Regions Of Secondary Structure Research Articles

Bioinformatics analysis on ORF1 protein of Torque teno virus (SANBAN isolate)

To analyze the sequence of ORF1 protein of Torque teno virus to prepare for the future hybrid experiments. The sequence of ORF1 protein of Torque teno virus was analyzed by bioinformatics using some web tools. The most likely cleavage site was between position 14aa and 15aa and signal peptide may be position 1aa-14aa. Two possible transmembrane helices from inside to outside and three possible transmembrane helices from outside to inside were found. The position 509 (NKTN) was the potential N-glycosylation site. The speculative molecular weight of TTV ORF1 protein, which may be a kind of unstable protein was 88 705.7 Da. 1aa-91aa and 278aa-361aa were localized in non-regular secondary structure region. TTV ORF1 protein may be a nuclear protein which contains two non-regular secondary structure region. 265aa to 486aa and 510aa to 679aa may be the two approciate fragments to construct the plasmids, which would be prepared for the future hybrid experiments to study the functional positions of the protein and the interactions between TTV and its hosts. Bioinformatics analysis would possibly make it easier to study the protein's function.

Hotspot-Centric De Novo Design of Protein Binders

Protein–protein interactions play critical roles in biology, and computational design of interactions could be useful in a range of applications. We describe in detail a general approach to de novo design of protein interactions based on computed, energetically optimized interaction hotspots, which was recently used to produce high-affinity binders of influenza hemagglutinin. We present several alternative approaches to identify and build the key hotspot interactions within both core secondary structural elements and variable loop regions and evaluate the method's performance in natural-interface recapitulation. We show that the method generates binding surfaces that are more conformationally restricted than previous design methods, reducing opportunities for off-target interactions.

DNA Binding Restricts the Intrinsic Conformational Flexibility of Methyl CpG Binding Protein 2 (MeCP2)

Mass spectrometry-based hydrogen/deuterium exchange (H/DX) has been used to define the polypeptide backbone dynamics of full-length methyl CpG binding protein 2 (MeCP2) when free in solution and when bound to unmethylated and methylated DNA. Essentially the entire MeCP2 polypeptide chain underwent H/DX at rates faster than could be measured (i.e. complete exchange in ≤10 s), with the exception of the methyl DNA binding domain (MBD). Even the H/DX of the MBD was rapid compared with that of a typical globular protein. Thus, there is no single tertiary structure of MeCP2. Rather, the full-length protein rapidly samples many different conformations when free in solution. When MeCP2 binds to unmethylated DNA, H/DX is slowed several orders of magnitude throughout the MBD. Binding of MeCP2 to methylated DNA led to additional minor H/DX protection, and only locally within the N-terminal portion of the MBD. H/DX also was used to examine the structural dynamics of the isolated MBD carrying three frequent mutations associated with Rett syndrome. The effects of the mutations ranged from very little (R106W) to a substantial increase in conformational sampling (F155S). Our H/DX results have yielded fine resolution mapping of the structure of full-length MeCP2 in the absence and presence of DNA, provided a biochemical basis for understanding MeCP2 function in normal cells, and predicted potential approaches for the treatment of a subset of RTT cases caused by point mutations that destabilize the MBD.

Infrared spectroscopy with multivariate analysis to interrogate endometrial tissue: a novel and objective diagnostic approach

Background:Endometrial cancer is the most common gynaecological malignancy in the United Kingdom. Diagnosis currently involves subjective expert interpretation of highly processed tissue, primarily using microscopy. Previous work has shown that infrared (IR) spectroscopy can be used to distinguish between benign and malignant cells in a variety of tissue types.Methods:Tissue was obtained from 76 patients undergoing hysterectomy, 36 had endometrial cancer. Slivers of endometrial tissue (tumour and tumour-adjacent tissue if present) were dissected and placed in fixative solution. Before analysis, tissues were thinly sliced, washed, mounted on low-E slides and desiccated; 10 IR spectra were obtained per slice by attenuated total reflection Fourier-transform IR (ATR-FTIR) spectroscopy. Derived data was subjected to principal component analysis followed by linear discriminant analysis. Post-spectroscopy analyses, tissue sections were haematoxylin and eosin-stained to provide histological verification.Results:Using this approach, it is possible to distinguish benign from malignant endometrial tissue, and various subtypes of both. Cluster vector plots of benign (verified post-spectroscopy to be free of identifiable pathology) vs malignant tissue indicate the importance of the lipid and secondary protein structure (Amide I and Amide II) regions of the spectrum.Conclusion:These findings point towards the possibility of a simple objective test for endometrial cancer using ATR-FTIR spectroscopy. This would facilitate earlier diagnosis and so reduce the morbidity and mortality associated with this disease.

