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PubMed2005

Stepwise molding of a highly selective ribonucleopeptide receptor.

The structural characteristics of RNA-peptide (RNP) complexes are suitable for molding of a ligand-binding pocket of the RNP complex in a stepwise manner. The first step involves molding of the RNA subunit by in vitro selection of an RNP pool originating from an RNA library and the peptide, as previously reported for the construction of an ATP-binding RNP complex from an RRE RNA-Rev peptide complex. The second step involves selection from an RNP library consisting of Rev peptides with randomized amino acid residues and the RNA subunit selected in the first molding. The ATP-binding pocket produced by sequential molding of RNA and peptide subunits shows higher affinity to ATP and a distinct specificity for ATP versus dATP as compared to the ATP-binding RNP receptor in which only the RNA subunit has been molded. The second step selection from the peptide-based RNP library allows expansion of the ATP recognition surface, consisting of both RNA and peptide subunits, to enhance the affinity and selectivity to discriminate ATP against dATP. Our approach of stepwise molding offers the advantage of increasing the diversity of the RNP library by utilizing characteristics of different biopolymers. The ribonucleopeptide-based, multi-subunit approach is also extendable to other biomacromolecular assemblies, which may yield artificial receptors and enzymes with increased specificity and more diverse chemical activities.

Adenosine Triphosphate↗
PubMed1999

Peptide dependency of alloreactive CD4+ T cell responses.

Alloreactivity, the capacity of a large number of T lymphocytes to react with foreign MHC molecules, represents the cellular basis for the rejection of tissue grafts. Although it was originally assumed that the TCR of alloreactive T cells focus their recognition on the polymorphic residues that differ between the MHC molecules of responder and stimulator cells, studies in the MHC class I system have clearly demonstrated that MHC-bound peptides can influence this interaction. It remains unclear, however, whether peptides play an equally important role for the recognition of MHC class II molecules by alloreactive CD4+ T cells. Another issue that remains unresolved is the overall frequency of peptide-dependent versus peptide-independent alloreactive T cells. We have addressed these questions with antigen-presenting cells (APC) from H2-M mutant mice that predominantly express a single MHC class II-peptide complex, H2-Ab bound by a peptide (CLIP) derived from the class II-associated invariant chain. APC from these mice were used as targets and stimulators for alloreactive CD4+ T cells. Results demonstrated that the vast majority of CD4+ alloreactive T cells recognize MHC class II molecules in a peptide-dependent fashion.

Amino Acid Sequence↗
PubMed2004

Bending of adenovirus origin DNA by nuclear factor I as shown by scanning force microscopy is required for optimal DNA replication.

Nuclear factor I (NFI) is a transcription factor that binds to the adenovirus type 5 (Ad5) origin of replication and recruits the adenovirus DNA polymerase, thereby stimulating initiation of DNA replication in vitro. Using scanning force microscopy, we demonstrate that NFI induces a 60 degrees bend upon binding to the origin. The A/T-rich region preceding the core recognition sequence of NFI influences the DNA bend angle, since substitution of A/T base pairs by G/C base pairs severely decreases bending. Mutations in the A/T-rich region do not affect binding of NFI to DNA. However, mutations that reduce the protein-induced bend lead to a loss of NFI-stimulated replication, indicating that DNA bending is functionally important. In contrast, basal initiation or DNA binding of the polymerase is not impaired by these origin mutations. We conclude that binding of NFI to the Ad5 origin causes structural changes in DNA that are essential for the stimulatory function of NFI in replication. We propose that NFI-induced origin bending facilitates the assembly of a functional initiation complex.

Adenoviruses, Human↗
PubMed1981

Antigen-dependent proliferation of cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes.

