Beta Domain Research Articles

Procrustean rotation in concert with principal component analysis of molecular dynamics trajectories: Quantifying global and local differences between conformational samples

Given the principal component analysis (PCA) of a molecular dynamics (MD) conformational trajectory for a model protein, we perform orthogonal Procrustean rotation to "best fit" the PCA squared-loading matrix to that of a target matrix computed for a related but different molecular system. The sum of squared deviations of the elements of the rotated matrix from those of the target, known as the error of fit (EOF), provides a quantitative measure of the dissimilarity between the two conformational samples. To estimate precision of the EOF, we perform bootstrap resampling of the molecular conformations within the trajectories, generating a distribution of EOF values for the system and target. The average EOF per variable is determined and visualized to ascertain where, locally, system and target sample properties differ. We illustrate this approach by analyzing MD trajectories for the wild-type and four selected mutants of the beta1 domain of protein G.

Crystallization and preliminary X-ray crystallographic characterization of a public CMV-specific TCR in complex with its cognate antigen

The T-cell response to human cytomegalovirus is characterized by a dramatic reduction of clonal diversity in patients undergoing chronic inflammation or immunodepression. In order to check whether all the selected high-avidity T-cell clones recognize the immunodominant pp65 peptide antigen pp65(495-503) (NLVPMVATV) presented by the major histocompatibility complex (MHC) molecule HLA-A2 in a similar manner, several public high-affinity T-cell receptors (TCRs) specific for the pp65(495-503)-HLA-A2 complex have been investigated. Expression, purification and crystallization were performed and preliminary crystallographic data were collected to 4.7 angstrom resolution for the RA15 TCR in complex with the pp65(495-503)-HLA-A2 complex. Comparison of the RA15-pp65(495-503)-HLA-A2 complex molecular-replacement solution with the structure of another high-affinity pp65(495-503)-HLA-A2-specific TCR, RA14, shows a shared docking mode, indicating that the clonal focusing could be accompanied by the selection of a most favoured peptide-readout mode. However, the position of the RA15 V beta domain is significantly shifted, suggesting a different interatomic interaction network.

Atomic resolution protein structure determination by three-dimensional transferred echo double resonance solid-state nuclear magnetic resonance spectroscopy

We show that quantitative internuclear (15)N-(13)C distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of (13)C sites in the beta1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3-(13)C or 2-(13)C-glycerol as the (13)C source. Quantitative dipolar trajectories are extracted from two-dimensional (15)N-(13)C planes, in which approximately 750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one (13)C and several (15)N each), yielding quantitative precision as good as 0.1 A for approximately 350 sites, better than 0.3 A for another 150, and approximately 1.0 A for 150 distances in the range of 5-8 A. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25+/-0.09 A), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79+/-0.03 A). Moreover, side chain heavy atoms are well restrained (0.76+/-0.06 A total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure.

Arsenic metalation of seaweed Fucus vesiculosus metallothionein: the importance of the interdomain linker in metallothionein.

The presence of metallothionein in seaweed Fucus vesiculosus has been suggested as the protecting agent against toxic metals in the contaminated waters it can grow in. We report the first kinetic pathway data for A3+ binding to an algal metallothionein, F. vesiculosus metallothionein (rfMT). The time and temperature dependence of the relative concentrations of apo-rfMT and the five As-containing species have been determined following mixing of As3+ and apo-rfMT using electrospray ionization mass spectrometry (ESI MS). Kinetic analysis of the detailed time-resolved mass spectral data for As3+ metalation allows the simulation of the metalation reactions showing the consumption of apo-rfMT, the formation and consumption of As1- to As4-rfMT, and subsequent, final formation of As5-rfMT. The kinetic model proposed here provides a stepwise analysis of the metalation reaction showing time-resolved occupancy of the Cys7 and the Cys9 domain. The rate constants (M(-1) s(-1)) calculated from the fits for the 7-cysteine gamma domain are k1gamma, 19.8, and k2gamma, 1.4, and for the 9-cysteine beta domain are k1beta, 16.3, k2beta, 9.1, and k3beta, 2.2. The activation energies and Arrhenius factors for each of the reaction steps are also reported. rfMT has a long 14 residue linker, which as we show from analysis of the ESI MS data, allows each of its two domains to bind As3+ independently of each other. The analysis provides for the first time an explanation of the differing metal-binding properties of two-domain MTs with linkers of varying lengths, suggesting further comparison between plant (with long linkers) and mammalian (with short linkers) metallothioneins will shed light on the role of the interdomain linker.

