Aa In Length Research Articles

Tryptophan zippers: stable, monomeric beta -hairpins.

A structural motif, the tryptophan zipper (trpzip), greatly stabilizes the beta-hairpin conformation in short peptides. Peptides (12 or 16 aa in length) with four different turn sequences are monomeric and fold cooperatively in water, as has been observed previously for some hairpin peptides. However, the folding free energies of the trpzips exceed substantially those of all previously reported beta-hairpins and even those of some larger designed proteins. NMR structures of three of the trpzip peptides reveal exceptionally well-defined beta-hairpin conformations stabilized by cross-strand pairs of indole rings. The trpzips are the smallest peptides to adopt an unique tertiary fold without requiring metal binding, unusual amino acids, or disulfide crosslinks.

Localization of CD4+ T cell epitope hotspots to exposed strands of HIV envelope glycoprotein suggests structural influences on antigen processing.

The spectrum of immunogenic epitopes presented by the H2-IA(b) MHC class II molecule to CD4(+) T cells has been defined for two different (clade B and clade D) HIV envelope (gp140) glycoproteins. Hybridoma T cell lines were generated from mice immunized by a sequential prime and boost regime with DNA, recombinant vaccinia viruses, and protein. The epitopes recognized by reactive T cell hybridomas then were characterized with overlapping peptides synthesized to span the entire gp140 sequence. Evidence of clonality also was assessed with antibodies to T cell receptor Valpha and Vbeta chains. A total of 80 unique clonotypes were characterized from six individual mice. Immunogenic peptides were identified within only four regions of the HIV envelope. These epitope hotspots comprised relatively short sequences ( approximately 20-80 aa in length) that were generally bordered by regions of heavy glycosylation. Analysis in the context of the gp120 crystal structure showed a pattern of uniform distribution to exposed, nonhelical strands of the protein. A likely explanation is that the physical location of the peptide within the native protein leads to differential antigen processing and consequent epitope selection.

The Leishmania major RNA Polymerase II Largest Subunit Lacks a Carboxy-Terminus Heptad Repeat Structure and its Encoding Gene is Linked with the Calreticulin Gene

The gene encoding the RNA polymerase II largest subunit (RPOIILS) has been isolated and sequenced from the kinetoplastid protozoan, Leishmania (Leishmania) major. The RPOIILS gene was shown to be present as a single copy and is composed of an uninterrupted open reading frame of 4.99 kb, specifying a protein 1663 aa in length with a predicted molecular mass of approximately 185 kDa. The carboxy terminus domain (CTD) of the RPOIILS from L. (L.) major, typical of the more evolutionary primitive protozoa, lacked a heptad repeat structure which is present in higher eukaryotes and some other protozoan phyla. Comparison of the predicted aa composition of the CTD from a diverse range of eukaryotic species revealed the abundance of Ser and Pro residues as the only discernible evolutionary conservative feature. A putative ATG start codon for an additional expressed sequence was located 1.1 kb downstream of the L. (L.) major RPOIILS gene stop codon. Nucleic acid database searches revealed the identity of this gene as that encoding the calcium binding protein calreticulin (CLT). The close proximity of the RPOIILS and CLT genes in L. (L.) major raises the possibility that these genes are transcribed as part of the same polycistronic unit.

Molecular characterization of the first non-mammalian p73 cDNA

The p53 gene is believed to be mutated or deficient in over 50% of human tumours, and is therefore considered to be instrumental in the process of carcinogenesis. Recently in humans, homologues of p53 (such as p73 and p63) have been isolated. In our studies in fish, we have been isolating tumour suppressor genes with a view to their potential use to study genotoxins in the aquatic environment. In this paper, we report the characterisation of the first non-mammalian p73 cDNA, isolated from barbel (Barbus barbus), a freshwater cyprinid fish indigenous to UK rivers. The deduced barbel p73 amino acid sequence has a high homology with human p73α: the proteins are 641 and 636 aa in length, respectively, and there is a 72% identity over the entire sequence length of the protein (over 90% in the putative DNA binding domain). The level of conservancy for p73 is considerably higher across class (from man to fish), than for p53 and it may therefore have particular value in studies on environmental mutagenesis. Northern analysis showed expression of three p73 mRNA transcripts/ homologues. The patterns of p73 tissue expression in the barbel differed from the expression of p53 mRNA, suggesting specific functional roles for the two genes.

