Crystal structure of the ectodomain of Methuselah, a Drosophila G protein-coupled receptor associated with extended lifespan.

The Drosophila mutant methuselah (mth) was identified from a screen for single gene mutations that extended average lifespan. Mth mutants have a 35% increase in average lifespan and increased resistance to several forms of stress, including heat, starvation, and oxidative damage. The protein affected by this mutation is related to G protein-coupled receptors of the secretin receptor family. Mth, like secretin receptor family members, has a large N-terminal ectodomain, which may constitute the ligand binding site. Here we report the 2.3-A resolution crystal structure of the Mth extracellular region, revealing a folding topology in which three primarily beta-structure-containing domains meet to form a shallow interdomain groove containing a solvent-exposed tryptophan that may represent a ligand binding site. The Mth structure is analyzed in relation to predicted Mth homologs and potential ligand binding features.

Mutational analysis of the transferrin receptor reveals overlapping HFE and transferrin binding sites.

The transferrin receptor (TfR) binds two proteins critical for iron metabolism: transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. Previous results demonstrated that Tf and HFE compete for binding to TfR, suggesting that Tf and HFE bind to the same or an overlapping site on TfR. TfR is a homodimer that binds one Tf per polypeptide chain (2:2, TfR/Tf stoichiometry), whereas both 2:1 and 2:2 TfR/HFE stoichiometries have been observed. In order to more fully characterize the interaction between HFE and TfR, we determined the binding stoichiometry using equilibrium gel-filtration and analytical ultracentrifugation. Both techniques indicate that a 2:2 TfR/HFE complex can form at submicromolar concentrations in solution, consistent with the hypothesis that HFE competes for Tf binding to TfR by blocking the Tf binding site rather than by exerting an allosteric effect. To determine whether the Tf and HFE binding sites on TfR overlap, residues at the HFE binding site on TfR were identified from the 2.8 A resolution HFE-TfR co-crystal structure, then mutated and tested for their effects on HFE and Tf binding. The binding affinities of soluble TfR mutants for HFE and Tf were determined using a surface plasmon resonance assay. Substitutions of five TfR residues at the HFE binding site (L619A, R629A, Y643A, G647A and F650Q) resulted in significant reductions in Tf binding affinity. The findings that both HFE and Tf form 2:2 complexes with TfR and that mutations at the HFE binding site affect Tf binding support a model in which HFE and Tf compete for overlapping binding sites on TfR.

Crystal structure of the hemochromatosis protein HFE and characterization of its interaction with transferrin receptor.

HFE is an MHC-related protein that is mutated in the iron-overload disease hereditary hemochromatosis. HFE binds to transferrin receptor (TfR) and reduces its affinity for iron-loaded transferrin, implicating HFE in iron metabolism. The 2.6 A crystal structure of HFE reveals the locations of hemochromatosis mutations and a patch of histidines that could be involved in pH-dependent interactions. We also demonstrate that soluble TfR and HFE bind tightly at the basic pH of the cell surface, but not at the acidic pH of intracellular vesicles. TfR:HFE stoichiometry (2:1) differs from TfR:transferrin stoichiometry (2:2), implying a different mode of binding for HFE and transferrin to TfR, consistent with our demonstration that HFE, transferrin, and TfR form a ternary complex.

Approaches to the study of Rox1 repression of the hypoxic genes in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a facultative aerobe that responds to changes in oxygen tension by changing patterns of gene expression. One set of genes that responds to this environmental cue is the hypoxic genes. Oxygen levels are sensed by changes in heme biosynthesis, which controls the transcription of the ROX1 gene, encoding a protein that binds to the regulatory region of each hypoxic gene to repress transcription. Several experimental molecular and genetic approaches are described here to study Rox1 repression. Derepression of the hypoxic genes is rapid, and one model for such a response requires that Rox1 have a short half-life. This was demonstrated to be the case by immunoblotting using a c-myc epitope-tagged protein. Rox1 repression is mediated through the general repressors Ssn6 and Tup1. To explore possible interactions among these proteins, all three were expressed and partially purified using a baculovirus expression system and histidine-tagged proteins. The effect of Ssn6 and Tup1 on the formation of Rox1-DNA complexes was explored using these purified proteins by both electrophoretic mobility shift and DNase I protection assays. We found that Rox1 DNA-binding activity decayed rapidly and that Ssn6 could stabilize and restore lost activity. Finally, genetic selections are described for the isolation of loss-of-function mutations in Rox1. Also, schemes are proposed for the reversion of such mutations. These selections have been extended to genetic analyses of the TUP1 and SSN6 genes.