The Evolutionary History of the Structure of 5S Ribosomal RNA

5S rRNA is the smallest nucleic acid component of the large ribosomal subunit, contributing to ribosomal assembly, stability, and function. Despite being a model for the study of RNA structure and RNA-protein interactions, the evolution of this universally conserved molecule remains unclear. Here, we explore the history of the three-domain structure of 5S rRNA using phylogenetic trees that are reconstructed directly from molecular structure. A total of 46 structural characters describing the geometry of 666 5S rRNAs were used to derive intrinsically rooted trees of molecules and molecular substructures. Trees of molecules revealed the tripartite nature of life. In these trees, superkingdom Archaea formed a paraphyletic basal group, while Bacteria and Eukarya were monophyletic and derived. Trees of molecular substructures supported an origin of the molecule in a segment that is homologous to helix I (alpha domain), its initial enhancement with helix III (beta domain), and the early formation of the three-domain structure typical of modern 5S rRNA in Archaea. The delayed formation of the branched structure in Bacteria and Eukarya lends further support to the archaeal rooting of the tree of life. Remarkably, the evolution of molecular interactions between 5S rRNA and associated ribosomal proteins inferred from a census of domain structure in hundreds of genomes established a tight relationship between the age of 5S rRNA helices and the age of ribosomal proteins. Results suggest 5S rRNA originated relatively quickly but quite late in evolution, at a time when primordial metabolic enzymes and translation machinery were already in place. The molecule therefore represents a late evolutionary addition to the ribosomal ensemble that occurred prior to the early diversification of Archaea.

The LIM-Homeodomain Protein ISL1 Activates Insulin Gene Promoter Directly through Synergy with BETA2

The LIM-homeodomain transcription factor ISL1 (islet factor 1) is essential for pancreatic islet cell and dorsal mesenchyme development. Mutations in ISL1 are associated with maturity-onset diabetes of the young and type 2 diabetes. Whether ISL1 plays a role in the insulin gene expression has not been fully elucidated. In the present study, we show that ISL1 can synergistically activate insulin gene transcription with BETA2 in pancreatic β cells. The protein–protein interactions of ISL1 and BETA2 are directly mediated by the LIM domains of ISL1 and the basic helix–loop–helix domain of BETA2. Deletion of the two LIM domains of ISL1 enhances the transcriptional activation of the insulin gene, indicating a key role for the homeodomain in activating the insulin promoter. Furthermore, ISL1 can bind with the A3/4 box in the rat insulin gene І promoter through its homeodomain. ISL1 expression is up-regulated at the mRNA level in type 2 diabetes (db/db mouse model) but down-regulated by dexamethasone in rat insulinoma cells. These results suggest that ISL1 is a transcriptional activator for insulin gene expression, and the interactions of ISL1 with BETA2 are required for the transcriptional activity of the insulin gene. Reduction in Isl1 gene expression appears to be involved in the impairment of insulin expression mediated by dexamethasone.

Differential Recognition of CD1d-α-Galactosyl Ceramide by the Vβ8.2 and Vβ7 Semi-invariant NKT T Cell Receptors

The semi-invariant natural killer T cell receptor (NKT TCR) recognizes CD1d-lipid antigens. Although the TCR alpha chain is typically invariant, the beta chain expression is more diverse, where three V beta chains are commonly expressed in mice. We report the structures of V alpha 14-V beta 8.2 and V alpha 14-V beta 7 NKT TCRs in complex with CD1d-alpha-galactosylceramide (alpha-GalCer) and the 2.5 A structure of the human NKT TCR-CD1d-alpha-GalCer complex. Both V beta 8.2 and V beta 7 NKT TCRs and the human NKT TCR ligated CD1d-alpha-GalCer in a similar manner, highlighting the evolutionarily conserved interaction. However, differences within the V beta domains of the V beta 8.2 and V beta 7 NKT TCR-CD1d complexes resulted in altered TCR beta-CD1d-mediated contacts and modulated recognition mediated by the invariant alpha chain. Mutagenesis studies revealed the differing contributions of V beta 8.2 and V beta 7 residues within the CDR2 beta loop in mediating contacts with CD1d. Collectively we provide a structural basis for the differential NKT TCR V beta usage in NKT cells.