Synthetic peptides of CD66a stimulate neutrophil adhesion to endothelial cells.

Four members of the carcinoembryonic Ag family, CD66a, CD66b, CD66c, and CD66d, are expressed on human neutrophils. CD66a, CD66b, CD66c, and CD66d Ab binding to the neutrophil surface triggers an activation signal that regulates the adhesive activity of CD11/CD18, resulting in an increase in neutrophil adhesion to HUVEC. To identify active sites on the CD66a Ag, molecular modeling was performed using IgG and CD4 as models, and 28 peptides of 14 aa in length were synthesized that were predicted to be present at loops and turns between beta-sheets. The peptides were tested for their ability to alter neutrophil adhesion to HUVEC. Three peptides, each from the N-terminal domain, increased neutrophil adhesion to HUVEC monolayers. This increase in neutrophil adhesion caused by CD66a peptides was associated with up-regulation of CD11/CD18 and down-regulation of CD62L on the neutrophil surface. Scrambled versions of these three peptides had no effect on neutrophil adhesion to the endothelial cells. The data suggest that peptide motifs from at least three regions of the N-terminal domain of CD66a are involved in the interaction of CD66a with other ligands and can initiate signal transduction in neutrophils.

Towards defining a minimal functional domain for NTPase and RNA helicase activities of the hepatitis C virus NS3 protein

Hepatitis C virus (HCV) possesses two separate enzymatic functions in the NS3 protein: a protease and an NTPase/RNA helicase. In order to determine the minimal domain for NTPase and RNA helicase activities of the HCV NS3 protein, serial deletion mutants were constructed. The NS3H protein, a fusion protein of 25 amino acids (aa) from an expression vector and the C-terminal 466 aa of the HCV NS3 protein, contains an NTPase/RNA helicase activity. We made deletion mutants of 10, 30, 50, 97, and 135 aa from the C-terminus and 16 and 32 aa from the N-terminus of the NS3H protein. The deleted protein lacking 50 aa from the C-terminus still possessed both activities, while the protein lacking 97 aa from the C-terminus lost an RNA helicase activity. The mutant lacking 16 amino acids from the N-terminus retained the enzymatic activities and the N-terminal 32 aa deleted mutant lost an NTPase/RNA helicase activity. A combinational deletion mutant lacking 16 aa the N-terminus and 50 aa from the C-terminus retained the enzymatic activities. These results show that the functional domain of the HCV NTPase/RNA helicase is about 400 aa in length and maps between NS3 residues 1209 and 1608.

Biochemical and genetic characterization of Rbf1p, a putative transcription factor of Candida albicans.

A Candida albicans gene encoding a novel DNA-binding protein that bound to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans was previously cloned and designated RBF1 (RPG-box-binding factor). In this report, determination of the functional domains of the protein is described. The DNA-binding domain was 140 aa in length, was centrally located between two glutamine-rich regions, and correlated with transcriptional activation in S. cerevisiae. The results, together with the previous finding that showed its predominant localization in the nucleus, suggest that this DNA-binding protein could be a transcription factor. Disruption of the functional RBF1 gene of C. albicans strains caused an alteration in cell morphology to the filamentous form on all solid and liquid media tested. Thus, we speculate that Rbf1p may be involved in the regulation of the transition between yeast and filamentous forms at the level of transcription.

