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Biomedical subjects

M Yeager

Publications and source records attributed to M Yeager.

At least 55 records · Page 3Linked to original sources

Molecular evolution of the vertebrate immune system.

Adaptive immunity is unique to the vertebrates, and the molecules involved (including immunoglobulins, T cell receptors and the major histocompatibility complex molecules) seem to have diversified very rapidly early in vertebrate history. Reconstruction of gene phylogenies has yielded insights into the evolutionary origin of a number of molecular systems, including the complement system and the major histocompatibility complex (MHC). These analyses have indicated that the C5 component of complement arose by gene duplication prior to the divergence of C3 and C4, which suggests that the alternative complement pathway was the first to evolve. In the case of the MHC, phylogenetic analysis supports the hypothesis that MHC class II molecules evolved before class I molecules. The fact that the MHC-linked proteasome components that specifically produce peptides for presentation by class I MHC appear to have originated before the separation of jawed and jawless vertebrates suggests that the MHC itself may have been present at this time. Immune system gene families have evolved by gene duplication, interlocus recombination and (in some cases) positive Darwinian selection favoring diversity at the amino acid level.

Animals↗

Coordinated amino acid changes in the evolution of mammalian defensins.

The mammalian defensin molecule is a short, highly cationic peptide cytotoxic to both microbial and mammalian cells which is cleaved from a precursor including a signal peptide and a highly anionic propiece. A phylogenetic analysis of 28 complete sequences from five mammalian species (mouse, rat, guinea pig, rabbit, and human) showed species-specific clusters of sequences, indicating that the genes duplicated after divergence of these species. Comparison of rates of synonymous and nonsynonymous nucleotide substitution suggested that gene duplication has often been followed by a period in which diversification of the mature defensins at the amino acid level has been selectively favored. In some comparisons, it appeared that amino acid differences in this region have appeared in a nonrandom fashion so as to change the pattern of residue charges. Because it has been hypothesized that the negative charge in the propiece serves to balance the positive charge in the mature defensin and thus to prevent cytotoxicity prior to cleavage, we used a maximum likelihood method of reconstructing ancestral states in order to test whether this balance has been maintained over evolutionary time in spite of rapid diversification of the mature defensin at the amino acid level. Reconstructed ancestral sequences always maintained a charge balance between mature defensin and propiece, and changes in the net positive charge of the mature defensin were balanced by corresponding changes in the propiece. The results support the hypothesis that, in the evolution of these proteins, amino acid changes have occurred in a coordinated fashion so as to preserve an adaptive phenotype.

Amino Acid Sequence↗

Comparative evolutionary rates of introns and exons in murine rodents.

Analysis of DNA sequences of 132 introns and 140 exons from 42 pairs of orthologous genes of mouse and rat was used to compare patterns of evolutionary change between introns and exons. The mean of the absolute difference in length (measured in base pairs) between the two species was nearly five times as high in the case of introns as in the case of exons. The average rate of nucleotide substitution in introns was very similar to the rate of synonymous substitution in exons, and both were about three times the rate of substitution at nonsynonymous sites in exons. G+C content of introns and exons of the same gene were correlated; but mean G+C content at the third positions of exons was significantly higher than that of introns or positions 1-2 of exons from the same gene. G+C content was conserved over evolutionary time, as indicated by strong correlations between mouse and rat; but the change in G+C content was greatest at position 3 of exons, intermediate in introns, and lowest at positions 1-2 in introns.

Animals↗

Projection structure of a gap junction membrane channel at 7 A resolution.

Electron cryo-microscopy and image analysis of frozen-hydrated, two-dimensional crystals of gap junction membrane channels formed by recombinant alpha 1 connexin (Cx43) reveal a ring of transmembrane alpha-helices that lines the aqueous pore and a second ring of alpha-helices in close contact with the membrane lipids.

Animals↗

Sequence convergence in the peptide-binding region of primate and rodent MHC class Ib molecules.