Solution structure of BTK-2, a novel hK v1.1 inhibiting scorpion toxin, from the eastern Indian scorpion Mesobuthus tamulus

The three dimensional structure of a 32 residue three disulfide scorpion toxin, BTK-2, from the Indian red scorpion Mesobuthus tamulus has been determined using isotope edited solution NMR methods. Samples for structural and electrophysiological studies were prepared using recombinant DNA methods. Electrophysiological studies show that the peptide is active against hK v1.1 channels. The structure of BTK-2 was determined using 373 distance restraints from NOE data, 66 dihedral angle restraints from NOE, chemical shift and scalar coupling data, 6 constraints based on disulfide linkages and 8 constraints based on hydrogen bonds. The root mean square deviation (r.m.s.d) about the averaged co-ordinates of the backbone (N, C α, C′) and all heavy atoms are 0.81 ± 0.23 Å and 1.51 ± 0.29 Å respectively. The backbone dihedral angles (ϕ and ψ) for all residues occupy the favorable and allowed regions of the Ramachandran map. The three dimensional structure of BTK-2 is composed of three well defined secondary structural regions that constitute the α–β–β structural motif. Comparisons between the structure of BTK-2 and other closely related scorpion toxins pointed towards distinct differences in surface properties that provide insights into the structure–function relationships among this important class of voltage-gated potassium channel inhibiting peptides.

Intrinsic disorder of Drosophila melanogaster hormone receptor 38 N‐terminal domain

Drosophila hormone receptor 38 (dHR38), an ortholog of the vertebrate NR4A subclass of nuclear receptors, responds to ecdysteroids, which mediate developmental transitions during the Drosophila life cycle. However, this response is independent of the ecdysteroid receptor, and it does not involve binding of ecdysteroids to dHR38. It has been suggested that ecdysteroids may indirectly activate dHR38, perhaps by recruiting specific proteins. There have been recent reports pointing out the decisive role that nuclear receptor N-terminal domains (NTDs) have in protein-protein interactions that are important for regulation of gene expression. It is reasonable to assume that dHR38-NTD may also be involved in some protein-protein interactions that are critical for the ecdysteroid signaling pathway. To facilitate the exploration of the molecular basis of these interactions, we developed and optimized a protocol for the efficient expression and purification of the recombinant dHR38-NTD. Using a diverse array of biochemical and biophysical methods, we carried out the first structural characterization of dHR38-NTD. The results of our study indicate that dHR38-NTD exhibits a characteristic reminiscent of pre-molten globule-like intrinsically disordered proteins existing in a partially unfolded conformation with regions of secondary structures. The dHR38-NTD structure, which apparently comprises some local, ordered, tertiary structure clusters, is pliable and can adopt more ordered conformations in response to changes in environmental conditions. Thus, dHR38-NTD, which exhibits the structural and functional characteristic of a pre-molten globule-like intrinsically disordered protein, could serve as a platform for multiple protein-protein interactions, possibly including interactions with proteins involved in an unusual ecdysteroid signaling pathway.

Muscleblind-like 1 (Mbnl1) Promotes Insulin Receptor Exon 11 Inclusion via Binding to a Downstream Evolutionarily Conserved Intronic Enhancer

The insulin receptor exists as two isoforms, IR-A and IR-B, which result from alternative splicing of exon 11 in the primary transcript. These two isoforms show a cell-specific distribution, and their relative proportions also vary during development, aging, and in different disease states. We have previously demonstrated that both intron 10 and the alternatively spliced exon 11 contain regulatory sequences that affect insulin receptor splicing both positively and negatively and that these sequences bind the serine/arginine-rich (SR) proteins SRp20 and SF2/ASF and the CELF protein CUG-BP1. In this study, we describe a new intronic splicing element within intron 11 that is highly conserved across species. Using minigenes carrying deletion mutations within intron 11, we demonstrated that this sequence functions as an intronic splicing enhancer. We subsequently used RNA affinity chromatography to identify Mbnl1 as a splicing factor that recognizes this enhancer. By ribonucleoprotein immunoprecipitation, we also established that Mbnl1 binds specifically to the INSR (insulin receptor gene) RNA. Overexpression or knockdown of Mbnl1 in hepatoma and embryonic kidney cells altered the levels of exon 11 inclusion. Finally, we showed that deletion of the intronic enhancer eliminates the ability of Mbnl1 to promote exon inclusion. Collectively, these findings demonstrate a role for Mbnl1 in controlling insulin receptor exon 11 inclusion via binding to a downstream intronic enhancer element.

