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Plasmodium falciparum: gene structure and hydropathy profile of the major merozoite surface antigen (gp195) of the Uganda-Palo Alto isolate

Chang SP, Kramer KJ, Yamaga KM, Kato A, Case SE, Siddiqui WA

Exp Parasitol, 1988 Oct;67(1):1-11.
PMID: 3049134

The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.

Matched MeSH terms: Amino Acid Sequence
Fulltext A novel sequence-based predictor for identifying and characterizing thermophilic proteins using estimated propensity scores of dipeptides

Charoenkwan P, Chotpatiwetchkul W, Lee VS, Nantasenamat C, Shoombuatong W

Sci Rep, 2021 Dec 10;11(1):23782.
PMID: 34893688 DOI: 10.1038/s41598-021-03293-w

Owing to their ability to maintain a thermodynamically stable fold at extremely high temperatures, thermophilic proteins (TTPs) play a critical role in basic research and a variety of applications in the food industry. As a result, the development of computation models for rapidly and accurately identifying novel TTPs from a large number of uncharacterized protein sequences is desirable. In spite of existing computational models that have already been developed for characterizing thermophilic proteins, their performance and interpretability remain unsatisfactory. We present a novel sequence-based thermophilic protein predictor, termed SCMTPP, for improving model predictability and interpretability. First, an up-to-date and high-quality dataset consisting of 1853 TPPs and 3233 non-TPPs was compiled from published literature. Second, the SCMTPP predictor was created by combining the scoring card method (SCM) with estimated propensity scores of g-gap dipeptides. Benchmarking experiments revealed that SCMTPP had a cross-validation accuracy of 0.883, which was comparable to that of a support vector machine-based predictor (0.906-0.910) and 2-17% higher than that of commonly used machine learning models. Furthermore, SCMTPP outperformed the state-of-the-art approach (ThermoPred) on the independent test dataset, with accuracy and MCC of 0.865 and 0.731, respectively. Finally, the SCMTPP-derived propensity scores were used to elucidate the critical physicochemical properties for protein thermostability enhancement. In terms of interpretability and generalizability, comparative results showed that SCMTPP was effective for identifying and characterizing TPPs. We had implemented the proposed predictor as a user-friendly online web server at http://pmlabstack.pythonanywhere.com/SCMTPP in order to allow easy access to the model. SCMTPP is expected to be a powerful tool for facilitating community-wide efforts to identify TPPs on a large scale and guiding experimental characterization of TPPs.

Matched MeSH terms: Amino Acid Sequence*
Fulltext A prawn core histone 4: derivation of N- and C-terminal peptides and their antimicrobial properties, molecular characterization and mRNA transcription

Chaurasia MK, Palanisamy R, Bhatt P, Kumaresan V, Gnanam AJ, Pasupuleti M, et al.

Microbiol Res, 2015 Jan;170:78-86.
PMID: 25271126 DOI: 10.1016/j.micres.2014.08.011

This study investigates the complete molecular characterization including bioinformatics characterization, gene expression, synthesis of N and C terminal peptides and their antimicrobial activity of the core histone 4 (H4) from freshwater giant prawn Macrobrachium rosenbergii (Mr). A cDNA encoding MrH4 was identified from the constructed cDNA library of M. rosenbergii during screening and the sequence was obtained using internal sequencing primers. The MrH4 coding region possesses a polypeptide of 103 amino acids with a calculated molecular weight of 11kDa and an isoelectric point of 11.5. The bioinformatics analysis showed that the MrH4 polypeptide contains a H4 signature at (15)GAKRH(19). Multiple sequence alignment of MrH4 showed that the N-terminal (21-42) and C-terminal (87-101) antimicrobial peptide regions and the pentapeptide or H4 signature (15-19) are highly conserved including in humans. The phylogenetic tree formed two separate clades of vertebrate and invertebrate H4, wherein MrH4 was located within the arthropod monophyletic clade of invertebrate H4 groups. Three-dimensional model of MrH4 was established using I-TASSER program and the model was validated using Ramachandran plot analysis. Schiffer-Edmundson helical wheel modeling was used to predict the helix propensity of N (21-42) and C (87-101) terminal derived Mr peptides. The highest gene expression was observed in gills and is induced by viral [white spot syndrome baculovirus (WSBV) and M. rosenbergii nodovirus (MrNV)] and bacterial (Aeromonas hydrophila and Vibrio harveyi) infections. The N and C terminal peptides were synthesized and their antimicrobial and hemolytic properties were examined. Both peptides showed activity against the tested Gram negative and Gram positive bacteria; however, the highest activity was noticed against Gram negative bacteria. Among the two peptides used in this study, C-terminal peptide yielded better results than the N-terminal peptide. Therefore, C terminal peptide can be recommended for the development of an antimicrobial agent.