Identification and cDNA cloning of a novel human mosaic protein, LGN, based on interaction with G αi2

We have used the yeast two-hybrid system to identify proteins that interact with the α-subunit of the heterotrimeric GTP-binding protein, G i2. We screened a human B cell cDNA library with full-length G αi2 and isolated four positive colonies, one of which expressed the 44-kDa COOH terminus of a previously unrecognized 677-amino acid (aa) protein. A full-length clone was isolated from a HeLa cell cDNA library. The deduced protein contains 10 Leu-Gly-Asn repeats, and thus we named it LGN. Computer analysis indicates that LGN is a mosaic protein with seven repeated sequences of about 40 aa in length at its N-terminal end, and four repeated sequences of about 34 aa at its C-terminal end. Each of the two repeat regions shows substantial similarity to proteins found in other organisms. RT-PCR analysis of human tissues showed that the mRNA of LGN was ubiquitously expressed. The specificity of interaction between G αi2 and LGN was confirmed by an in vitro binding assay using recombinant proteins. These data indicate that the yeast two-hybrid system can identify novel proteins, such as LGN, that interact with G α proteins. As a mosaic protein, LGN shows similarity with portions of proteins from many species and thus may define a new protein family.

Halolysin R4, a serine proteinase from the halophilic archaeon Haloferax mediterranei; gene cloning, expression and structural studies

A gene encoding a halophilic serine proteinase, halolysin R4, from a halophilic archaeon Haloferax mediterranei strain R4 was cloned, its nucleotide sequence determined, and expressed in Haloferax volcanii WFD11. The deduced amino-acid sequence (403 aa in length) showed the highest similarity to halolysin 172P1, produced by another halophilic archaeon, strain 172P1 (now designated as Natrialba asiatica). Both halolysins belong to the thermitase branch of class I subtilases, but show long C-terminal extensions of 117 and 123 amino acids, respectively. Removal of this ‘tail’ region from halolysin R4 abolished proteinase activity, indicating it provides an essential (but as yet unknown) function. Substitution of the two cysteine residues in the C-terminal extension with serine decreased enzyme stability in hypotonic solutions, possibly owing to disruption of potential disulfide bonds or perturbation of calcium binding site(s).

A New Form of Particulate Single and Multiple Immunogen Delivery System Based on Recombinant Bluetongue Virus-Derived Tubules

The development of particular vector systems for the presentation of immunogenic epitopes provides a powerful approach for the delivery of antigens. These include the core-like particles formed by recombinant bluetongue virus (BTV) capsid proteins VP3 and VP7 synthesized in insect cells by recombinant baculoviruses. Previously we have reported localization of an antigenic site on the surface of tubular structures formed by the nonstructural protein NS1 of BTV, and their potential use for epitope presentation. In this study foreign sequences ranging from 44 to 116 aa in length and representing 44 aa sequence fromClostridium difficiletoxin A, 48 aa of the hepatitis B virus preS2 region, and the whole of bovine leukemia virus p15 protein were inserted at the C-terminus of BTV-10 NS1. The chimeric NS1 genes were expressed using recombinant baculoviruses and the ability of the mutated NS1 proteins to form tubules was investigated. All chimeric constructs formed tubular structures which carried the foreign antigenic sequences exposed on the surface of the tubules and were highly immunogenic. When Sf cells were coinfected with three recombinant baculoviruses expressing chimeric NS1 proteins with different epitopes, their simultaneous assembly into the same tubule was demonstrated. This observation opens up the possibility of using recombinant NS1 tubules as carriers for the delivery of multiple epitopes.

Identification of calmodulin-binding peptide consensus sequences from a phage-displayed random peptide library

The calcium-binding protein, calmodulin (CaM), was used to screen a phage library displaying random peptides 26 amino acids (aa) in length. Twenty CaM-binding peptides were identified, 17 of which contained one of three consensus sequence motifs: + W-OλR, WRAAV or WRXXAAAL, where +, -, O,λ and X are positively charged, negatively charged, hydrophobic, leucine or valine, and any residue, respectively. The Trp residue in these motifs is located within 14 aa of the N-terminus of the displayed peptide. Previous studies [Dedman et al., J. Biol. Chem. 268 (1993) 23025–23030] using a library displaying random peptides 15 aa in length identified CaM-binding peptides which contained a Trp-Pro dipeptide motif. These results suggest that the type of CaM-binding motif identified can vary between different types of combinatorial peptides

Genomic organization and isoforms of the mouse ELP gene.