A proposed structural model of domain 1 of fasciclin III neural cell adhesion protein based on an inverse folding algorithm.

Fasciclin III is an integral membrane protein expressed on a subset of axons in the developing Drosophila nervous system. It consists of an intracellular domain, a transmembrane region, and an extracellular region composed of three domains, each predicted to form an immunoglobulin-like fold. The most N-terminal of these domains is expected to be important in mediating cell-cell recognition events during nervous system development. To learn more about the structure/function relationships in this cellular recognition molecule, a model structure of this domain was built. A sequence-to-structure alignment algorithm was used to align the protein sequence of the fasciclin III first domain to the immunoglobulin McPC603 structure. Based on this alignment, a model of the domain was built using standard homology modeling techniques. Side-chain conformations were automatically modeled using a rotamer search algorithm and the model was minimized to relax atomic overlaps. The resulting model is compact and has chemical characteristics consistent with related globular protein structures. This model is a de novo test of the sequence-to-structure alignment algorithm and is currently being used as the basis for mutagenesis experiments to discern the parts of the fasciclin III protein that are necessary for homophilic molecular recognition in the developing Drosophila nervous system.

Expression and crystallization of a soluble form of Drosophila fasciclin III.

A truncated form of Drosophila fasciclin III has been engineered by site-directed mutagenesis. Secreted fasciclin III is expressed at 35 to 40 mg/l in insect cells with baculovirus carrying the recombinant gene. Single crystals of purified soluble fasciclin III have been grown by vapor diffusion versus polyethylene glycol 8000/sodium citrate at low pH. The space group is P6(1)22 or its enantiomorph P6(5)22, with unit cell dimensions a = b = 140 A, c = 260 A. Cryo-preserved crystals diffract to reciprocal lattice spacings beyond 3.0 A.

Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting.

Fasciclin I is a membrane-associated glycoprotein that is regionally expressed on a subset of fasciculating axons during neuronal development in insects; it is expressed on apposing cell surfaces, suggesting a role in specific cell adhesion. In this paper we show that Drosophila fasciclin I is a novel homophilic cell adhesion molecule. When the nonadhesive Drosophila S2 cells are transfected with the fasciclin I cDNA, they form aggregates that are blocked by antisera against fasciclin I. When cells expressing fasciclin I are mixed with cells expressing fasciclin III, another Drosophila homophilic adhesion molecule, the mixture sorts into aggregates homogeneous for either fasciclin I- or fasciclin III-expressing cells. The ability of these two novel adhesion molecules to mediate cell sorting in vitro suggests that they might play a similar role during neuronal development.

Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1.

Drosophila neuroglian is an integral membrane glycoprotein that is expressed on a variety of cell types in the Drosophila embryo, including expression on a large subset of glial and neuronal cell bodies in the central and peripheral nervous systems and on the fasciculating axons that extend along them. Neuroglian cDNA clones were isolated by expression cloning. cDNA sequence analysis reveals that neuroglian is a member of the immunoglobulin superfamily. The extracellular portion of the protein consists of six immunoglobulin C2-type domains followed by five fibronectin type III domains. Neuroglian is closely related to the immunoglobulin-like vertebrate neural adhesion molecules and, among them, shows most extensive homology to mouse L1. Its homology to L1 and its embryonic localization suggest that neuroglian may play a role in neural and glial cell adhesion in the developing Drosophila embryo. We report here on the identification of a lethal mutation in the neuroglian gene.

Fasciclin III: a novel homophilic adhesion molecule in Drosophila.