The antigenic requirements for in vitro proliferation of several cloned continuous lines of H-2-restricted influenza virus-specific cytotoxic T lymphocytes (CTL) has been examined. The cloned CTL lines were established from individual splenic CTL precursors obtained from A/JAPAN/305/57 (H2N2)-immune F1 (C57BL/6 X BALB/c) donors. The lines were isolated (by limiting dilution in liquid culture) and expanded in the presence of A/JAPAN/305/57-infected irradiated syngeneic (F1) spleen cells and T cell growth factor (TCGF) of rat spleen origin. Optimal proliferation (and long-term in vitro cultivation) of these H-2-restricted CTL lines required both specific antigenic stimulation in the form of virus-infected syngeneic spleen cells and an exogenous source of TCGF. In addition, the antigenic requirements for proliferation of these lines directly reflected the pattern of H-2-restricted influenza virus-specific recognition at the level of target cell recognition and lysis.

Animals↗
PubMed2004

The role of DNA structure and dynamics in the recognition of bovine papillomavirus E2 protein target sequences.

The papillomavirus E2 transcription and replication factors bind to the DNA consensus ACCGN(4)CGGT sequence (E2-BS), through both direct and indirect readout mechanisms. The two symmetric half-sites ACCG.CGGT are highly conserved in the genomes and are hydrogen bound with E2. Although E2 does not contact the N4 spacer, the affinities are modulated by the base composition of this DNA part. Nevertheless, the origin of either the global recognition mechanism or the spacer effect remains unclear, particularly in the case of the bovine papillomavirus type 1 E2 (BPV-1-E2) system, used as model to study the papillomaviruses. We present, herein, studies carried out on oligomers differently recognized by the BPV-1-E2 protein and based on molecular dynamic simulations including counterions and water. The sequences contain the conserved half-sites but three different spacers (CCAT, ACGT and AAAC), resulting in very high, high and low affinity targets for BPV-1-E2. In order to estimate how much the free DNAs resemble the bound conformations, comparisons are made with two DNAs extracted from E2-BS-BPV-1 crystallographic complexes, representative of high and moderate affinity structures. The analysis of 15 ns trajectories reveals that the ACCG/CGGT half-sites, whatever the spacer, have the same behavior and adopt average stable base-pair parameters very close to those of the bound conformations. In contrast, the three different free spacers strongly differ in their BI <--> BII backbone dynamics. The low affinity AAAC spacer exhibits stable BI backbone conformations, the high affinity ACGT spacer is characterized by a dramatic instability of the CpG phosphate groups, and the CpA and GpG backbones in the very high affinity CCAT.ATGG spacer are trapped in BII conformations. All resemble more of the moderate affinity complex DNA than the high affinity one. Nevertheless, the particular behavior of the CCAT and ACGT backbones allows the emergence of BII-rich spacers, a configuration reproducing both local and global helical features of the bound DNA conformation of the high affinity complex and favoring the minor groove curvature required in the complex. In particular, the CCAT-containing site spends almost half of the time in this form that well mimics the bound one. Thus, we propose that the E2 protein could take advantage of the invariant favorable structures of the half-sites to form a pre-complex, but would require a specific spacer intrinsic malleability to lock the interaction. Finally, the backbone conformational states, by their ability to translate information coded in the sequence into structural properties, provide insight into the mechanisms that contribute to fine binding site selection and specific nucleic acid ligand recognition.

Base Sequence↗
PubMed1995

Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition.

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH2 (peptide 1), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH2 (peptide 2), which closely corresponds to the original Flag sequence. Enrichment of the peptides with 15N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes.

Amino Acid Sequence↗
PubMed1983

A new procedure for the synthesis of polyethylene glycol-protein adducts; effects on function, receptor recognition, and clearance of superoxide dismutase, lactoferrin, and alpha 2-macroglobulin.

A new, simplified technique for the synthesis of polyethylene glycol (PEG) derivatives of proteins utilizing 1,1'-carbonyldiimidazole for PEG activation, is described. PEG derivatives of superoxide dismutase, alpha 2-macroglobulin, alpha 2-macroglobulin-trypsin, and lactoferrin were prepared and studied. Superoxide dismutase coupled to PEG preserved 95% of its original activity while its plasma half-life increased from 3.5 min to 9 or more hours depending on the PEG derivative studied. PEG-derivatized alpha 2-macroglobulin showed decreased protease binding activity but PEG derivatives of performed alpha 2-macroglobulin-trypsin demonstrated no loss of activity. The plasma clearance of PEG-alpha 2-macroglobulin-trypsin was prolonged significantly compared to native alpha 2-macroglobulin-trypsin, particularly when a high-molecular-weight PEG was coupled to the protease inhibitor complex. The plasma clearance half-life of lactoferrin was increased 5- to 20-fold by this modification. Trinitrobenzenesulfonic acid titration studies demonstrated that epsilon-amino groups of lysine residues are modified by the coupling of carbonyldiimidazole-activated PEG to proteins.