Analysis was made on the genomic structure, functions, and expression of the mouse ELP gene, which codes for the embryonal long terminal repeat binding protein. Extensive screening of the cDNA library of embryonal carcinoma cells (EC cells) identified four isoforms of ELP: ELP1 (the original ELP isolate), ELP2, ELP3, and Ad4BP/SF1. Analysis of the genomic sequences revealed that these ELP isoforms were generated by alternative promoter usage and differential splicing. The mRNAs of isoforms initiated at four transcription start sites distributed on three exons. Sequence analysis of the four isoforms identified three polypeptides. The N-terminal portion of ELP1 and ELP2 was longer than ELP3, and Ad4BP/SF1 by 77 aa. The DNA-binding domain and region II were shared by all four isoforms. The C-terminal portion shared by ELP2, ELP3, and Ad4BP/SF1 was 131 aa in length, and that specific to ELP1 was 57 aa in length. The ELP3 and Ad4BP/SF1 isoforms were identical for the coding sequence, but the two differed at the 5' noncoding region. Region II and III domains of nuclear receptors were thought to be involved in ligand-binding and transcriptional activation. ELP1, which lacked region III, functioned as a repressor. The isoforms carrying intact region II and region III functioned as transactivators. Expression of the four isoforms was studied in mouse tissues and in tissue culture cells by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Complex patterns of expression of these isoforms were observed in various tissues. All four ELP isoforms were expressed only in EC cells.

A Binding Interface on the I Domain of Lymphocyte Function-associated Antigen-1 (LFA-1) Required for Specific Interaction with Intercellular Adhesion Molecule 1 (ICAM-1)

Previous studies have shown that lymphocyte function-associated antigen-1 (LFA-1) molecules containing the human alpha (CD11a) and human beta (CD18) subunits but not the murine alpha and human beta subunits can bind to human intercellular adhesion molecule 1 (ICAM-1). Using human/mouse LFA-1 alpha subunit chimeras, we mapped regions required for binding to ICAM-1 N-terminal to amino acid (aa) residue 350. Ligand binding sites were mapped in greater detail by scanning this region with murine sequences from 56 down to 17 aa in length and finally by introducing single or few murine aa residue replacements into the human sequence. Replacement of two non-contiguous regions of aa residues 119-153 and 218-248 in the me domain with the corresponding mouse sequences abolished most binding to human ICAM-1, without affecting alpha beta subunit association or expression on the surface of transfected COS cells. Specific residues within the I domain found to be important were Met-140, Glu-146, Thr-243, and Ser-245. Using the recently solved structure of the Mac-1 (CD11b) I domain as a model (Lee, J.-O., Rieu, P., Arnaout, M.A., and Liddington, R. (1995) Cell 80, 631-638), these residues are shown to be located on the surface of the I domain surrounding the site to which Mg2+ is chelated, and fine a ligand binding interface. Mapping of the epitopes of a panel of mouse anti-human and rat anti-mouse monoclonal antibodies gave concordant results. Epitopes were mapped to two different regions in the N-terminal domain, four regions within the I domain, and two regions between the I domain and the EF hand-like repeats. Monoclonal antibodies to epitopes within the mid- to C-terminal portion of the I domain and the N-terminal portion of the region between the I domain and the EF hand-like repeats gave good inhibition of LFA-1-dependent homotypic aggregation with cells that express either ICAM-1 or ICAM-3 as the major LFA-1 ligand.

Comparative Nucleotide and Amino Acid Sequence Analysis of the Sequence-Specific RNA-Binding Rotavirus Nonstructural Protein NSP3

NSP3, an acidic nonstructural protein, encoded by gene 7 has been implicated as the key player in the assembly of the 11 viral plus-strand RNAs into the early replication intermediates during rotavirus morphogenesis. To date, the sequence of NSP3 from only three animal rotaviruses (SA11, SA114F, and bovine UK) has been determined and that from a human strain has not been reported. To determine the genetic diversity among gene 7 alleles from group A rotaviruses, the nucleotide sequence of the NSP3 gene from 13 strains belonging to nine different G serotypes, from both humans and animals, has been determined. Based on the amino acid sequence identity as well as phylogenetic analysis, NSP3 from group A rotaviruses falls into three evolutionarily related groups, i.e., the SA11 group, the Wa group, and the S2 group. The SA11/SA114F gene appears to have a distant ancestral origin from that of the others and codes for a polypeptide of 315 amino acids (aa) in length. NSP3 from all other group A rotaviruses is only 313 aa in length because of a 2-amino-acid deletion near the carboxy-terminus. While the SA114F gene has the longest 3′ untranslated region (UTR) of 132 nucleotides, that from other strains suffered deletions of varying lengths at two positions downstream of the translational termination codon. In spite of the divergence of the nucleotide (nt) sequence in the protein coding region, a stretch of about 80 nt in the 3′ UTR is highly conserved in the NSP3 gene from all the strains. This conserved sequence in the 3′ UTR might play an important role in the regulation of expression of the NSP3 gene.