Matched MeSH terms: Amino Acid Sequence
In-silico analysis and mRNA modulation of detoxification enzymes GST delta and kappa against various biotic and abiotic oxidative stressors

Chaurasia MK, Ravichandran G, Nizam F, Arasu MV, Al-Dhabi NA, Arshad A, et al.

Fish Shellfish Immunol, 2016 Jul;54:353-63.
PMID: 27109581 DOI: 10.1016/j.fsi.2016.04.031

This study reports the comprehensive comparative information of two different detoxification enzymes such as glutathione S-transferases (GSTs) delta and kappa from freshwater giant prawn Macrobrachium rosenbergii (designated as MrGSTD and MrGSTK) by investigating their in-silico characters and mRNA modulation against various biotic and abiotic oxidative stressors. The physico-chemical properties of these cDNA and their polypeptide structure were analyzed using various bioinformatics program. The analysis indicated the variation in size of the polypeptides, presence or absence of domains and motifs and structure. Homology and phylogenetic analysis revealed that MrGSTD shared maximum identity (83%) with crustaceans GST delta, whereas MrGSTK fell in arthropods GST kappa. It is interesting to note that MrGSTD and MrGSTK shared only 21% identity; it indicated their structural difference. Structural analysis indicated that MrGSTD to be canonical dimer like shape and MrGSTK appeared to be butterfly dimer like shape, in spite of four β-sheets being conserved in both GSTs. Tissue specific gene expression analysis showed that both MrGSTD and MrGSTK are highly expressed in immune organs such as haemocyte and hepatopancreas, respectively. To understand the role of mRNA modulation of MrGSTD and MrGSTK, the prawns were inducted with oxidative stressors such as bacteria (Vibrio harveyi), virus [white spot syndrome virus (WSSV)] and heavy metal, cadmium (Cd). The analysis revealed an interesting fact that both MrGSTD and MrGSTK showed higher (P

Matched MeSH terms: Amino Acid Sequence
Fulltext Characterization of affinity-purified isoforms of Acinetobacter calcoaceticus Y1 glutathione transferases

Chee CS, Tan IK, Alias Z

ScientificWorldJournal, 2014;2014:750317.
PMID: 24892084 DOI: 10.1155/2014/750317

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively.

Matched MeSH terms: Amino Acid Sequence
Identification of a 48kDa tubulin or tubulin-like C6/36 mosquito cells protein that binds dengue virus 2 using mass spectrometry

Chee HY, AbuBakar S

Biochem Biophys Res Commun, 2004 Jul 16;320(1):11-7.
PMID: 15207695

Binding of dengue virus 2 (DENV-2) to C6/36 mosquito cells protein was investigated. A 48 kDa DENV-2-binding C6/36 cells protein (D2BP) was detected in a virus overlay protein-binding assay. The binding occurred only to the C6/36 cells cytosolic protein fraction and it was inhibited by free D2BP. D2BP was shown to bind to DENV-2 E in the far-Western-binding studies and using mass spectrometry (MS) and MS/MS, peptide masses of the D2BP that matched to beta-tubulin and alpha-tubulin chains were identified. These findings suggest that DENV-2 through DENV-2 E binds directly to a 48 kDa tubulin or tubulin-like protein of C6/36 mosquito cells.