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from Asclepias fruticosa latex

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZalpha vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.

A Common Biosynthetic Pathway Governs the Dimerization and Secretion of Inhibin and Related Transforming Growth Factor β (TGFβ) Ligands

The assembly and secretion of transforming growth factor beta superfamily ligands is dependent upon non-covalent interactions between their pro- and mature domains. Despite the importance of this interaction, little is known regarding the underlying regulatory mechanisms. In this study, the binding interface between the pro- and mature domains of the inhibin alpha-subunit was characterized using in vitro mutagenesis. Three hydrophobic residues near the N terminus of the prodomain (Leu(30), Phe(37), Leu(41)) were identified that, when mutated to alanine, disrupted heterodimer assembly and secretion. It is postulated that these residues mediate dimerization by interacting non-covalently with hydrophobic residues (Phe(271), Ile(280), Pro(283), Leu(338), and Val(340)) on the outer convex surface of the mature alpha-subunit. Homology modeling indicated that these mature residues are located at the interface between two beta-sheets of the alpha-subunit and that their side chains form a hydrophobic packing core. Mutation of these residues likely disturbs the conformation of this region, thereby disrupting non-covalent interactions with the prodomain. A similar hydrophobic interface was identified spanning the pro- and mature domains of the inhibin beta(A)-subunit. Mutation of key residues, including Ile(62), Leu(66), Phe(329), and Pro(341), across this interface was disruptive for the production of both inhibin A and activin A. In addition, mutation of Ile(62) and Leu(66) in the beta(A)-propeptide reduced its ability to bind, or inhibit the activity of, activin A. Conservation of the identified hydrophobic motifs in the pro- and mature domains of other transforming growth factor beta superfamily ligands suggests that we have identified a common biosynthetic pathway governing dimer assembly.

Metalation of metallothioneins

Metalloproteins represent approximately 30% of all proteins known, yet our understanding of the structures of these metalloproteins, the metal content, and the mechanism for metalation are still very limited. One of the most studied metalloproteins is the ubiquitous metallothionein (MT), which in mammals contains two metal-binding domains: a 9-cysteine beta domain and a 11-cysteine alpha domain. Metals are coordinated in MT via the cysteinyl thiols present in the primary amino acid sequence and the geometry is controlled by the metal ion. This short review discusses the use of optical spectroscopy to study the metalation of MT with particular emphasis on the benefits and pitfalls involved. Further, the new properties of MT that have been revealed using electrospray ionization mass spectrometry in recent metalation studies will also be discussed.

Dynamic Influence of the Two Membrane-Proximal Immunoglobulin-Like Domains upon the Peptide-Binding Platform Domain in Class I and Class II Major Histocompatibility Complexes: Normal Mode Analysis

Major histocompatibility complexes (MHCs) mainly fall into class I and class II. The two classes have similar structures, with two membrane-proximal immunoglobulin-like domains and a peptide-binding platform domain, though their organizations are different. We simulated the dynamics of a whole and partial model deficient in either of the two membrane-proximal domains for class I and class II using normal mode analysis. Our study showed that the influence of the two membrane-proximal domains upon the dynamics of the platform domain were decisively different between class II and class I. Both membrane-proximal domains (the alpha2 and beta2 domains) of class II MHC, especially the alpha2 domain, influenced the most important pocket that accommodates a large hydrophobic anchor side chain of the N-terminal side of the bound peptide, though the pocket was not in the alpha2 domain neighborhood. By contrast, the two membrane-proximal domains (the alpha3 and beta2m domains) of class I MHC had little influence upon the most important pocket that accommodates the N-terminal residue of the bound peptide. These results suggest that the two membrane-proximal domains of class II MHC have a greater influence upon peptide-binding than those of class I MHC.

