain. Both the in vivo and in vitro conversion of PrPC into PrPSc is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrPSc, but not PrPC, suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrPC with PrPSc. Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrPSc propagation without inducing immune response side effects.

Characterization of Acid-soluble Collagen from the Skin of Hammerhead Shark (Sphyrna lewini )

The characteristics of the acid-soluble collagen (ASC) from the skin of hammerhead shark (Sphyrna lewini) (ASC-H) were investigated and compared with those of calf skin collagen (CSC). ASC-H with a yield of 4.23 ± 0.54% (based on the wet weight of skins) contained Gly (227 residues/1,000 residues) as the major amino acid and had imino acids of 205 residues/1,000 residues. Amino acid composition, sodium dodecyl sulfate polyacrylamide gel electrophoresis pattern and Fourier transform spectroscopy (FTIR), confirmed that ASC-H was mainly composed of type I collagen. The peptide map of ASC-H was different from that of CSC, suggesting the differences in amino acid sequences and conformation. Td of ASC-H was 16.89C, which was similar to those of cold-water fishes but significantly lower than those of tropical fish species and mammals. ASC-H exhibited high solubility in pH (1-4) and low NaCl concentrations (≤3%). In addition, the lyophilized collagen displayed loose, fibrous and porous ultrastructure. Practical Applications At present, large quantities of by-products, accounting for 50–70% of the original raw material, are generated during the process of aquatic products processing industry. Therefore, optimal use of these by-products is a promising way to protect the environment and produce value-added products to increase the revenue of fish processors. Recently, many scientists have focused their interests on isolation and characterization of collagens extracted from skins of aquatic organisms. However, no information regarding acid-soluble collagen (ASC) from skins of Hammerhead shark (Sphyrna lewini) has been reported. The aim of this study was to isolate and characterize ASC from skin of Hammerhead shark in comparison with type I collagen from calf skin (CSC). Therefore, the collagen extracted from the skin of hammerhead shark could bring considerably economic benefits as a substitute for mammalian collagen.

The Structure of the Mercury Transporter MerF in Phospholipid Bilayers: A Large Conformational Rearrangement Results from N-Terminal Truncation

The three-dimensional structure of the 81-residue mercury transporter MerF determined in liquid crystalline phospholipid bilayers under physiological conditions by Rotationally Aligned (RA) solid-state NMR has two long helices, which extend well beyond the bilayer, with a well-defined interhelical loop. Truncation of the N-terminal 12 residues, which are mobile and unstructured when the protein is solubilized in micelles, results in a large structural rearrangement of the protein in bilayers. In the full-length protein, the N-terminal helix is aligned nearly parallel to the membrane normal and forms an extension of the first transmembrane helix. By contrast, this helix adopts a perpendicular orientation in the truncated protein. The close spatial proximity of the two Cys-containing metal binding sites in the three-dimensional structure of full-length MerF provides insights into possible transport mechanisms. These results demonstrate that major changes in protein structure can result from differences in amino acid sequence (e.g., full-length vs truncated proteins) as well as the use of a non-native membrane mimetic environment (e.g., micelles) vs liquid crystalline phospholipid bilayers. They provide further evidence of the importance of studying unmodified membrane proteins in near-native bilayer environments in order to obtain accurate structures that can be related to their functions.

Evolution of Three Parent Genes and Their Retrogene Copies inDrosophilaSpecies

Retrogenes form a class of gene duplicate lacking the regulatory sequences found outside of the mRNA-coding regions of the parent gene. It is not clear how a retrogene's lack of parental regulatory sequences affects the evolution of the gene pair. To explore the evolution of parent genes and retrogenes, we investigated three such gene pairs in the family Drosophilidae; in Drosophila melanogaster, these gene pairs are CG8331 and CG4960, CG17734 and CG11825, and Sep2 and Sep5. We investigated the embryonic expression patterns of these gene pairs across multiple Drosophila species. Expression patterns of the parent genes and their single copy orthologs are relatively conserved across species, whether or not a species has a retrogene copy, although there is some variation in CG8331 and CG17734. In contrast, expression patterns of the retrogene orthologs have diversified. We used the genome sequences of 20 Drosophila species to investigate coding sequence evolution. The coding sequences of the three gene pairs appear to be evolving predominantly under negative selection; however, the parent genes and retrogenes show some distinct differences in amino acid sequence. Therefore, in general, retrogene expression patterns and coding sequences are distinct compared to their parents and, in some cases, retrogene expression patterns diversify.

Presence of antigen-specific somatic allelic mutations and splice variants do not predict for immunological response to genetic vaccination

BackgroundAntigen-specific anti-tumor vaccines have demonstrated clinical efficacy, but immunological and clinical responses appear to be patient-dependent. We hypothesized that naturally-occurring differences in amino acid sequence of a host’s target antigen might predict for immunological outcome from genetic vaccination by presentation of epitopes different from the vaccine.MethodsUsing peripheral blood cells from 33 patients who had been treated with a DNA vaccine encoding prostatic acid phosphatase (PAP), we sequenced the exons encoding PAP and PSA genes from somatic DNA to identify single nucleotide polymorphisms. In addition, mRNA was collected to detect alternative splice variants of PAP.ResultsWe detected four synonymous coding mutations of PAP among 33 patients; non-synonymous coding mutations were not identified. Alternative splice variants of PAP were detected in 22/27 patients tested. The presence of detectable splice variants was not predictive of immunological outcome from vaccination. Immune responses to peptides encoded by these splice variants were common (16/27) prior to immunization, but not associated with immune responses elicited with vaccination.ConclusionsThese results suggest that antigen-specific immune responses detectable after treatment with this genetic vaccine are specific for the host-encoded antigen and not due to epitope differences between the vaccine and a particular individual’s somatic coding sequence.