Matched MeSH terms: Amino Acid Sequence
Molecular surveillance of coxsackievirus A16 reveals the emergence of a new clade in mainland China

Chen L, Yao XJ, Xu SJ, Yang H, Wu CL, Lu J, et al.

Arch Virol, 2018 Nov 29.
PMID: 30498962 DOI: 10.1007/s00705-018-4112-3

Coxsackievirus A16 (CV-A16) of the genotypes B1a and B1b have co-circulated in mainland China in the past decades. From 2013 to 2017, a total of 3,008 specimens from 3,008 patients with mild hand, foot, and mouth disease were collected in the present study. Viral RNA was tested for CV-A16 by a real-time RT-PCR method, and complete VP1 sequences and full-length genome sequences of CV-A16 strains from this study were determined by RT-PCR and sequencing. Sequences were analyzed using a series of bioinformatics programs. The detection rate for CV-A16 was 4.1%, 25.9%, 10.6%, 28.1% and 12.9% in 2013, 2014, 2015, 2016 and 2017, respectively. Overall, the detection rate for CV-A16 was 16.5% (497/3008) in this 5-year period in Shenzhen, China. One hundred forty-two (142/155, 91.6%) of the 155 genotype B1 strains in the study belonged to subgenotype B1b, and 13 (13/155, 8.4%) strains belonged to subgenotype B1a. Two strains (CVA16/Shenzhen174/CHN/2017 and CVA16/Shenzhen189/CHN/2017) could not be assigned to a known genotype. Phylogenetic analysis of these two strains and other Chinese CV-A16 strains indicated that these two CV-A16 strains clustered independently in a novel clade whose members differed by 8.4%-11.8%, 8.4%-12.1%, and 14.6%-14.8% in their nucleotide sequences from those of Chinese B1a, B1b, and genotype D strains, respectively. Phylogenetic analysis of global CV-A16 strains further indicated that the two novel CV-A16 strains from this study grouped in a previously uncharacterized clade, which was designated as the subgenogroup B3 in present study. Meanwhile, phylogenetic reconstruction revealed two other new genotypes, B1d and B4, which included a Malaysian strain and two American strains, respectively. The complete genome sequences of the two novel CV-A16 strains showed the highest nucleotide sequence identity of 92.3% to the Malaysian strain PM-15765-00 from 2000. Comparative analysis of amino acid sequences of the two novel CV-A16 strains and their relatives suggested that variations in the nonstructural proteins may play an important role in the evolution of modern CV-A16.

Matched MeSH terms: Amino Acid Sequence
Screening for nasopharyngeal carcinoma with an ELISA using the Epstein-Barr virus nuclear antigen, EBNA 1: a complementary test to the IgA/VCA immunofluorescence assay

Cheng HM, Foong YT, Mathew A, Sam CK, Dillner J, Prasad U

J Virol Methods, 1993 Apr;42(1):45-51.
PMID: 7686558

An ELISA using the Epstein-Barr virus nuclear antigen 1 (EBNA 1) was found to detect selectively specific IgA in sera from patients with nasopharyngeal carcinoma (NPC). The antigen, p107, was a 20-amino acid synthetic peptide, representing a major epitope of EBNA 1.267/294 (90.8%) of NPC patients had IgA antibodies to p107 but in normal individuals, only 41/577 (7.1%) had IgA/p107. In sera from patients with other cancers, 11/77 (14.3%) had IgA/p107 reactivity. 124 IgA/VCA positive and 86 IgA/VCA negative NPC sera were also tested for IgA/p107 binding in ELISA. The majority of IgA/VCA positive sera (117) also contained IgA/p107 antibodies. Of interest was the detection of 74/86 IgA/p107 reactive sera in the IgA/VCA negative group. The results suggest that the IgA/p107 ELISA could become a useful, complementary screening assay to the IgA/VCA immunofluorescence test for detection of NPC.