Kinetic Analysis of Arsenic−Metalation of Human Metallothionein: Significance of the Two-Domain Structure

Metallothionein (MT) is ubiquitous in Nature, underlying MT's importance in the cellular chemistry of metals. Mammalian MT consists of two metal-binding domains while microorganisms like cyanobacteria consist of a single metal-binding domain MT. The evolution of a two-domain protein has been speculated on for some time; however, no conclusive evidence explaining the evolutionary necessity of the two-domain structure has been reported. The results presented in this report provide the complete kinetic analysis and subsequent mechanism of the As(3+)-metalation of the two-domain beta alpha hMT and the isolated single domain fragments using time- and temperature-resolved electrospray ionization mass spectrometry. The mechanism for beta alpha hMT binding As(3+) is noncooperative and involves six sequential bimolecular reactions in which the alpha domain binds As(3+) first followed by the beta domain. At room temperature (295 K) and pH 3.5, the sequential individual rate constants, k(n) (n = 1-6) for the As(3+)-metalation of beta alpha hMT starting at k(1beta alpha) are 25, 24, 19, 14, 8.7, and 3.7 M(-1)s(-1). The six rate constants follow an almost linear trend directly dependent on the number of unoccupied sites for the incoming metal. Analysis of the temperature-dependent kinetic electrospray ionization mass spectra data allowed determination of the activation energy for the formation of As(1)-H(17)-beta alpha hMT (14 kJ mol(-1)) and As(2-6)-beta alpha hMT (22 kJ mol(-1)). On the basis of the increased rate of metalation for the two-domain protein when compared with the isolated single-domain, we propose that there is an evolutionary advantage for the two-domain MT structures in higher organism, which allows MT to bind metals faster and, therefore, be a more efficient metal scavenger.

Localization of chondroitin sulfate proteoglycan versican in adult brain with special reference to large projection neurons

Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versican has two glycosaminoglycan attachment regions, named the GAG alpha and GAG beta domains, which are both regulated by alternative splicing and yield four protein isoforms. We have investigated the expression and localization of versican in the developing and adult brain by using anti-versican GAG alpha and GAG beta antibodies. Western analysis revealed that GAG alpha-reactive isoform was dominant in the adult brain. Immunohistochemical study demonstrated that GAG alpha immunoreactivity was detectable from neonatal periods to adulthood, whereas GAG beta immunoreactivity completely disappeared within 3 weeks of birth. In the adult brain, GAG alpha immunoreactivity was seen in the white matter regions and was also localized in the gray matter including somata and dendrites of cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells. In contrast, GAG alpha immunoreactivity was not localized on parvalbumin-positive interneurons and cerebellar stellate cells. Furthermore, GAG alpha immunoreactivity was not co-localized with perineuronal net markers such as Wisteria floribunda agglutinin lectin and phosphacan. Thus, versican was localized on large projection neurons rather than small interneurons. To confirm the binding mechanism of versican to neurons, hyaluronan and chondroitin sulfates were enzymatically removed from brain sections before the immunolabeling of versican. These treatments had no effect on the labeling pattern of versican, suggesting that other versican-interactive molecules are involved in the binding of versican to neurons.

The cadmium binding domains in the metallothionein isoform Cd7-MT10 from Mytilus galloprovincialis revealed by NMR spectroscopy

The metal-thiolate connectivity of recombinant Cd(7)-MT10 metallothionein from the sea mussel Mytilus galloprovincialis has been investigated for the first time by means of multinuclear, multidimensional NMR spectroscopy. The internal backbone dynamics of the protein have been assessed by the analysis of (15)N T (1) and T (2) relaxation times and steady state {(1)H}-(15)N heteronuclear NOEs. The (113)Cd NMR spectrum of mussel MT10 shows unique features, with a remarkably wide dispersion (210 ppm) of (113)Cd NMR signals. The complete assignment of cysteine Halpha and Hbeta proton resonances and the analysis of 2D (113)Cd-(113)Cd COSY and (1)H-(113)Cd HMQC type spectra allowed us to identify a four metal-thiolate cluster (alpha-domain) and a three metal-thiolate cluster (beta-domain), located at the N-terminal and the C-terminal, respectively. With respect to vertebrate MTs, the mussel MT10 displays an inversion of the alpha and beta domains inside the chain, similar to what observed in the echinoderm MT-A. Moreover, unlike the MTs characterized so far, the alpha-domain of mussel Cd(7)-MT10 is of the form M(4)S(12) instead of M(4)S(11), and has a novel topology. The beta-domain has a metal-thiolate binding pattern similar to other vertebrate MTs, but it is conformationally more rigid. This feature is quite unusual for MTs, in which the beta-domain displays a more disordered conformation than the alpha-domain. It is concluded that in mussel Cd(7)-MT10, the spacing of cysteine residues and the plasticity of the protein backbone (due to the high number of glycine residues) increase the adaptability of the protein backbone towards enfolding around the metal-thiolate clusters, resulting in minimal alterations of the ideal tetrahedral geometry around the metal centres.