In addition to the universally expressed and highly polymorphic class Ia genes, the major histocompatibility complex (MHC) of placental mammals includes class Ib genes that are characterized by restricted expression and low levels of sequence polymorphism. The functional importance of class Ib genes as well as their actual function has long been controversial. Phylogenetic analyses have suggested that there are no orthologous relationships among class Ib loci of mammals belonging to different orders, suggesting that these loci have evolved independently since the placental mammals diverged. Here, we present evidence of convergent evolution at the molecular sequence level in the putative peptide-binding regions (PBRs) of human and mouse class Ib genes. So far, there are few if any convincing examples of convergent evolution at the amino acid sequence level, and such evolution is believed to be likely to occur only as a result of strong positive selection. Because the present case involves the functionally important PBR and because the primate and rodent molecules are known to bind similar peptides, this study represents both a convincing case of molecular-level convergence and evidence that MHC class Ib molecules, although not orthologous, may evolve similar functions convergently.

Amino Acid Sequence↗

The cytoplasmic tail of NSP4, the endoplasmic reticulum-localized non-structural glycoprotein of rotavirus, contains distinct virus binding and coiled coil domains.

The final steps in the assembly of rotavirus occur in the lumen of the endoplasmic reticulum (ER). Targeting of the immature inner capsid particle (ICP) to this compartment is mediated by the cytoplasmic tail of NSP4, a non-structural virus glycoprotein located in the ER membrane. To delineate structural and functional features of NSP4, soluble fragments of the cytoplasmic tail have been expressed and purified. Our analysis combines a functional assay for ICP binding with biochemical and CD spectroscopic studies to examine the secondary and quaternary structure. The ICP-binding domain is located within the C-terminal 20 amino acids of the polypeptide. A second region, distinct from this receptor domain, adopts an alpha-helical coiled coil structure and mediates the oligomerization of the virus binding domains into a homotetramer. The domain organization of the cytoplasmic fragments of NSP4 suggests a novel structure for an icosahedral virus receptor protein in which C-terminal binding sites for immature rotavirus particles are connected to an alpha-helical coiled coil stalk which projects from the ER membrane.

Biopolymers↗

Structure of gap junction intercellular channels.

Gap junctions are formed by a multigene family of polytopic membrane channel proteins, connexins, that have four hydrophobic transmembrane domains and their N and C termini located on the cytoplasmic membrane face. The C-terminal tail plays important roles in channel regulation by pH and phosphorylation. Conserved cysteine residues stabilize the conformation of the extracellular loops that mediate the 'docking' between connexons in the intercellular channel. Over the past year, electron cryocrystallography of two-dimensional crystals of a truncated recombinant alpha 1 (Cx43) has revealed that the transmembrane boundary of the intercellular channel is lined with alpha helices. Furthermore, a ring of alpha helices resides at the interface with the membrane lipids. A three-dimensional analysis based on images recorded from tilted crystals should reveal the location and secondary structure of additional transmembrane domains, as well as provide important structural details about the interactions between connexins within a hemi-channel and connexon-connexon interactions in the extracellular gap.

Amino Acid Sequence↗

Mass spectrometry and viral analysis.

BACKGROUND: Electrospray ionization (ESI) mass spectrometry is a powerful new approach for analyzing biomolecules and biomolecular complexes. Previous studies have provided evidence that non-covalent biomolecular complexes can be observed by ESI mass spectrometry; it is not clear, however, whether the native conformation of the biomolecules is maintained throughout the ionization and analysis process. We set out to address this question using live viruses. RESULTS: Viral ions have been generated in the gas phase using electrospray ionization mass spectrometry. These ions have been collected, following ion filtering through the mass analyzer, and then analyzed by transmission electron microscopy. Transmission electron microscopy revealed that rice yellow mottle virus and tobacco mosaic virus retained their respective spherical and rod-like ultrastructure. The viability of the isolated tobacco mosaic virus was confirmed by inoculation and infection of tobacco plants. CONCLUSIONS: These results demonstrate the utility of electrospray for supramolecular complexes with molecular weights of over 40 million Da and offer conclusive evidence that native biomolecular structures can be conserved through the electrospray process.

Mass Spectrometry↗

Peptide binding function and the paradox of HLA disease associations.

Many studies have found associations between HLA loci and susceptibility or resistance to both infectious and autoimmune diseases, but often subsequent studies fail to find the same association. Such inconsistencies are not surprising if we consider what is known of the population biology and evolution of HLA genes, especially the consequences of natural selection favouring heterozygosity in the peptide binding regions (PBR) of HLA molecules. Because of past recombination event and/or convergent evolution, alleles that are not closely related in overall sequence may come to resemble each other in the PBR. Because peptide binding is likely to be important for the role of HLA molecules in both infectious and autoimmune disease, a strategy of searching for HLA disease associations that groups alleles in functional categories based on PBR motifs may prove more successful than conventional strategies. Likewise, in developing approaches for molecular typing, it may be advisable to develop methods that group alleles on the basis of shared PBR motifs rather than simply on the basis of shared primer sites in less functionally important regions.

