Drosophila Ten-a is a maternal pair-rule and patterning gene

The Ten-a gene of Drosophila melanogaster encodes several alternative variants of a full length member of the Odz/Tenm protein family. A number of Ten-a mutants created by inexact excisions of a resident P-element insertion are embryonic lethal, but show no pair-rule phenotype. In contrast, these mutants, and deficiencies removing Ten-a, do enhance the segmentation phenotype of a weak allele of the paralog gene odz (or Ten-m) to the odz amorphic phenotype. Germ line clone derived Ten-a(-) embryos display a pair-rule phenotype which phenocopies that of odz. Post segmentation eye patterning phenotypes of Ten-a mutants establish it as a pleiotropic patterning co-partner of odz.     

Drosophila Ten-m and filamin affect motor neuron growth cone guidance

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.     

Teneurins: a novel family of neuronal cell surface proteins in vertebrates, homologous to the Drosophila pair-rule gene product Ten-m

We have characterized chicken teneurin-1 and teneurin-2, two homologues of the Drosophila pair-rule gene product Ten-m and Drosophila Ten-a. The high degree of conservation between the vertebrate and invertebrate proteins suggests that these belong to a novel family. We propose to name the vertebrate members of this family teneurins, because of their predominant expression in the nervous system. The expression of teneurin-1 and -2 was investigated by in situ hybridization. We show that teneurin-1 and -2 are expressed by distinct populations of neurons during the time of axonal growth. The most prominent site of expression of chicken teneurins is the developing visual system. Recombinant teneurin-2 was expressed to assay its molecular and functional properties. We show that it is a type II transmembrane protein, which can be released from the cell surface by proteolytic cleavage at a furin site. The expression of teneurin-2 in neuronal cells led to a significant increase in the number of filopodia and to the formation of enlarged growth cones. The expression pattern of teneurins in the developing nervous system and the ability of teneurin-2 to reorganize the cellular morphology indicate that these proteins may have an important function in the formation of neuronal connections.     

The murine Ten-m/Odz genes show distinct but overlapping expression patterns during development and in adult brain

The mouse TEN-M/ODZ proteins belong to a new family of type II transmembrane proteins with unknown function. The family consists of four members, which are expressed highly in brain and less in many other tissues. In the present study we have generated specific RNA probes and antibodies to characterize the expression of the 4 Ten-m/Odz genes in the developing and adult central nervous system (CNS) of mice. Ten-m/Odz3 and Ten-m/Odz4 mRNAs were first detectable at E7.5, Ten-m/Odz2 expression started at the 37 somite (E 10.5) stage, while Ten-m/Odz1 mRNA is not found before E15.5. In the adult mouse CNS mRNAs of the 4 Ten-m/Odzs were expressed in distinct patterns, which partially overlapped. Immunostaining and in situ hybridization localized proteins and mRNAs of Ten-m/Odzs in adjacent areas suggesting that TEN-M/ODZ proteins might be transported from the cell body along the axon or that they are shed from the cell surface and diffuse into distant regions.     

All four members of the Ten-m/Odz family of transmembrane proteins form dimers

Ten-m/Odz/teneurins are a new family of four distinct type II transmembrane molecules. Their extracellular domains are composed of an array of eight consecutive EGF modules followed by a large globular domain. Two of the eight modules contain only 5 instead of the typical 6 cysteine residues and have the capability to dimerize in a covalent, disulfide-linked fashion. The structural properties of the extracellular domains of all four mouse Ten-m proteins have been analyzed using secreted, recombinant molecules produced by mammalian HEK-293 cells. Electron microscopic analysis supported by analytical ultracentrifugation data revealed that the recombinant extracellular domains of all Ten-m proteins formed homodimers. SDS-PAGE analysis under nonreducing conditions as well as negative staining after partial denaturation of the molecules indicated that the globular COOH-terminal domains of Ten-m1 and -m4 contained subdomains with a pronounced stability against denaturing agents, especially when compared with the homologous domains of Ten-m2 and -m3. Cotransfection experiments of mammalian cells with two different extracellular domains revealed that Ten-m molecules have also the ability to form heterodimers, a property that, combined with alternative splicing events, allows the formation of a multitude of molecules with different characteristics from a limited set of genes.     