Cloning and structure of a chicken zinc finger cDNA: restricted expression in developing neural crest cells.

The cloning, DNA sequence analysis and expression pattern of a chicken cDNA, cKr2, is described. cKr2 is a 4492-bp cDNA that encodes a 1173-amino-acid (aa) protein with two domains: the N-terminal portion contains 16 zinc fingers (Zf) of the 2Cys + 2His class, while the C-terminal domain contains a stretch of 181 aa which consists of seven consecutive sequence repeats each being 24 aa in length. The aa sequence repeats harbor a putative DNA-binding helix-turn-helix motif. Northern and Western blotting experiments indicate cKr2 expression from days 2 to 12 of development. In situ hybridization and immunohistochemical analyses using an anti-cKr2 antibody and the HNK-1 antibody, a marker of neural crest cells, revealed cKr2 activity in cephalic and trunk neural crest-derived cells. cKr2 is expressed predominantly in differentiating Schwann cells lining the nerves which innervate the branchial arches and limb buds, in cells of the sympathetic ganglia and aortic plexus, and in putative neuroblasts of dorsal root ganglia. The nuclear localization of the cKr2-encoded protein is consistent with its presumed role as a transcription factor.

Potential therapeutic recombinant proteins comprised of peptides containing recombined T cell epitopes

The complete primary structure of Fel d I 2 has been determined and shown to be comprised of two separate polypeptide chains (designated chain 1 and chain 2). Overlapping peptides covering the entire sequence of both chains of Fel d I have been used to map the major areas of human T cell reactivity. The present study describes three non-contiguous T cell reactive regions of < 30 aa in length that were assembled in all six possible configurations using PCR and recombinant DNA methods. These six recombinant proteins comprised of defined non-contiguous T cell epitope regions artificially combined into single polypeptide chains have been expressed in E. coli, highly purified, and examined for their ability to bind to human cat-allergic IgE and for human T cell reactivity. Several of these recombined T cell epitope-containing polypeptides exhibit markedly reduced IgE binding as compared to the native Fel d I. Importantly, the human T cell reactivity to individual T cell epitope-containing regions is maintained even though each was placed in an unnatural position as compared to the native molecule. In addition, T cell responses to potential junctional epitopes were not detected. It was also demonstrated in mice that s.c. injection of T cell epitope-containing polypeptides inhibits the T cell response to the individual peptides upon subsequent challenge in vitro. Thus, these recombined T cell epitope-containing polypeptides, which harbor multiple T cell reactive regions but have significantly reduced reactivity with allergic human IgE, constitute a novel potential approach for desensitization to important allergens.

Sequences of the ribonucleotide reductase-encoding genes of equine herpesvirus 4

The equine herpesvirus 4 (EHV-4) genes encoding the two subunits of the enzyme ribonucleotide reductase (RR) were cloned and their nucleotide (nt) sequences determined. The large subunit (RR1) is predicted to comprise 789 amino acids (aa), which compares with lengths of 790, 775 and 1137 aa for the RR1 proteins encoded by equine herpesvirus 1 (EHV-1) gene 21, varicella zoster virus (VZV) gene 19 and herpes simplex virus type 1 (HSV-1) UL39, respectively. In common with VZV RR1, the EHV-4 RR1 protein lacks the N-terminal domain of HSV-1 RR1 which possesses protein kinase activity. EHV-4 RR1 demonstrates identities of 88, 52 and 29% with the RR1 proteins of EHV-1, VZV and HSV-1, respectively. The small subunit (RR2) is predicted to be 320 aa in length, which compares with lengths of 321, 306 and 340 aa for the RR2 proteins encoded by EHV-1 gene 20, VZV gene 18 and HSV-1 UL40, respectively. The EHV-4 RR2 protein exhibits identities of 90, 60 and 55% with the RR2 proteins of EHV-1, VZV and HSV-1, respectively.