Animals↗

Interallelic recombination has not played a major role in the history of the HLA-C locus.

The human major histocompatibility complex class I loci HLA-A and HLA-B have previously been shown to differ with regard to the rate of interallelic recombination, with a much higher rate being observed in the case of HLA-B. Until recently, there were very few HLA-C transcripts available for analysis. In the present study, we analyzed C locus alleles from human (n = 24), chimpanzee (n = 1), and gorilla (n = 5). Both phylogenetic analysis and a method for detecting clusters of sites indicative of recombination showed relatively little evidence of interallelic recombination. Thus, in spite of the close evolutionary relationship between HLA-C and HLA-B, HLA-C is evolving more similarly to the HLA-A locus than is HLA-B, further emphasizing the uniqueness of HLA-B.

Animals↗

Electron microscopic image analysis of cardiac gap junction membrane crystals.

Cardiac gap junctions play an important functional role in the myocardium by electrically coupling adjacent cells, thereby providing a low resistance pathway for cell-to-cell propagation of the action potential. Two-dimensional crystallization of biochemically isolated rat ventricular gap junctions has been accomplished by an in situ method in which membrane suspensions are sequentially dialyzed against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are partially extracted without solubilizing the protein, and the increased protein concentration facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Projection density maps show that the connexons in cardiac gap junctions are formed by a hexameric cluster of alpha 1 connexin subunits. Protease cleavage of alpha 1 connexin from 43 to 30 kDa releases approximately 13kDa from the carboxy-tail, and the projection density maps are not significantly altered. Uranyl acetate stain penetrates the ion channel, whereas phosphotungstic acid is preferentially deposited over the lipid regions. This differential staining can be used to selectively probe the central channel of the connexon and the interface between the connexon and the lipid. The hexameric design of alpha 1 connexons appears to be a recurring quaternary motif for the multigene family of gap junction proteins.

Amino Acid Sequence↗

Projection structure of the CHIP28 water channel in lipid bilayer membranes at 12-A resolution.

Osmotic water transport across plasma membranes in erythrocytes and several epithelial cell types is facilitated by CHIP28, a water-selective membrane channel protein. In order to examine the structure of CHIP28 in membranes, large (1.5-2.5-microns diameter), highly ordered, two-dimensional (2-D) crystals of purified and deglycosylated erythrocyte CHIP28 were generated by reconstitution of detergent-solubilized protein into synthetic lipid bilayers via detergent dialysis. Fourier transforms computed from low-dose electron micrographs of such crystals preserved in negative stain display order to 12-A resolution. The crystal lattice is tetragonal (a = b = 99.2 +/- 1.4 A) with plane group symmetry p4g. A projection density map at 12-A resolution defines the molecular boundary and organization of the CHIP28 monomers in the membrane plane. The unit cell contains four CHIP28 dimers, each composed of two oblong-shaped (37 x 25 A ) monomers with opposite orientations. The CHIP28 monomers associate to form tetrameric structures around the 4-fold axes normal to the membrane plane where stain is excluded. The 2-D crystals of CHIP28 display order extending beyond the limit typically achieved by negative staining and therefore may be amenable to high-resolution structure analysis by cryo-electron microscopy.

Aquaporin 1↗

In situ two-dimensional crystallization of a polytopic membrane protein: the cardiac gap junction channel.

In situ crystallization of rat ventricular gap junctions was accomplished by sequential dialysis of membranes against low concentrations of deoxycholate and dodecyl-beta-D-maltoside. Lipids are removed without solubilizing the protein, and the increased protein concentration in the membrane plane facilitates two-dimensional crystallization in the native membrane environment. The two-dimensional crystals have a nominal resolution of 16 A and display plane group symmetry p6 with a = b = 85 A and gamma = 120 degrees. Electron crystallography reveals that the cardiac gap junction membrane channel is formed by a hexameric cluster of protein subunits, and this hexameric design appears to be a recurring quaternary motif for the multigene family of gap junction proteins. Exposure of membranes to low concentrations of detergents may provide an approach for in situ two-dimensional crystallization of other connexins as well as other membrane proteins, especially those that are labile when solubilized as protein-detergent micelles.