Animals↗
PubMedDate not supplied

Binding behavior of aliphatic oligopeptides by bridged and metallobridged bis(beta-cyclodextrin)s bearing an oxamido bis(2-benzoic) carboxyl linker.

beta-Cyclodextrin dimers bearing an oxamido bis(2-benzoic) carboxyl linker (1) or its metal complexes (2 and 3) were newly synthesized, and their inclusion complexation behavior with a series of representative aliphatic oligopeptides, i.e., Leu-Gly, Gly-Leu, Gly-Pro, Glu-Glu, Gly-Gly, Gly-Gly-Gly, and Glu(Cys-Gly), was elucidated by means of UV/vis, circular dichroism, fluorescence, and 2D NMR spectroscopy in Tris-HCl buffer solution (pH 7.4) at 25 degrees C. The results obtained indicated that metallobridged bis(beta-cyclodextrin)s 2 or 3 could significantly enhance the original molecular binding abilities of parent bis(beta-cyclodextrin) 1 toward model substrates through the cooperative binding of two cyclodextrin moieties and the additional chelation effect supplied by the coordinated metal centers. It is interesting that hosts 2 and 3 displayed an entirely different fluorescence behavior upon complexation with guest oligopeptides. Among the guest peptides examined, 3 showed the highest complex formation constant of 68 200 M(-)(1) for Glu-Glu, up to 510-fold as compared with 1 (135 M(-)(1)), while 1 gave excellent molecular selectivity for Glu(Cys-Gly)/Glu-Glu pair, up to 51-fold. The molecular binding ability and selectivity were discussed from the viewpoints of the induced-fit and multiple recognition mechanism between host and guest.

Amides↗
PubMed1995

How is pheromone specificity encoded in proteins?

Pheromone specificity in the Lepidoptera is encoded in protein components of the antennal sensillum lymph and dendritic membrane. In this paper, we highlight recent work on the molecular determinants of pheromone binding affinity of pheromone binding proteins (PBPs) of three genera. First, we describe new cDNA sequences for Lymantria dispar (Lymantriidae) and Agrotis segetum (Noctuidae). These data enrich the conclusions derived from our functional studies. Secondly, we indicate how preparation of multimilligram quantities of the recombinant PBP 'Apol-3' (originally from Antheraea polyphemus) has provided a platform (i) to determine the ligand binding sites using photoaffinity labeling, (ii) to conduct structural analysis by CD and NMR, and (iii) to measure binding affinities using a new binding assay. Thirdly, we describe the use of expression-cassette PCR technology to prepare two related PBPs from Antheraea perneyi to test binding affinities of naturally-occurring homologous PBPs. Our results support a model in which ligand specificity for chain length, double bond position, and terminal functionality is partially encoded in the PBPs. We propose that the final decoding is accomplished when the PBP-pheromone complex activates a G-protein coupled seven-transmembrane domain receptor that contains recognition sites for both the presented pheromone and the presenting PBP.

Amino Acid Sequence↗
PubMed1997

High-resolution structure of a polyomavirus VP1-oligosaccharide complex: implications for assembly and receptor binding.