Ten-m3 Is Required for the Development of Topography in the Ipsilateral Retinocollicular Pathway

Background

The alignment of ipsilaterally and contralaterally projecting retinal axons that view the same part of visual space is fundamental to binocular vision. While much progress has been made regarding the mechanisms which regulate contralateral topography, very little is known of the mechanisms which regulate the mapping of ipsilateral axons such that they align with their contralateral counterparts.

Results

Using the advantageous model provided by the mouse retinocollicular pathway, we have performed anterograde tracing experiments which demonstrate that ipsilateral retinal axons begin to form terminal zones (TZs) in the superior colliculus (SC), within the first few postnatal days. These appear mature by postnatal day 11. Importantly, TZs formed by ipsilaterally-projecting retinal axons are spatially offset from those of contralaterally-projecting axons arising from the same retinotopic location from the outset. This pattern is consistent with that required for adult visuotopy. We further demonstrate that a member of the Ten-m/Odz/Teneurin family of homophilic transmembrane glycoproteins, Ten-m3, is an essential regulator of ipsilateral retinocollicular topography. Ten-m3 mRNA is expressed in a high-medial to low-lateral gradient in the developing SC. This corresponds topographically with its high-ventral to low-dorsal retinal gradient. In Ten-m3 knockout mice, contralateral ventrotemporal axons appropriately target rostromedial SC, whereas ipsilateral axons exhibit dramatic targeting errors along both the mediolateral and rostrocaudal axes of the SC, with a caudal shift of the primary TZ, as well as the formation of secondary, caudolaterally displaced TZs. In addition to these dramatic ipsilateral-specific mapping errors, both contralateral and ipsilateral retinocollicular TZs exhibit more subtle changes in morphology.

Conclusions

We conclude that important aspects of adult visuotopy are established via the differential sensitivity of ipsilateral and contralateral axons to intrinsic guidance cues. Further, we show that Ten-m3 plays a critical role in this process and is particularly important for the mapping of the ipsilateral retinocollicular pathway.     

Phylogenetic analysis of teneurin genes and comparison to the rearrangement hot spot elements of E. coli

Teneurins are a novel family of transmembrane proteins conserved between invertebrates and vertebrates. There are two members in Drosophila, one in C. elegans and four members in mouse. Here, we describe the analysis of the genomic structure of the human teneurin-1 gene. The entire human teneurin-1 (TEN1) gene is contained in eight PAC clones representing part of the chromosomal locus Xq25. Interestingly, many X-linked mental retardation syndromes (XLMR) and non-specific mental retardation (MRX) are mapped to this region. The location of the human TEN1 together with the neuronal expression makes TEN1 a candidate gene for XLMR and MRX. We also identified large parts of the human teneurin-2 sequence on chromosome 5 and sections of human teneurin-4 at chromosomal position 11q14. Database searches resulted in the identification of ESTs encoding parts of all four human members of the teneurin family. Analysis of the genomic organization of the Drosophila ten-a gene revealed the presence of exons encoding a long form of ten-a, which can be aligned with all other teneurins known. Sequence comparison and phylogenetic trees of teneurins show that insects and vertebrates diverged before the teneurin ancestor was duplicated independently in the two phyla. This is supported by the presence of conserved intron positions between teneurin genes of man, Drosophila and C. elegans. It is therefore not possible to class any of the vertebrate teneurins with either Drosophila Ten-a or Ten-m. The C-terminal part of all teneurins harbours 26 repetitive sequence motifs termed YD-repeats. YD-repeats are most similar to the repeats encoded by the core of the rearrangement hot spot (rhs) elements of Escherichia coli. This makes the teneurin ancestor a candidate gene for the source of the rhs core acquired by horizontal gene transfer.     

ten-a overexpression causes abnormal pattern in the Drosophila compound eye

Ten-a is one of the two Drosophila proteins that belong to the Ten M protein family. This protein is a type Ⅱ transmembrane protein and is expressed mainly in the embryonic CNS, in the larval eye imaginal disc and in the compound eye of the pupa. Here, we investigate the role of ten-α during development of the compound eye by using the Gal4/ UAS system to induce ten-α overexpression in the developing eye. We found that overexpression of ten-α can perturb eye development during all stages examined. In an early stage, overexpression of ten-α in eye primordial cells caused small and rough eyes and interfered with photoreceptor cell recruitment, resulting in some ommatidia having fewer or extra photoreceptor cells. Conversely, ten-α overexpression daring ommatidial formation caused severe eye defects due to absence of many cellular components. Interestingly, overexpression of ten-α in the late stage developing ommatidial cluster affected the number of pigment cells, caused cone cells proliferation in many ommatidia, and caused some photoreceptor cell defects. These results suggest that ten-α may be a novel gene required for normal eye morphogenesis.     