ExSer: A standalone tool to mine protein data bank (PDB) for secondary structural elements

Detailed structural analysis of protein necessitates investigation at primary, secondary and tertiary levels, respectively. Insight into protein secondary structures pave way for understanding the type of secondary structural elements involved (α-helices, β-strands etc.), the amino acid sequence that encode the secondary structural elements, number of residues, length and, percentage composition of the respective elements in the protein. Here we present a standalone tool entitled "ExSer" which facilitate an automated extraction of the amino acid sequence that encode for the secondary structural regions of a protein from the protein data bank (PDB) file. ExSer is freely downloadable from http://code.google.com/p/tool-exser/

Characterization of RNase HII substrate recognition using RNase HII–argonaute chimaeric enzymes from Pyrococcus furiosus

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth beta-sheet and the fifth alpha-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes.

Site-Specific Protein Labeling using Reversible Transition Metal Ion Binding

Fluorescence spectroscopy is a powerful tool for studying the structure and conformational dynamics of protein molecules both in isolation and in their cellular context. Fluorescence experiments frequently employ small, cysteine-reactive fluorophores. However, it can be difficult to obtain specific labeling of a desired cysteine in proteins with multiple cysteines or in a protein's native environment in which many cysteine-containing proteins are present. To obtain specific labeling, we have developed a method where a desired cysteine can be reversibly protected by binding transition metal ions (e.g. Cd2+ and Zn2+) while background cysteines are blocked with non-fluorescent covalent modifiers. Following removal of transition metal ions, the deprotected cysteine is then available to specifically react with a fluorophore. In order to protect specific cysteines, the affinity for metal binding was increased by placing metal binding residues nearby in regions of known secondary structure (e.g. an alpha-helix) to act as coordination partners in a metal binding site. The placement of histidine residues next to or one turn away from a cysteine in an alpha-helix increased the metal binding affinity of that cysteine. This allows for protection of a particular cysteine at much lower metal concentrations. Using pairs of cysteines, rather than a combination of cysteines and histidines, further stabilized metal binding. Finally, we demonstrate the ability of this technique to selectively label a particular cysteine in mixtures of proteins containing cysteines. These motifs are simple, can be introduced into proteins with minimal perturbation, and allow for selective labeling of sites within a protein, specific proteins in a mixture, or proteins in a native environment. This technique has great potential for use in patch-clamp fluorometry studies of ion channels in which non-specific labeling of patch-associated proteins makes it difficult to resolve fluorescence from channel proteins.

An Automated Approach to Segregate and Identify Functional or Disordered Loop Regions in Protein Structures using their Ramachandran Maps

The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved 3D structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least two-fold higher for unstructured proteins (PDB codes: 2SOB, 1LXL, 2HDL & 1VZS) in comparison with folded proteins (PDB codes: 1BGF & 1MUN). Additionally it was observed that deviations in MSRP were highest amongst loop regions, while it was lowest amongst alpha-helical regions. The above results validate use of MSRP parameter as a tool to identify & investigate functionally active loops and unstructured regions in protein structures.

A novel approach to segregate and identify functional loop regions in protein structures using their Ramachandran maps

The loops which connect or flank helices/sheets in protein structures are known to be functionally important. However, ironically they also belong to the part of protein whose structure is least accurately predicted. Here, a new method to isolate and analyze loop regions in protein structure is proposed using the spatial coordinates of the solved three-dimensional structure. The extent of dispersion among points of successive amino acid residues in the Ramachandran map of protein region is utilized to calculate the Mean Separation between these points in the Ramachandran Plot (MSRP). Based on analysis of 2935 protein secondary structure regions obtained using DSSP software, spanning a range from 2 to 64 residues, taken from a set of 170 proteins, it is shown that helices (MSRP < 17) and strands (MSRP < 64) stand effectively demarcated from the loop regions (MSRP > 130). Analysis of 43 DNA binding and 98 ligand binding proteins revealed several loop regions with clear change in MSRP subsequent to binding. The population of such loops correlated with the magnitude of backbone displacement in the protein subsequent to binding. Can changes in MSRP quantify the temporal oscillations in dihedral angles among structured/unstructured regions in proteins? Molecular dynamics simulations (10 ns) revealed that deviations in MSRP among different snapshots in the trajectory were at least twofold higher for unstructured proteins in comparison with ordered proteins. The above results validate the use of MSRP parameter as a tool to identify and investigate functionally active loops and unstructured regions in protein structures. Proteins 2010. (c) 2009 Wiley-Liss, Inc.