The crystal structure of a recombinant polyomavirus VP1 pentamer (residues 32-320) in complex with a branched disialylated hexasaccharide receptor fragment has been determined at 1.9 A resolution. The result extends our understanding of oligosaccharide receptor recognition. It also suggests a mechanism for enhancing the fidelity of virus assembly. We have previously described the structure of the complete polyomavirus particle complexed with this receptor fragment at 3.65 A. The model presented here offers a much more refined view of the interactions that determine carbohydrate recognition and allows us to assign additional specific contacts, in particular those involving the (alpha2,6)-linked, branching sialic acid. The structure of the unliganded VP1 pentamer, determined independently, shows that the oligosaccharide fits into a preformed groove and induces no measurable structural rearrangements. A comparison with assembled VP1 in the virus capsid reveals a rearrangement of residues 32-45 at the base of the pentamer. This segment may help prevent the formation of incorrectly assembled particles by reducing the likelihood that the C-terminal arm will fold back into its pentamer of origin.

Amino Acid Sequence↗
PubMed2007

Asthma phenotypes.

PURPOSE OF REVIEW: Asthma is a heterogeneous disorder presenting with many phenotypes. Precise phenotypic definition has eluded the medical research community for years, despite recognition of different disease subtypes. Improved phenotypic characterization and knowledge of underlying pathobiology is necessary for linkage of specific genotypes with clinical disease manifestations. RECENT FINDINGS: Phenotyping has been difficult because asthma is likely to be comprised of overlapping syndromes with varying origins and heterogeneous pathobiology. Currently, the field is too reliant on classification by trigger or symptoms. Since genotypic and phenotypic heterogeneity are inherent in asthma, patients presenting with different asthma phenotypes may need tailored therapies. Studies have begun to link genetics with disease mechanism and therapeutic response. As disease etiology, onset, progression and severity vary greatly among patients, however, the relative contribution of genetic factors may be difficult to ascertain. Definition of the full array of complex biological consequences of molecular target modulation is a prerequisite for therapies based on this concept. SUMMARY: The advent of targeted therapies for asthma and clinical trials based on phenotype and genotype have raised interest in more accurate description of asthma phenotypes. Therapies based on phenotypic and genotypic characteristics may be useful in asthma management. A variety of factors, however, must be addressed before such approaches become standard.

Aspirin↗
PubMed1983

[Topochemical aspects of pyrimidine specificity of ribonuclease A].

To get insight into the origin of pyrimidine specificity of ribonuclease A, a study of the enzyme interaction with the substrate analogs having a modified nucleobase was undertaken. Pyridine and pyrimidine cyclophosphates were obtained by phosphorylation of 2'(3')-O-dibutyl stannylidene derivatives of nonprotected nucleosides in high yields. The results of kinetic and NMR studies suggested that a substrate should be locked in anti-conformation in the productive enzyme-substrate complex as it was shown for the crystalline complexes of the enzyme with pyrimidine nucleotides by X-ray analysis. The interaction between carbonyl group in position 2 of substrate nucleobase and proton accepting group of the protein (NH of Thr45) was found to be a prerequisite for the specific recognition of a substrate by the enzyme. The rate constants for transformation of lactam form (slow) and lactim form (fast) of pyrimidine substrates were estimated.

Allosteric Regulation↗
PubMed2006

Chemical identification of MHC-influenced volatile compounds in mouse urine. I: Quantitative Proportions of Major Chemosignals.

The genes of the major histocompatibility complex (MHC) are highly polymorphic loci that encode cell surface proteins, class I and II molecules. They present peptide antigens to T cells and thereby control immunological self/nonself recognition. Increasing evidence indicates that MHC genes also influence odor and mating preferences; however, it is unclear how. Here we report the results of chemical analyses of male mouse urinary odors collected from a variety of mouse strains, including MHC-congenics, recombinants, mutants, and transgenics (i.e., beta2 microglobulin "knockouts," which lack class I expression, and transporters associated with antigen processing (TAP) knock-outs). After the identification of volatile odor components by gas chromatography/mass spectrometry, the odor profiles of urine samples were analyzed quantitatively by using stir bar sorptive extraction and gas chromatography/atomic emission detection. Results showed that MHC genes influenced the amounts of testosterone-mediated pheromones, sulfur-containing compounds, and several carbonyl metabolites. This is the first report to quantitatively link known mouse pheromones to classical, antigen-binding MHC loci. Surprisingly, these compounds were not influenced by TAP genes, even though these loci are MHC-linked and play a role in peptide presentation. Whereas identification of MHC-determined odorants does not reveal their metabolic origin, some constituents were also present in blood serum, and their levels were not altered by antibiotics.