Molecular characterization and expression analysis of β2-microglobulin in large yellow croaker Pseudosciaena crocea

Beta(2)-microglobulin (beta(2)m), a protein necessary for proper folding, peptide binding, and surface display of class I antigens plays an important role in immune response. The full-length cDNA containing beta(2)m was cloned from the spleen cDNA library of large yellow croaker Pseudosciaena crocea (Pscr-beta ( 2 ) m) by expressed sequence tag (EST) analysis. The Pscr-beta ( 2 ) m is 926 nucleotides (nt) long, including an open reading frame (ORF) of 348 nt encoding a polypeptide of 116 amino acids (aa). The deduced Pscr-beta ( 2 ) m possessed all characteristic domains of beta(2)m in other species, including a 16-aa leader peptide and a typical immunoglobulin (Ig) and major histocompatibility complex protein (MHC) signature YSCRVTH at residues 81-87. Homology modeling showed that the 3D structure of Pscr-beta ( 2 ) m protein is similar to that of human beta(2)m, except for a beta-strand (G) being lost in Pscr-beta ( 2 ) m due to amino acid deletion (positions 94-95). Tissue expression profile analysis revealed that the Pscr-beta ( 2 ) m was constitutively expressed in all tissues examined, such as kidney, spleen, liver, gills, heart, intestine, brain, and muscle, although at different levels. Upon stimulation with poly(I:C) or inactivated trivalent bacterial vaccine, the expression of Pscr-beta ( 2 ) m was significantly up-regulated in intestine, kidney and spleen at 24 h post-induction, and increase of Pscr-beta ( 2 ) m transcripts was also observed in liver post-induction with poly(I:C). Real-time PCR further revealed that the expression of Pscr-beta ( 2 ) m in intestine, kidney and spleen tissues was differentially regulated by poly(I:C) and bacterial vaccine during 72 h of induction. These results suggested that Pscr-beta ( 2 ) m might be involved in both antiviral and antibacterial mechanisms in large yellow croaker.

The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway

The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.

Ovine progressive pneumonia provirus levels associate with breed and Ovar-DRB1

Previous studies initiated defining the role of host genetics in influencing the outcome of exposure to ovine progressive pneumonia virus. However, specific genes influencing host control of virus replication and disease progression have not been identified. This study, using 383 ewes of the Columbia, Polypay, and Rambouillet breeds, tested the hypothesis that host control of OPPV as measured by provirus levels in the peripheral blood associates with certain breeds and MHC class II Ovis aries (Ovar)-DRB1 expressed alleles. Rambouillet ewes were less likely to have measurable provirus levels as compared to Columbia ewes at ages 5 and 6 (P value < 0.02), and they exhibited lower provirus levels when compared to both Columbia and Polypay ewes of the same ages (P value < 0.05). The presence of DRB1*0403- or DRB1*07012-expressed alleles were significantly associated (P value = 0.019 and 0.0002, respectively) with lower OPP provirus levels but only were only found in 11% of the ewe flock. Analysis of each segregating amino acid in the beta1 domain of DR beta-chain revealed that amino acids Y31, T32, N37, T51, Q60, or N74 significantly associated (P value range = 0.0003-0.018) with lower OPP provirus levels, whereas amino acids H32, A38, or I67 associated (P value range = 0.013-0.043) with higher OPP provirus levels. These results suggest that Ovar-DRB1 contributes as one host genetic factor that controls OPP provirus levels, but does not fully account for the breed-specific OPP proviral differences.

Beta2-glycoprotein I inhibits vascular endothelial growth factor and basic fibroblast growth factor induced angiogenesis through its amino terminal domain.