Matched MeSH terms: Amino Acid Sequence
Linear epitopes of the replication-activator protein of Epstein-Barr virus recognised by specific serum IgG in nasopharyngeal carcinoma

Cheng HM, Foong YT, AbuSamah AJ, Dillner J, Sam CK, Prasad U

Cancer Immunol Immunother, 1995 Apr;40(4):251-6.
PMID: 7750123

The linear antigenic epitopes of the Epstein-Barr virus replication activator protein (ZEBRA), recognised by specific serum IgG in nasopharyngeal carcinoma (NPC), were determined. This was achieved by synthesizing the entire amino acid sequence of ZEBRA as a set of 29, 22-residue peptides with an overlap of 14 amino acids. The ZEBRA peptides were tested in enzyme-linked immunosorbent assay (ELISA) for IgG binding in sera from 37 selected NPC patients who had IgG antibodies to the native ZEBRA protein. The most immunogenic epitope was peptide 1 at the amino-terminal end with 36 of the sera reactive against it. Further analysis of peptide 1, using the multipin peptide-scanning technique, defined a 10-amino-acid sequence FTPDPYQVPF, which was strongly bound by IgG. Two other regions of ZEBRA were also identified as immunodominant IgG epitopes, namely peptide 11 (amino acids 82-103) and peptide 19/20 (amino acids 146-175) with 8-13 of the NPC sera reactive against the peptides. The number of peptides reactive with individual NPC serum varies from 1 to 6 or more and there is some correlation between a greater number of peptide (at least 4) bound and a higher (at least 1:40) titre of serum IgA to viral capsid antigen. The immunodominant ZEBRA peptide 1 could be utilised in IgG ELISA for the detection of NPC.

Matched MeSH terms: Amino Acid Sequence
Application of streptavidin mass spectrometric immunoassay tips for immunoaffinity based antibody phage display panning

Chin CF, Ler LW, Choong YS, Ong EB, Ismail A, Tye GJ, et al.

J Microbiol Methods, 2016 Jan;120:6-14.
PMID: 26581498 DOI: 10.1016/j.mimet.2015.11.007

Antibody phage display panning involves the enrichment of antibodies against specific targets by affinity. In recent years, several new methods for panning have been introduced to accommodate the growing application of antibody phage display. The present work is concerned with the application of streptavidin mass spectrometry immunoassay (MSIA™) Disposable Automation Research Tips (D.A.R.T's®) for antibody phage display. The system was initially designed to isolate antigens by affinity selection for mass spectrometry analysis. The streptavidin MSIA™ D.A.R.T's® system allows for easy attachment of biotinylated target antigens on the solid surface for presentation to the phage library. As proof-of-concept, a domain antibody library was passed through the tips attached with the Hemolysin E antigen. After binding and washing, the bound phages were eluted via standard acid dissociation and the phages were rescued for subsequent panning rounds. Polyclonal enrichment was observed for three rounds of panning with five monoclonal domain antibodies identified. The proposed method allows for a convenient, rapid and semi-automated alternative to conventional antibody panning strategies.

Matched MeSH terms: Amino Acid Sequence
Fulltext Thermal stability engineering of Glomerella cingulata cutinase

Chin IS, Abdul Murad AM, Mahadi NM, Nathan S, Abu Bakar FD

Protein Eng. Des. Sel., 2013 May;26(5):369-75.
PMID: 23468570 DOI: 10.1093/protein/gzt007

Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.

Matched MeSH terms: Amino Acid Sequence
Molecular epidemiology of Newcastle disease viruses in Vietnam

Choi KS, Kye SJ, Kim JY, To TL, Nguyen DT, Lee YJ, et al.