Animals↗
PubMed2005

Novel classification of idiopathic inflammatory myopathies based on overlap syndrome features and autoantibodies: analysis of 100 French Canadian patients.

Our objective was to improve the currently imperfect classifications of idiopathic inflammatory myopathies (IIM). In clinical practice, overlap features are common in IIM. This provided a rationale for positioning overlap clinical features at the core of a new classification system. We conducted a longitudinal study of 100 consecutive adult French Canadian patients with IIM. Clinical and laboratory data were obtained by retrospective chart review. Sera were analyzed for autoantibodies (aAbs) by protein A-assisted immunoprecipitation and double immunodiffusion. Overlap aAbs encompassed aAbs to synthetases, systemic sclerosis-associated aAbs, anti-signal recognition particle (SRP) and anti-nucleoporins. Patients were classified both at IIM diagnosis, based on data at presentation, and at the end of follow-up, based on cumulative findings. Three classifications were used: 1) the Bohan and Peter original classification, 2) a new version of that classification as modified by us, and 3) a novel clinicoserologic classification. As investigators were blinded to aAb results, the modified classification is strictly a clinical classification. Its core concept is the attribution of diagnostic significance to the presence of overlap features, that is, their presence resulted in a diagnosis of overlap myositis (OM). This approach allowed direct comparison with the original Bohan and Peter classification. By integrating aAb results to the modified classification, we also defined the clinicoserologic classification, which allowed to examine the added value of aAbs to diagnostic, therapeutic and prognostic stratification. Whereas polymyositis (PM) was the most common IIM according to the original classification, accounting for 45% of the cohort at diagnosis, its frequency fell to 14% with the modified classification. Conversely, while the frequency of myositis associated with connective tissue disease was 24% according to the original classification, the frequency of OM was 60% when using the modified classification. At last follow-up, the frequency of PM fell further to only 9%, while the frequency of OM rose to 67%. Systemic sclerosis was the most common connective tissue disease associated with IIM, accounting for 42.6% of OM patients and 29% of the cohort. The frequencies of overlap aAbs in the cohort and in OM patients were 48% and 70.5% (n =48/68), respectively. The presence of overlap aAbs at IIM diagnosis identified additional OM patients unrecognized by the modified classification. The sensitivity of the modified classification for OM at diagnosis was 87%, suggesting that clinicians may rely on the modified classification for identification of most OM patients, while awaiting results of aAb assays. The new classifications predicted the response to prednisone and IIM course. Using stringent definitions, IIM was classified as responsive or refractory after an adequate initial corticosteroid therapy, and the disease course as monophasic or chronic after a single adequate trial of prednisone. PM was always chronic and was associated with the highest rate (50%) of refractoriness to initial corticosteroid treatment. Dermatomyositis was almost always chronic (92% rate); however, its responsiveness to initial corticosteroid treatment was high (87%). OM was almost always responsive to corticosteroids (89%-100% rates). When OM patients were divided according to aAb subsets, anti-synthetase, SRP, or nucleoporin aAbs were markers for chronic myositis, whereas aAbs to U1RNP, Pm-Scl, or Ku were markers for monophasic myositis. We conclude that the original Bohan and Peter classification should be abandoned as it leads to misclassification of patients. Much of IIM is composed of OM. The proposed modified and clinicoserologic classifications have diagnostic, prognostic, and therapeutic value.

Adolescent↗
PubMed2004

Substrate recognition and catalysis by the Holliday junction resolving enzyme Hje.