Strategies for Extracting Structural Information from 2D IR Spectroscopy of Amyloid: Application to Islet Amyloid Polypeptide

The 37-residue human islet amyloid polypeptide (hIAPP or amylin) self-assembles into fibers, the assembly of which has been associated with the disease mechanism of type II diabetes. Infrared spectroscopy in conjunction with isotope labeling is proving to be a powerful tool for studying the aggregation process of hIAPP and other amyloid forming proteins with residue specific structure and kinetic information, but the relationship between the spectroscopic observables and the structure is not fully established. We report a detailed analysis of the linear and 2D IR spectra of hIAPP fibers isotope labeled at seven different residue positions. The features of the 2D IR spectra, including the frequencies, linewidths, intensities, and polarization dependence of the diagonal and cross-peaks, rely heavily on the position of the isotope labeled residue. In order to understand how these measured parameters depend on fiber secondary and tertiary structure, we have simulated 1D and 2D IR spectra utilizing idealized structural models in addition to a recently published solid-state NMR based model of the amyloid fibril. The analysis provides a more rigorous foundation for interpreting the infrared spectra of amyloids. In addition, we demonstrate that 2D IR spectra can be employed to distinguish between residues in beta-sheets versus those in turn regions, and that transitional residues between secondary structures can be identified by the suppression of their cross-peaks in 2D IR spectra. This latter approach is not limited to amyloid fibrils and will be generally useful in identifying regions of secondary structure in proteins using 2D IR spectroscopy and isotope labeling.

Short-distance probes for protein backbone structure based on energy transfer between bimane and transition metal ions

The structure and dynamics of proteins underlies the workings of virtually every biological process. Existing biophysical methods are inadequate to measure protein structure at atomic resolution, on a rapid time scale, with limited amounts of protein, and in the context of a cell or membrane. FRET can measure distances between two probes, but depends on the orientation of the probes and typically works only over long distances comparable with the size of many proteins. Also, common probes used for FRET can be large and have long, flexible attachment linkers that position dyes far from the protein backbone. Here, we improve and extend a fluorescence method called transition metal ion FRET that uses energy transfer to transition metal ions as a reporter of short-range distances in proteins with little orientation dependence. This method uses a very small cysteine-reactive dye monobromobimane, with virtually no linker, and various transition metal ions bound close to the peptide backbone as the acceptor. We show that, unlike larger fluorophores and longer linkers, this donor-acceptor pair accurately reports short-range distances and changes in backbone distances. We further extend the method by using cysteine-reactive metal chelators, which allow the technique to be used in protein regions of unknown secondary structure or when native metal ion binding sites are present. This improved method overcomes several of the key limitations of classical FRET for intramolecular distance measurements.

Double-Stranded RNA Binding Domain (dsRBD) of PKR Shows Variable Dynamics in the Presence of Bacteriophage Pf1: An NMR Insight and its Possible Implications

The human Protein Kinase R (PKR) is one of the important and critical components of the innate immune response against viral infection. It regulates distinct cellular functions and controls the fate of an RNA molecule in the cell. PKR dephosphorylation is characterised by inhibitory interactions between the kinase domain and the RNA binding domains (RBDs), but the complete structural details and dynamics of the latent state and its activation are not yet clear. Several studies on the activation of double stranded RNA binding domain (dsRBD) of Protein Kinase R (dsRBD-PKR) have been attempted during the last few decades. In order to further investigate on its activation mechanism, we have analysed the backbone ( 1 H- 15 N)-dynamics and chemical shift perturbation studies of dsRBD-PKR in the presence of bacteriophage Pf1. Pf1 is known to orient the protein in the presence of external magnetic field and, can be further used to get useful information about the protein properties such as the different dynamic behaviour in multidomain proteins like dsRBD-PKR. These NMR based results improve our basic understanding of the domain flexibilities of dsRBD-PKR where some secondary structural regions of dsRBM1 (dsRNA binding motif 1) show a different behaviour than the one from dsRBM2 (dsRNA binding motif 2) these observations further lead to a better understanding of PKR mechanism where dsRBM1 has more affinity towards RNA as compared to dsRBM2.