Journal Article↗

Three-dimensional structure of the rotavirus haemagglutinin VP4 by cryo-electron microscopy and difference map analysis.

The three-dimensional structure of the rotavirus spike haemagglutinin viral protein 4 (VP4) has been determined to a resolution of 26 A by cryo-electron microscopy and difference analysis of intact virions and smooth (spikeless) particles. Native and spikeless virions were mixed prior to cryo-preservation so that both structures could be determined from the same micrograph, thereby minimizing systematic errors. This mixing strategy was crucial for difference map analysis since VP4 only accounts for approximately 1% of the virion mass. The VP4 spike is multi-domained and has a radial length of approximately 200 A with approximately 110 A projecting from the surface of the virus. Interactions between VP4 and cell surface receptors are facilitated by the bi-lobed head, which allows multi-site interactions, as well as the uniform distribution of the VP4 heads at maximum radius. The bi-lobed head is attached to a square-shaped body formed by two rods that have a slight left-handed helical twist. These rods merge with an angled, rod-like domain connected to a globular base approximately 85 A in diameter. The anchoring base displays pseudo 6-fold symmetry. This surprising finding may represent a novel folding motif in which a single polypeptide of VP4 contributes similar but non-equivalent domains to form the arms of the hexameric base. The VP4 spike penetrates the virion surface approximately 90 A and interacts with both outer (VP7) and inner (VP6) capsid proteins. The extensive VP4-VP7 and VP4-VP6 interactions imply a scaffolding function in which VP4 may participate in maintaining precise geometric register between the inner and outer capsids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Early steps in reovirus infection are associated with dramatic changes in supramolecular structure and protein conformation: analysis of virions and subviral particles by cryoelectron microscopy and image reconstruction.

Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (approximately 800-A diam). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 A that is formed by 60 tetrameric and 60 hexameric clusters of ellipsoidal subunits of sigma 3. The vertex-associated cell attachment protein, sigma 1, also undergoes a striking change from a poorly visualized, more compact form, to an extended, flexible fiber. This conformational change may maximize interactions of sigma 1 with cell surface receptors. Transcription of viral mRNAs is mediated by the core particle (approximately 600-A diam), generated from the ISVP after penetration and uncoating. The transition from ISVP to core involves release of the 12 sigma 1 fibers and the remaining outer capsid layer formed by 200 trimers of rod-shaped mu 1 subunits that span radii from 306 to 395 A. In the virion and ISVP, flower-shaped pentamers of the lambda 2 protein are centered at the vertices. In the ISVP-to-core transition, domains of the lambda 2 subunits rotate and swing upward and outward to form a turret-like structure extending from radii 305 to 400 A, with a diameter of 184 A, and a central channel 84 A wide. This novel conformational change allows the potential diffusion of substrates for transcription and exit of newly synthesized mRNA segments.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

Demonstration of a second ligand for the low affinity receptor for immunoglobulin E (CD23) using recombinant CD23 reconstituted into fluorescent liposomes.

Recombinant full-length human CD23 has been incorporated into fluorescent liposomes to demonstrate the existence of a ligand for CD23 that is different from the previously known ligand, immunoglobulin E (IgE). The novel ligand for CD23 is expressed on subsets of normal T cells and B cells as well as on some myeloma cell lines. The interaction of full-length CD23 with its ligand is specifically inhibited by anti-CD23 monoclonal antibodies and by IgE, and it is Ca2+ dependent. Moreover, tunicamycin treatment of a CD23-binding cell line, RPMI 8226, significantly reduced the binding of CD23 incorporated into fluorescent liposomes, and a sugar, fucose-1-phosphate, was found to inhibit CD23-liposome binding to RPMI 8226 cells, suggesting the contribution of sugar structures on the CD23 ligand. In addition, CD23-transfected COS cells were shown to form specific conjugates with the cell line RPMI 8226. These data demonstrate that CD23 interacts with a ligand, which is different from IgE, and that CD23 can be considered as a new surface adhesion molecule involved in cell-cell interactions.

Antibodies, Monoclonal↗