Trop Anim Health Prod, 2014 Jan;46(1):271-7.
PMID: 24061688 DOI: 10.1007/s11250-013-0475-3

Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in Southeast Asia. In the present study, 12 field isolates of NDV were recovered from dead village chickens in Vietnam between 2007 and 2012, and were characterized. All the field isolates were classified as velogenic. Based on the sequence analysis of the F variable region, two distinct genetic groups (Vietnam genetic groups G1 and G2) were recognized. Phylogenetic analysis revealed that all the 12 field isolates fell into the class II genotype VII cluster. Ten of the field isolates, classified as Vietnam genetic group G1, were closely related to VIIh viruses that had been isolated from Indonesia, Malaysia, and Cambodia since the mid-2000s, while the other two field isolates, of Vietnam genetic group G2, clustered with VIId viruses, which were predominantly circulating in China and Far East Asia. Our results indicate that genotype VII viruses, especially VIIh viruses, are predominantly responsible for the recent epizootic of the disease in Vietnam.

Matched MeSH terms: Amino Acid Sequence
Fulltext Why hypothetical protein KPN00728 of Klebsiella pneumoniae should be classified as chain C of succinate dehydrogenase?

Choi SB, Normi YM, Wahab HA

Protein J, 2009 Dec;28(9-10):415-27.
PMID: 19859792 DOI: 10.1007/s10930-009-9209-9

Twenty percent of genes that encode for hypothetical proteins from Klebsiella pneumoniae MGH78578 were identified, leading to KPN00728 and KPN00729 after bioinformatics analysis. Both open reading frames showed high sequence homology to Succinate dehydrogenase Chain C (SdhC) and D (SdhD) from Escherichia coli. Recently, KPN00729 was assigned as SdhD. KPN00728 thus remains of particular interest as no annotated genes from the complete genome sequence encode for SdhC. We discovered KPN00728 has a missing region with conserved residues important for ubiquinone (UQ) and heme group binding. Structure and function prediction of KPN00728 coupled with secondary structure analysis and transmembrane topology showed KPN00728 adopts SDH-(subunit C)-like structure. To further probe its functionality, UQ was docked on the built model (consisting KPN00728 and KPN00729) and formation of hydrogen bonds between UQ and Ser27, Arg31 (KPN00728) and Tyr84 (KPN00729) further reinforces and supports that KPN00728 is indeed SDH. This is the first report on the structural and function prediction of KPN00728 of K. pneumoniae MGH78578 as SdhC.

Matched MeSH terms: Amino Acid Sequence
Fulltext Cytotoxicity of Snake Venoms and Cytotoxins From Two Southeast Asian Cobras (Naja sumatrana, Naja kaouthia): Exploration of Anticancer Potential, Selectivity, and Cell Death Mechanism

Chong HP, Tan KY, Tan CH

Front Mol Biosci, 2020;7:583587.
PMID: 33263003 DOI: 10.3389/fmolb.2020.583587

Venoms of cobras (Naja spp.) contain high abundances of cytotoxins, which contribute to tissue necrosis in cobra envenomation. The tissue-necrotizing activity of cobra cytotoxins, nevertheless, indicates anticancer potentials. This study set to explore the anticancer properties of the venoms and cytotoxins from Naja sumatrana (equatorial spitting cobra) and Naja kaouthia (monocled cobra), two highly venomous species in Southeast Asia. The cytotoxicity, selectivity, and cell death mechanisms of their venoms and cytotoxins (NS-CTX from N. sumatrana: NS-CTX; N. kaouthia: NK-CTX) were elucidated in human lung (A549), prostate (PC-3), and breast (MCF-7) cancer cell lines. Cytotoxins were purified through a sequential fractionation approach using cation-exchange chromatography, followed by C18 reverse-phase high-performance liquid chromatography (HPLC) to homogeneity validated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by liquid chromatography-tandem mass spectrometry (LCMS/MS). The cobra venoms and their respective cytotoxins exhibited concentration-dependent growth inhibitory effects in all cell lines tested, with the cytotoxins being more potent compared to the corresponding whole venoms. NS-CTX and NK-CTX are, respectively, P-type and S-type isoforms of cytotoxin, based on the amino acid sequences as per LCMS/MS analysis. Both cytotoxins exhibited differential cytotoxic effects in the cell lines tested, with NS-CTX (P-type cytotoxin) being significantly more potent in inhibiting the growth of the cancer cells. Both cytotoxins demonstrated promising selectivity only for the A549 lung cancer cell line (selectivity index = 2.17 and 2.26, respectively) but not in prostate (PC-3) and breast (MCF-7) cancer cell lines (selectivity index < 1). Flow cytometry revealed that the A549 lung cancer cells treated with NS-CTX and NK-CTX underwent necrosis predominantly. Meanwhile, the cytotoxins induced mainly caspase-independent late apoptosis in the prostate (PC-3) and breast (MCF-7) cancer cells lines but lacked selectivity. The findings revealed the limitations and challenges that could be faced during the development of new cancer therapy from cobra cytotoxins, notwithstanding their potent anticancer effects. Further studies should aim to overcome these impediments to unleash the anticancer potentials of the cytotoxins.