Sequence characteristics of potato virus Y recombinants

Potato virus Y (PVY) is one of the most economically important plant pathogens. The PVY genome has a high degree of genetic variability and is also subject to recombination. New recombinants have been reported in many countries since the 1980s, but the origin of these recombinant strains and the physical and evolutionary mechanisms driving their emergence are not clear at the moment. The replicase-mediated template-switching model is considered the most likely mechanism for forming new RNA virus recombinants. Two factors, RNA secondary structure (especially stem-loop structures) and AU-rich regions, have been reported to affect recombination in this model. In this study, we investigated the influence of these two factors on PVY recombination from two perspectives: their distribution along the whole genome and differences between regions flanking the recombination junctions (RJs). Based on their distributions, only a few identified RJs in PVY genomes were located in lower negative FORS-D, i.e. having greater secondary-structure potential and higher AU-content regions, but most RJs had more negative FORS-D values upstream and/or higher AU content downstream. Our whole-genome analyses showed that RNA secondary structures and/or AU-rich regions at some sites may have affected PVY recombination, but in general they were not the main forces driving PVY recombination.

Differential Display Detects Host Nucleic Acid Motifs Altered in Scrapie-Infected Brain

The transmissible spongiform encephalopathies (TSEs) including scrapie have been attributed to an infectious protein or prion. Infectivity is allied to conversion of the endogenous nucleic-acid-binding protein PrP to an infectious modified form known as PrPsc. The protein-only theory does not easily explain the enigmatic properties of the agent including strain variation. It was previously suggested that a short nucleic acid, perhaps host-encoded, might contribute to the pathoetiology of the TSEs. No candidate host molecules that might explain transmission of strain differences have yet been put forward. Differential display is a robust technique for detecting nucleic acid differences between two populations. We applied this technique to total nucleic acid preparations from scrapie-infected and control brain. Independent RNA preparations from eight normal and eight scrapie-infected (strain 263K) hamster brains were randomly amplified and visualized in parallel. Though the nucleic acid patterns were generally identical in scrapie-infected versus control brain, some rare bands were differentially displayed. Molecular species consistently overrepresented (or underrepresented) in all eight infected brain samples versus all eight controls were excised from the display, sequenced, and assembled into contigs. Only seven ros contigs (RNAs over- or underrepresented in scrapie) emerged, representing <4 kb from the transcriptome. All contained highly stable regions of secondary structure. The most abundant scrapie-only ros sequence was homologous to a repetitive transposable element (LINE; long interspersed nuclear element). Other ros sequences identified cellular RNA 7SL, clathrin heavy chain, visinin-like protein-1, and three highly specific subregions of ribosomal RNA (ros1–3). The ribosomal ros sequences accurately corresponded to LINE; retrotransposon insertion sites in ribosomal DNA (p<0.01). These differential motifs implicate specific host RNAs in the pathoetiology of the TSEs.

ProteinArchitect: Protein Evolution above the Sequence Level

BackgroundWhile many authors have discussed models and tools for studying protein evolution at the sequence level, molecular function is usually mediated by complex, higher order features such as independently folding domains and linear motifs that are based on or embedded in a particular arrangment of features such as secondary structure elements, transmembrane domains and regions with intrinsic disorder. This ‘protein architecture’ can, in its most simplistic representation, be visualized as domain organization cartoons that can be used to compare proteins in terms of the order of their mostly globular domains.MethodologyHere, we describe a visual approach and a webserver for protein comparison that extend the domain organization cartoon concept. By developing an information-rich, compact visualization of different protein features above the sequence level, potentially related proteins can be compared at the level of propensities for secondary structure, transmembrane domains and intrinsic disorder, in addition to PFAM domains. A public Web server is available at www.proteinarchitect.net, while the code is provided at protarchitect.sourceforge.net.Conclusions/SignificanceDue to recent advances in sequencing technologies we are now flooded with millions of predicted proteins that await comparative analysis. In many cases, mature tools focused on revealing hits with considerable global or local similarity to well-characterized proteins will not be able to lead us to testable hypotheses about a protein's function, or the function of a particular region. The visual comparison of different types of protein features with ProteinArchitect will be useful when assessing the relevance of similarity search hits, to discover subgroups in protein families and superfamilies, and to understand protein regions with conserved features outside globular regions. Therefore, this approach is likely to help researchers to develop testable hypotheses about a protein's function even if is somewhat distant from the more characterized proteins, by facilitating the discovery of features that are conserved above the sequence level for comparison and further experimental investigation.