Drosophila fasciclin III is an integral membrane glycoprotein that is expressed on a subset of neurons and fasciculating axons in the developing CNS, as well as in several other tissues during development. Here we report on the isolation of a full-length cDNA encoding an 80 kd form of fasciclin III. We have used this cDNA, under heat shock control, to transfect the relatively nonadhesive Drosophila S2 cell line. Examination of these transfected cells indicates that fasciclin III is capable of mediating adhesion in a homophilic, Ca2+-independent manner. Sequence analysis reveals that fasciclin III encodes a transmembrane protein with no significant homology to any known protein, including the previously characterized families of vertebrate cell adhesion molecules. The distribution of this adhesion molecule on subsets of fasciculating axons and growth cones during Drosophila development suggests that fasciclin III plays a role in growth cone guidance.

Characterization and cloning of fasciclin I and fasciclin II glycoproteins in the grasshopper.

Monoclonal antibodies were previously used to identify two glycoproteins, called fasciclin I and II (70 and 95 kDa, respectively), which are expressed on different subsets of axon fascicles in the grasshopper (Schistocerca americana) embryo. Here the monoclonal antibodies were used to purify these two membrane-associated glycoproteins for further characterization. Fasciclin II appears to be an integral membrane protein, whereas fasciclin I is an extrinsic membrane protein. The amino acid sequences of the amino terminus and fragments of both proteins were determined. Using synthetic oligonucleotide probes and antibody screening, we isolated genomic and cDNA clones. Partial DNA sequences of these clones indicate that they encode fasciclins I and II.

Neural-specific carbohydrate moiety shared by many surface glycoproteins in Drosophila and grasshopper embryos.

Antiserum against horseradish peroxidase (anti-HRP Ab) labels the surfaces of neurons in both Drosophila and grasshopper (Jan and Jan, 1982). Here we show that the anti-HRP Ab (1) immunoprecipitates at least 17 different membrane glycoproteins from the Drosophila embryo CNS (and a similar array from grasshopper), and (2) recognizes a neural-specific carbohydrate moiety expressed by most if not all of these proteins. Although the anti-HRP Ab stains all axon pathways, 2 of the anti-HRP glycoproteins, fasciclin I and II, are expressed on specific subsets of axon pathways in the grasshopper embryo.

Expression of fasciclin I and II glycoproteins on subsets of axon pathways during neuronal development in the grasshopper.

The "labeled pathways" hypothesis predicts that axon fascicles in the embryonic neuropil are differentially labeled by surface recognition molecules used for growth cone guidance. To identify candidates for such recognition molecules, we generated monoclonal antibodies (MAbs) that recognize surface antigens expressed on subsets of axon fascicles in the grasshopper embryo. The 3B11 and 8C6 MAbs immunoprecipitate 70- and 95-kd membrane glycoproteins called fasciclin I and II, respectively, which are expressed on different subsets of axon fascicles during development. These two glycoproteins are expressed regionally on particular portions of embryonic axons in correlation with their patterns of fasciculation, dynamically during the period of axon outgrowth in a manner consistent with a role in growth cone guidance, and at other times and places during embryogenesis, suggesting multiple developmental roles.

Characterization and cloning of fasciclin III: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophila.

To identify candidates for neuronal recognition molecules in Drosophila, we used monoclonal antibodies to search for surface glycoproteins expressed on subsets of axon bundles (or fascicles) during development. Here we report on the characterization and cloning of fasciclin III, which is expressed on a subset of neurons and axon pathways in the Drosophila embryo. Fasciclin III is also expressed at other times and places including transient segmentally repeated patches in the neuroepithelium and segmentally repeated stripes in the body epidermis. Antisera generated against each of four highly related forms of the protein were used for cDNA expression cloning to identify a single gene, which was confirmed to encode fasciclin III by tissue in situ hybridization and genetic deficiency analysis.

Association between the human thymic differentiation antigens T6 and T8.

The human T lymphocyte cell surface antigen T8 has been found to be associated on thymocytes with a protein of 46 kDa through disulfide bridges. Analysis by isoelectric focusing, and of peptides obtained by limited proteolysis and chemical cleavage demonstrated that this 46-kDa protein was the cell surface antigen T6, which is expressed on cortical thymocytes. The findings are discussed in the context of the importance of the T8 and T6 molecules in thymic differentiation.