Matched MeSH terms: Amino Acid Sequence
Proteomics profiling of epidermal mucus secretion of a cichlid (Symphysodon aequifasciata) demonstrating parental care behavior

Chong K, Joshi S, Jin LT, Shu-Chien AC

Proteomics, 2006 Apr;6(7):2251-8.
PMID: 16385477

The discus fish (Symphysodon aequifasciata) is a cichlid demonstrating advanced mode of parental care towards fry. Both male and female fish utilized epidermal mucus secreted from specialized epidermal cells to feed developing fry. We utilized proteomics to compare protein profile from parental and nonparental fish. Gel analysis revealed a total of 35 spots that were up-regulated in parental mucus. In tandem, another 18 spots were uniquely expressed in parental mucus. MS analysis of these spots identified proteins such as fructose biphosphate aldolase, nucleoside diphosphate kinase, and heat shock proteins, which are essential to support energy provision, cell repair and proliferation, stress mediation, and defense mechanism in parental fish during parental-care period. Concurrently, the detection of several antioxidant-related proteins such as thioredoxin peroxidase and hemopexin suggests a need to overcome oxidative stress during hypermucosal production in parental-care behavior. A C-type lectin was also found to be uniquely expressed in parental mucus and could have important role in providing antimicrobial defense to both parental fish and fry. In summary, our study shows that discus mucus proteome undergoes changes in protein expression during parental-care period.

Matched MeSH terms: Amino Acid Sequence
Fulltext Identification of highly conserved, serotype-specific dengue virus sequences: implications for vaccine design

Chong LC, Khan AM

BMC Genomics, 2019 Dec 24;20(Suppl 9):921.
PMID: 31874646 DOI: 10.1186/s12864-019-6311-z

BACKGROUND: The sequence diversity of dengue virus (DENV) is one of the challenges in developing an effective vaccine against the virus. Highly conserved, serotype-specific (HCSS), immune-relevant DENV sequences are attractive candidates for vaccine design, and represent an alternative to the approach of selecting pan-DENV conserved sequences. The former aims to limit the number of possible cross-reactive epitope variants in the population, while the latter aims to limit the cross-reactivity between the serotypes to favour a serotype-specific response. Herein, we performed a large-scale systematic study to map and characterise HCSS sequences in the DENV proteome.
METHODS: All reported DENV protein sequence data for each serotype was retrieved from the NCBI Entrez Protein (nr) Database (txid: 12637). The downloaded sequences were then separated according to the individual serotype proteins by use of BLASTp search, and subsequently removed for duplicates and co-aligned across the serotypes. Shannon's entropy and mutual information (MI) analyses, by use of AVANA, were performed to measure the diversity within and between the serotype proteins to identify HCSS nonamers. The sequences were evaluated for the presence of promiscuous T-cell epitopes by use of NetCTLpan 1.1 and NetMHCIIpan 3.2 server for human leukocyte antigen (HLA) class I and class II supertypes, respectively. The predicted epitopes were matched to reported epitopes in the Immune Epitope Database.
RESULTS: A total of 2321 nonamers met the HCSS selection criteria of entropy 0.8. Concatenating these resulted in a total of 337 HCSS sequences. DENV4 had the most number of HCSS nonamers; NS5, NS3 and E proteins had among the highest, with none in the C and only one in prM. The HCSS sequences were immune-relevant; 87 HCSS sequences were both reported T-cell epitopes/ligands in human and predicted epitopes, supporting the accuracy of the predictions. A number of the HCSS clustered as immunological hotspots and exhibited putative promiscuity beyond a single HLA supertype. The HCSS sequences represented, on average, ~ 40% of the proteome length for each serotype; more than double of pan-DENV sequences (conserved across the four serotypes), and thus offer a larger choice of sequences for vaccine target selection. HCSS sequences of a given serotype showed significant amino acid difference to all the variants of the other serotypes, supporting the notion of serotype-specificity.
CONCLUSION: This work provides a catalogue of HCSS sequences in the DENV proteome, as candidates for vaccine target selection. The methodology described herein provides a framework for similar application to other pathogens.