Prediction of the B cell epitopes of human heparanase protein and determination of their immunogenicity

Objective To predict the secondary structure and the B cell epitopes of human heparanase protein, and to identify its immunogenicity. Methods The flexible regions of secondary structure and the B cell epitopes of human heparanase amino acid sequence were predicted by DNAStar and Bcepred software. The multiple antigenic peptides (MAP) of the epitopes were synthesized in 8-branch form. Rabbits were immunized with the 8-branch MAPs mixed with a universal T-helper epitope human IL-1β peptide (VQGEESNDK, amino acid 163-171 ). The immunogenicity of the synthesized peptides was evaluated by ELISA, Western blot and immunohistochemistry. Results Amino acid 1 -15 ( MAP1), 279-293 (MAP2) and 175-189(MAP3) of large-subunit of human heparanase protein was predicted as the most potential epitopes of human heparanase protein. All the three synthesized MAPs induced high titer of antibodies. ELISA, Western blot and immunohistochemistry analysis showed all the three MAPs could produce high titer serum antibodies, antibodies induced by MAP1 and MAP2 had high specific binding activity , and MAP2 antibody showed the strongest binding activity with liver cancer tissues. Conclusion The large-subunit No. 1-15, 279-293 amino acid of human heparanase protein may be the B cell preponderant epitopes and the strongest immunogenicity may be No. 279-293 peptide, which provided a theoretic basis for the antibody and vaccine development of heparanase subunit peptide. Key words: Heparanase; B cell epitopes; Epitope prediction; Immunogenicity

Screening and identification of novel B cell epitopes in human heparanase and their anti-invasion property for hepatocellular carcinoma.

The aim of this study was to screen and identify novel B cell epitopes within the human heparanase protein and to investigate the impact of self-developed anti-heparanase polypeptide antibodies on growth and invasion of HCCLM6 human hepatocellular carcinoma cells in vitro. The flexible regions of secondary structure and the B cell epitopes of the human heparanase amino acid sequence were predicted by DNAStar and Bcepred software.The multiple antigenic peptides (MAP) of the epitopes were synthesized in eight-branched form. Rabbits were immunized with the eight-branched MAPs mixed with the universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acid 163-171). The immunogenicity of the synthesized peptides was evaluated by ELISA, western blot and immunohistochemistry. The impact of the self-developed rabbit anti-heparanase polyclonal antibodies on growth and invasion ability of HCCMLM6 cells were analyzed in a cell culture model. The cells were first treated with one of the three antibodies, respectively, and then measured by using MTT, flow cytometry, plate clone formation, invasion assay and heparan sulfate degrading enzyme assay. The three amino acid sequences 1-15 (MAP1), 279-293 (MAP2), and 175-189 (MAP3) in the large subunit of the human heparanase protein were predicted as its most potential epitopes. ELISA, western blot and immunohistochemistry analysis showed that all three MAPs were capable to induce high titer of serum antibodies. Antibodies induced by MAP1 and MAP2 were high specific. Furthermore, anti-MAP2 antibodies showed the strongest avidity towards liver cancer tissues. Under the treatment with the three anti-heparanase antibodies, respectively, the growth, cell cycle and clone formation of the cells remained unchanged when compared with a treatment with normal rabbit IgG. However, an inhibition of cell invasiveness and heparanase activity could be detected under the treatment with anti-MAP1- or anti-MAP2-antibody (with a terminal concentration of 100 mug/ml). The cell invasiveness was decreased by 54 and 38%, respectively, the heparanase activity by 43 and 39%, respectively. The multiple antigenic peptides MAP1 (AC 1-15) and MAP2 (AC 279-293) may be the dominant B cell epitopes in the human heparanase protein. The induced polypeptide antibodies can effectively inhibit the heparanase activity of HCCLM6 liver cancer cells and therefore influence their invasion ability, which provides a theoretic basis for the development of anti-heparanase antibodies and their clinical use as vaccine.