Role of HLA-A motifs in identification of potential CTL epitopes in human papillomavirus type 16 E6 and E7 proteins.

We have measured the binding affinity for five HLA-A alleles: HLA-A1 (A*0101), A2.1 (A*0201), A3 (A*0301), A11 (A*1101), and A24 (A*2401); of a set of all possible nonamer peptides (n = 240) of human papillomavirus type 16 E6 and E7 proteins. High affinity binding peptides were identified for each of the alleles, thus allowing us to select several candidates for CTL-based vaccines. Moreover, this unbiased set of peptides allowed an evaluation of the predictive value of HLA motifs derived either from the analysis of sequencing of pools of naturally processed peptides or from the binding analysis of polyalanine nonameric peptides that differed in the amino acids (aa) present at the anchor positions. Whereas pool sequencing-derived motifs were present in only 27% of high affinity binders, the more expanded motif, based on analysis of different aa substitutions at the anchor positions, was present in 73% of high affinity binders. Furthermore, it was found that the presence of anchor residues in a peptide was in itself not sufficient to determine binding to MHC class I molecules, because the majority of motif-containing peptides failed to bind to the relevant MHC. Finally, specific HLA motifs were used to predict peptide binders of 8, 10, and 11 aa in length. Several high affinity binding peptides were identified for each of the various peptide lengths, indicating a significant size heterogeneity in peptides capable of high affinity binding to HLA-A molecules.

Endogenous peptides with distinct amino acid anchor residue motifs bind to HLA-A1 and HLA-B8.

Distinct amino acid (aa) residue motifs for peptides binding to HLA-A1 and HLA-B8 were identified by sequence analyses of reversed-phase HPLC fractions containing endogenous peptides derived from these HLA molecules. Fifteen different primary sequences were determined for HLA-A1-associated peptides, 12 of which were nine aa in length. Common features among these peptide sequences were Tyr at the COOH-terminus, a negatively charged aa (usually Glu) at position 3 (P3), and Pro at P4. Twenty-seven different primary sequence assignments were made for HLA-B8-associated peptides, most of which were eight aa in length. Lys, and in a few cases Arg, predominated at P3 and P5; Leu and Pro predominated at P2, and Leu was the preferred COOH-terminal residue. Unlike all other human class I molecules whose peptide-binding properties have been studied, both HLA-A1 and HLA-B8 endogenous peptide sequences have a dominant anchor residue at P3, and these aa are opposite in charge to the aa at position 156 of the peptide-binding site. Synthetic peptides corresponding to endogenous peptide sequences bound to their respective HLA molecules in vitro, indicating that they derive from peptides bound to HLA and not from copurifying contaminants. Eight of the HLA-A1 and HLA-B8 endogenous peptide sequences matched intracellularly expressed proteins found in protein sequence data bases. The HLA-A1 peptide-binding motif was then used to identify potential antigenic peptides from influenza A viral proteins that bound to HLA-A1 in vitro.

Cloning and characterization of a human protein phosphatase 1-encoding cDNA

While sequence information is available for a number of eukaryotic protein phosphatase 1 (PPl)-encoding genes, the cloning and characterization of a complete human pp1 gene has not been reported. We have used two conserved regions within the pp1 family of genes to synthesize oligodeoxyribonucleotide primers for the amplification of a 438-bp sequence from human mRNA. This DNA fragment was sequenced to verify that it corresponded to a pp1 cDNA and it was used to screen a human cDNA library to isolate a full-length clone. The deduced amino acid (aa) sequence identified a protein of 330 aa in length. Comparison with the rabbit pp1 cDNA sequence showed some nucleotide differences, largely at the third position of the codon, with complete concordance at the aa level. Northern blot analysis revealed an mRNA of approximately 1.6kb.