Matched MeSH terms: Amino Acid Sequence
Nucleotide sequence and phylogenetic analysis of a segment of a highly virulent strain of infectious bursal disease virus

Chong LK, Omar AR, Yusoff K, Hair-Bejo M, Aini I

Acta Virol., 2001;45(4):217-26.
PMID: 11885928

The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.

Matched MeSH terms: Amino Acid Sequence
Molecular characterization and expression of a putative ATPase domain from Eimeria tenella

Chong SP, Jangi MS, Wan KL

J. Biochem. Mol. Biol. Biophys., 2002 Apr;6(2):123-8.
PMID: 12186768

VCP (Valosin-Containing Protein), a member of the AAA (ATPases Associated to a variety of cellular Activities) family of proteins, possesses a duplicated highly conserved ATPase domain. An expressed sequence tag (EST), representing a clone from the Eimeria tenella merozoite cDNA library, was found to have high similarity to VCP genes from other organisms. A complete sequence derived from the corresponding clone (designated eth060) shows amino acid identity of 42-62% with other members of the VCP subfamily. Sequence analysis identified a putative ATPase domain in the eth060 sequence. This domain was PCR-amplified using gene-specific primers and cloned into a pBAD/Thio-TOPO expression vector. Expression in Escherichia coli demonstrated that the putative ATPase domain, which consists of 414 amino acid residues, produced a fusion protein of approximately 60 kDa in size.

Matched MeSH terms: Amino Acid Sequence
Phylogeny and characterization of three nifH-homologous genes from Paenibacillus azotofixans

Choo QC, Samian MR, Najimudin N

Appl Environ Microbiol, 2003 Jun;69(6):3658-62.
PMID: 12788777

In this paper, we report the cloning and characterization of three Paenibacillus azotofixans DNA regions containing genes involved in nitrogen fixation. Sequencing analysis revealed the presence of nifB1H1D1K1 gene organization in the 4,607-bp SacI DNA fragment. This is the first report of linkage of a nifB open reading frame upstream of the structural nif genes. The second (nifB2H2) and third (nifH3) nif homologues are confined within the 6,350-bp HindIII and 2,840-bp EcoRI DNA fragments, respectively. Phylogenetic analysis demonstrated that NifH1 and NifH2 form a monophyletic group among cyanobacterial NifH proteins. NifH3, on the other hand, clusters among NifH proteins of the highly divergent methanogenic archaea.

Matched MeSH terms: Amino Acid Sequence
Minireview: applied structural bioinformatics in proteomics

Choong YS, Tye GJ, Lim TS

Protein J, 2013 Oct;32(7):505-11.
PMID: 24096348 DOI: 10.1007/s10930-013-9514-1

The limited sequence similarity of protein sequences with known structures has led to an indispensable need for computational technology to predict their structures. Structural bioinformatics (SB) has become integral in elucidating the sequence-structure-function relationship of a protein. This report focuses on the applications of SB within the context of protein engineering including its limitation and future challenges.

Matched MeSH terms: Amino Acid Sequence

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