Two archaeal Holliday junction resolving enzymes, Holliday junction cleavage (Hjc) and Holliday junction endonuclease (Hje), have been characterized. Both are members of a nuclease superfamily that includes the type II restriction enzymes, although their DNA cleaving activity is highly specific for four-way junction structure and not nucleic acid sequence. Despite 28% sequence identity, Hje and Hjc cleave junctions with distinct cutting patterns--they cut different strands of a four-way junction, at different distances from the junction centre. We report the high-resolution crystal structure of Hje from Sulfolobus solfataricus. The structure provides a basis to explain the differences in substrate specificity of Hje and Hjc, which result from changes in dimer organization, and suggests a viral origin for the Hje gene. Structural and biochemical data support the modelling of an Hje:DNA junction complex, highlighting a flexible loop that interacts intimately with the junction centre. A highly conserved serine residue on this loop is shown to be essential for the enzyme's activity, suggesting a novel variation of the nuclease active site. The loop may act as a conformational switch, ensuring that the active site is completed only on binding a four-way junction, thus explaining the exquisite specificity of these enzymes.

Archaeal Proteins↗
PubMed1996

Selectively 13C-enriched DNA: evidence from 13C1' relaxation rate measurements of an internal dynamics sequence effect in the lac operator.

In order to study some internal dynamic processes of the lac operator sequence, the 13C-labeled duplex 5'd(C0G1C2T3C4A5C6A7A8T9T10).d(A10A9T8T7G6T5G4A3G2C1G0)3' was used. The spreading of both the 1H1' and 13C1' resonances brought about an excellent dispersion of the 1H1'-13C1' correlations. The spin-lattice relaxation parameters R(Cz), R(Cx,y) and R(Hz --> Cz) were measured for each residue of the two complementary strands, except for the 3'-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387-7401; and (1990) J. Am. Chem. Soc., 112, 4989-4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1'-H1' fragments exhibited both slow (tau s = 1.5 ns) and fast (tau f = 20 ps) restricted libration motions (Ss2 = 0.74 to 1.0 and Sf2 = 0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5'-terminal residues showed large internal motions (S2 = 0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters Sf2 and Ss2 along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (Sf2 = 0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(Cz) and R(Hz --> Cz) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(Cx,y) for the purine residues in the (5' --> 3') G4A3 and A10A9T8T7 sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213-216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.

Bacterial Proteins↗
PubMed1994

Monoclonal antibodies defining functional sites on the toxin superantigen staphylococcal enterotoxin B.

Four monoclonal antibodies (mAbs) were produced binding to four nonoverlapping epitopes on the superantigen staphylococcal enterotoxin B (SEB). The mAbs were tested for their ability to detect SEB bound to major histocompatibility complex (MHC) class II, to inhibit SEB binding to MHC class II, to inhibit SEB stimulation of T cell hybridomas, to bind to various nonfunctional mutants of SEB, and to capture and present SEB and its mutants to T cells in the absence of MHC class II. We concluded that two mAbs, B344 and B327, bound to epitopes not required for superantigen function, one mAb, 2B33, blocked an MHC interaction site on SEB, and the fourth mAb, B87, blocked the T cell recognition site on SEB. Moreover, two mAbs (B344 and 2B33) were capable of presenting SEB, although much less efficiently than APC, to CD4- but not CD4+ T cell hybridomas. The results confirm the functional domains on SEB originally defined by mutation and show that MHC class II is not always an essential component of the superantigen ligand.

Animals↗
PubMed2003

Recognition of novel viral sequences that associate with the dynein light chain LC8 identified through a pepscan technique.

Recent data from multiple laboratories indicate that upon infection, many different families of viruses hijack the dynein motor machinery and become transported in a retrograde manner towards the cell nucleus. In certain cases, one of the dynein light chains, LC8, is involved in this interaction. Using a library of overlapping dodecapeptides synthesized on a cellulose membrane (pepscan technique) we have analyzed the interaction of the dynein light chain LC8 with 17 polypeptides of viral origin. We demonstrate the strong binding of two herpesvirus polypeptides, the human adenovirus protease, vaccinia virus polymerase, human papillomavirus E4 protein, yam mosaic virus polyprotein, human respiratory syncytial virus attachment glycoprotein, human coxsackievirus capsid protein and the product of the AMV179 gene of an insect poxvirus to LC8. Our data corroborate the manipulation of the dynein macromolecular complex of the cell during viral infection and point towards the light chain LC8 as one of the most frequently used targets of virus manipulation.

Adenoviridae↗