Analysis of the structure of the human T cell surface antigen T8 by limited proteolysis and chemical cleavage.

The structure of the human T lymphocyte surface antigen T8 (Leu 2) has been explored utilizing limited proteolysis on viable cells and cellular lysates. The positions of the cleavage sites of trypsin and papain were placed relative to the single CNBr cleavage point. Additional data allowed the location of the amino and carboxyl termini relative to the enzymatic and chemical cleavage sites. This information, together with earlier evidence concerning the position of a membrane binding site, allowed the construction of a model illustrating the vectorial orientation of the molecule on the cell. Within this model, the approximate positions of disulfide linkages were indicated based on the results of nonreduced/reduced two-dimensional sodium sulfide-polyacrylamide gel electrophoresis. Carbohydrate moieties were localized using cleavage with trifluoromethanesulfonic acid, a reagent which cleaves both N-linked and O-linked oligosaccharides. Finally, the implications of the proteolysis experiments in relation to the function of T8 were discussed.

Purification and N-terminal amino acid sequence of the human T cell surface antigen T8.

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic T lymphocytes (CTL) that are involved in their effector function. Among these cell surface components, T8 is of particular interest because it is required during the recognition of target cells by a subset of CTL. An understanding of its role during CTL:target adhesion requires detailed biochemical structural analysis of the T8 molecule. This has been hindered by the small amounts of protein currently available. Here we describe the development of a purification scheme that will permit the accumulation of larger quantities of T8. We studied the binding of T8 to several lectins and determined that one of these, wheat germ agglutinin, bound T8 quantitatively. Experiments designed to test the properties of T8 in a phase separation system with the use of Triton X-114 were performed. These indicated that T8 partitions into the aqueous phase rather than the detergent phase during this procedure. With this in mind, we developed a protocol that resulted in a significant purification of T8 after affinity chromatography. Upon preparative SDS-PAGE followed by electroelution, the T8 antigen was purified to homogeneity and used for N-terminal acid acid sequencing. This analysis yielded the amino terminal 22 amino acids of T8. On purification, it was observed that the protein existed as two bands of Mr 33 and 34 kilodaltons after SDS-PAGE analysis. The relationship between these chains was investigated by limited radio-sequencing and tryptic peptide map analysis; our results indicated that the two polypeptides were identical. The two chains were treated with trifluoromethane sulfonic acid to determine whether carbohydrates accounted for the difference in m.w. This reagent, which cleaves both N-linked and O-linked sugars, cleaved approximately 2000 daltons of oligosaccharides from the T8 molecule. These oligosaccharides are most likely of the O-linked rather than the N-linked variety.

The T8 antigen is a multimeric complex of two distinct subunits on human thymocytes but consists of homomultimeric forms on peripheral blood T lymphocytes.

Monoclonal antibodies allow for the detection of structures on the cell surface of human cytotoxic thymus-derived lymphocytes, which are involved in their function. Among these cell surface components, T8 is of particular interest, since it is required during the recognition of target cells by a subset of human cytotoxic T lymphocytes. The only other cell types on which T8 has been detected are functionally inert thymocytes. Here we demonstrate that T8 isolated from peripheral blood T lymphocytes is different from thymocyte T8. On peripheral blood T lymphocytes T8 was found in multimeric forms of a 34-kDa glycoprotein. These forms were also found on thymocytes, but in addition multimeric complexes of the 34-kDa T8 with a 46-kDa protein were detected. Preliminary chemical analyses showed that the 34-kDa and the 46-kDa forms differ in both their protein and carbohydrate structures. The 46-kDa structure was found to be distinct from the major thymocyte antigen T10. The interchain disulfide bridges between the 34-kDa T8 polypeptide are located in the 24-kDa CNBr fragment, which contains a hydrophobic region. Two (14 kDa and 20 kDa) of the three CNBr fragments of the 46-kDa T8 subunit were found to be involved in interchain disulfide bridging with the 34-kDa T8 species. We suggest that the switch from heteromultimers to homomultimers may occur concomitantly with the last step in T lymphocyte maturation.