A variant of blueberry necrotic ring blotch virus associated with red lesions in blueberry

The complete genome of a variant of the multi-segmented (+) RNA virus blueberry necrotic ring blotch virus (BNRBV), which has not been assigned to a genus, was obtained from foliar red lesions on southern highbush blueberries grown in Alachua Co., Florida. The genome organization of this variant, BNRBV-RL, is the same as that of BNRBV: four genomic segments and seven ORFs (one ORF on each of RNA 1, RNA 2, and RNA 4 and as many as four ORFs on RNA 3). BLAST analysis revealed nucleic acid sequence identities of 89%, 90%, 90% and 86% to BNRBV RNA 1, RNA 2, RNA 3 and RNA 4, respectively. Phylogenetic analysis of the amino acid sequence of the putative RdRp domain indicated that BNRBV-RL was closely related to BNRBV and less related to citrus leprosis virus type C and three other mite-transmitted viruses. The nucleotide and amino acid sequence differences between BNRBV-RL and BNRBV combined with differences in symptom expression in blueberry would suggest that BNRBV-RL is a strain of BNRBV.

Mining the antibodyome for HIV-1–neutralizing antibodies with next-generation sequencing and phylogenetic pairing of heavy/light chains

Next-generation sequencing of antibody transcripts from HIV-1-infected individuals with broadly neutralizing antibodies could provide an efficient means for identifying somatic variants and characterizing their lineages. Here, we used 454 pyrosequencing and identity/divergence grid sampling to analyze heavy- and light-chain sequences from donor N152, the source of the broadly neutralizing antibody 10E8. We identified variants with up to 28% difference in amino acid sequence. Heavy- and light-chain phylogenetic trees of identified 10E8 variants displayed similar architectures, and 10E8 variants reconstituted from matched and unmatched phylogenetic branches displayed significantly lower autoreactivity when matched. To test the generality of phylogenetic pairing, we analyzed donor International AIDS Vaccine Initiative 84, the source of antibodies PGT141-145. Heavy- and light-chain phylogenetic trees of PGT141-145 somatic variants also displayed remarkably similar architectures; in this case, branch pairings could be anchored by known PGT141-145 antibodies. Altogether, our findings suggest that phylogenetic matching of heavy and light chains can provide a means to approximate natural pairings.

Crystal structure of Dioclea violacea lectin and a comparative study of vasorelaxant properties with Dioclea rostrata lectin

Lectins from Diocleinae subtribe belong to the family of legume lectins and are characterized by high identity between their amino acids sequences. It has been shown that punctual differences in amino acid sequences, such as one single amino acid or an alternative conformation, represent changes in biological activities caused by these lectins. Therefore, a more detailed understanding of three-dimensional structures of these proteins is essential for accurate analyzing the relationship between structure and function. In this study lectins purified from the seeds of Dioclea violacea (DVL) and Dioclea rostrata (DRL) were compared with regard to crystal structure and vasorelaxant properties. Differences in structure of lectins were found to be reflected in differences in vasorelaxant effects based on their high specificity and selectivity for cell glycans. Binding activity was related to the position of specific residues in the carbohydrate recognition domain (CRD). DVL complexed structure was solved by X-ray crystallography and was compared to native DVL and DRL. Therefore, DVL was co-crystallized with X-Man, and a molecular modeling with X-Man complexed with DVL was done to compare the complexed and native forms adjusted fit. The relatively narrow and deep CRD in DVL promotes little interaction with carbohydrates; in contrast, the wider and shallower CRD in DRL favors interaction. This seems to explain differences in the level of relaxation induced by DVL (43%) and DRL (96%) in rat aortic rings.

Evolutionary aspects of a unique internal mitochondrial targeting signal in nuclear-migrated rps19 of sugar beet (Beta vulgaris L.)

The endosymbiotic theory postulates that many genes migrated from endosymbionts to the nuclear genomes of their hosts. Some migrated genes lack presequences directing proteins to mitochondria, and their mitochondrial targeting signals appear to be inscribed in the core coding regions as internal targeting signals (ITSs). ITSs may have evolved after sequence transfer to nuclei or ITSs may have pre-existed before sequence transfer. Here, we report the molecular cloning of a sugar beet gene for ribosomal protein S19 (Rps19; the first letter is capitalized when the gene is a nuclear gene). We show that sugar beet Rps19 (BvRps19) is an ITS-type gene. Based on amino-acid sequence comparison, dicotyledonous rps19s (the first letter is lower-cased when the gene is a mitochondrial gene), such as tobacco rps19 (Ntrps19), resemble an ancestral form of BvRps19. We investigated whether differences in amino-acid sequences between BvRps19 and Ntrps19 were involved in ITS evolution. Analyses of the intracellular localization of chimaeric GFP-fusion proteins that were transiently expressed in Welsh onion cells showed that Ntrps19-gfp was not localized in mitochondria. When several BvRps19-type amino acid substitutions, none of which was seen in any other angiosperm rps19, were introduced into Ntrps19-gfp, the modified Ntrps19-gfp became localized in mitochondria, supporting the notion that an ITS in BvRps19 evolved following sequence transfer to nuclei. Not all of these substitutions were seen in other ITS-type Rps19s, suggesting that the ITSs of Rps19 are diverse.