Beta-2 glycoprotein I (beta(2)GPI) is a plasma glycoprotein which interacts with various proteins of the coagulation and fibrinolysis system. beta(2)GPI has recently been shown to have anti-angiogenic properties. We undertook this study to investigate the specific domain of beta(2)GPI involved in the anti-angiogenic function and its effect on downstream signaling of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF). Various preparations of beta(2)GPI were used on human umbilical vein endothelial cells (HUVECs) in the absence or presence of VEGF and bFGF. The effect on HUVECs' proliferation, migration and tubule formation in Matrigel matrix was investigated. The effect of beta(2)GPI on the mRNA expression of VEGF receptors and phosphorylation of signaling molecules was also studied. beta(2)GPI is shown in this study to be an anti-angiogenic molecule in vitro by inhibiting VEGF and bFGF-induced proliferation, migration and papillary-like tubule formation of HUVECs. This inhibition was achieved by native, proteolytically clipped and domain deletion mutants, domain I-IV (DI-IV) but not domain II-V (DII-V) of beta(2)GPI. Native beta(2)GPI was found to downregulate the expression of the VEGF receptor KDR/Flk-1 on endothelial cells and to block the phosphorylation of VEGF's downstream effector molecules in the MAPK/ERK and PI3K/Akt/GSK3beta pathways. These results indicate that beta(2)GPI has anti-angiogenic functions which depend on the presence of domain I. This anti-angiogenic activity may have important implications for the therapeutic manipulation of angiogenesis in various disease states.

Polymorphism of Alternative Splicing of Major Histocompatibility Complex Transcripts in Wild Tiger Salamanders

Alternative splicing (AS) of mRNA transcripts is increasingly recognized as a source of transcriptome diversity. To date, most AS studies have focused either on comparisons across taxa or on intragenomic comparisons across gene families. We generated a novel data set that represents one of the first population genetic comparisons of AS across individuals. In ambystomatid salamanders, AS of the major histocompatibility complex (MHC) class IIbeta gene (Amti-DAB) produces two transcripts, one full-length and one truncated. The full-length transcript is functional, but the truncated transcript is missing the critical beta1 domain that forms half of the peptide binding region in the intact MHC class II molecule. We captured wild salamander larvae (Ambystoma tigrinum tigrinum) and genotyped them at Amti-DAB via DNA sequencing. From these same larvae, we extracted RNA from gill and spleen and evaluated the relative expression level of Amti-DAB in each tissue. Across individuals, 21% of the transcripts were truncated (alternatively spliced), and the absolute level of alternative transcript expression was higher in gill. The high level of nucleotide variation among seven Amti-DAB alleles provides the ability to detect substitutions (or linked DNA polymorphisms) that might have influenced AS. The data reveal no correlation between AS and haplotype, allele, or zygosity. However, indirect evidence (comparative expression patterns across 3 million years of evolution) suggests that the truncated Amti-DAB transcript may be functional and maintained by natural selection.

Myasthenia Gravis and the Tops and Bottoms of AChRs

The main immunogenic region (MIR), against which half or more of the autoantibodies to acetylcholine receptors (AChRs) in myasthenia gravis (MG) or experimental autoimmune MG (EAMG) are directed, is located at the extracellular end of alpha1 subunits. Rat monoclonal antibodies (mAbs) to the MIR efficiently compete with MG patient autoantibodies for binding to human muscle AChRs. Antibodies bound to the MIR do not interfere with cholinergic ligand binding or AChR function, but target complement and trigger antigenic modulation. Rat mAbs to the MIR also bind to human ganglionic AChR alpha3 subunits, but MG patient antibodies do not. By making chimeras of alpha1 subunits with alpha7 subunits or ACh binding protein, the structure of the MIR and its functional effects are being investigated. Many mAbs to the MIR bind only to the native conformation of alpha1 subunits because they bind to sequences that are adjacent only in the native structure. The MIR epitopes recognized by these mAbs are not recognized by most patient antibodies whose epitopes must be nearby. The presence of the MIR epitopes in alpha1/alpha7 chimeras greatly promotes AChR expression and sensitivity to activation. EAMG can be suppressed by treatment with denatured, bacterially expressed mixtures of extracellular and cytoplasmic domains of human alpha1, beta1, gamma, delta, and epsilon subunits. A mixture of only the cytoplasmic domains not only avoids the potential liability of provoking formation antibodies to pathologically significant epitopes on the extracellular surface, but also potently suppresses the development of EAMG.