High level expression of a synthetic gene coding for IgG-binding domain B of Staphylococcal protein A

A gene coding for one of the IgG-binding domains of Staphylococcal protein A, designated domain B, was chemically synthesized. This gene was tandemly repeated to give dimeric and tetrameric domain B genes by the use of two restriction enzymes which gave blunt ends. The genes were highly expressed in Escherichia coli to afford a large amount of dimeric and tetrameric domain B proteins. The single domain B protein was efficiently produced as a fusion protein with a salmon growth hormone fragment. The fusion protein was converted to monomeric domain B by cyanogen bromide cleavage. The CD spectra of the monomeric, dimeric and tetrameric domain B proteins were essentially the same as that of native form protein A, showing that their secondary structures were very similar. The dimeric and tetrameric domain B proteins formed precipitates with IgG as protein A. This system permits the efficient production of mutated single and multiple IgG-binding domains which can be used to study structural changes and protein A-immunoglobulin interactions.     

A kinetic and equilibrium study of ligand binding to the monomeric and dimeric haem-containing globins of two chitons.

The radular muscles of the amphineuran molluscs Amaurochiton glaucus and Sipharochiton pelliserpentis contain both a dimeric and a monomeric form of myoglobin. The dimeric form of the protein is composed of two polypeptide chains covalently linked to each other via one or more disulphide bonds. The dimeric protein shows co-operative O2-binding curves. Kinetic investigations indicate that CO binding is co-operative in the dimeric protein, subsequent to full photolysis, but mono-exponential following 10% photolysis. O2 recombination following part photolysis is mono-exponential in the dimeric form, whereas O2 dissociation kinetics indicates the presence of chain heterogeneity. The monomeric form of the protein exhibits mono-exponential time courses in all the experimental situations explored. Although the rate constants associated with the reactions of individual dimer and monomer molecular species are very different, the two species of chiton investigated show remarkably similar properties when compared with each other. All the reactions studied are pH-independent in the range pH 6-8. Amino acid analysis indicates that the monomeric units that combine to form the dimeric species are not identical with the naturally occurring monomeric form. A comparison is made between the chiton myoglobins and other similar O2-binding proteins.     

Relationship between stability of folding intermediates and amyloid formation for the yeast prion Ure2p: a quantitative analysis of the effects of pH and buffer system

The dimeric yeast protein Ure2 shows prion-like behaviour in vivo and forms amyloid fibrils in vitro. A dimeric intermediate is populated transiently during refolding and is apparently stabilized at lower pH, conditions suggested to favour Ure2 fibril formation. Here we present a quantitative analysis of the effect of pH on the thermodynamic stability of Ure2 in Tris and phosphate buffers over a 100-fold protein concentration range. We find that equilibrium denaturation is best described by a three-state model via a dimeric intermediate, even under conditions where the transition appears two-state by multiple structural probes. The free energy for complete unfolding and dissociation of Ure2 is up to 50 kcal mol(-1). Of this, at least 20 kcal mol(-1) is contributed by inter-subunit interactions. Hence the native dimer and dimeric intermediate are significantly more stable than either of their monomeric counterparts. The previously observed kinetic unfolding intermediate is suggested to represent the dissociated native-like monomer. The native state is stabilized with respect to the dimeric intermediate at higher pH and in Tris buffer, without significantly affecting the dissociation equilibrium. The effects of pH, buffer, protein concentration and temperature on the kinetics of amyloid formation were quantified by monitoring thioflavin T fluorescence. The lag time decreases with increasing protein concentration and fibril formation shows pseudo-first order kinetics, consistent with a nucleated assembly mechanism. In Tris buffer the lag time is increased, suggesting that stabilization of the native state disfavours amyloid nucleation.     

A role for the intersubunit disulfides of seminal RNase in the mechanism of its antitumor action.

The dimeric structure of seminal ribonuclease (BS-RNase) is maintained by noncovalent interactions and by two intersubunit disulfide bridges. Another unusual feature of this enzyme is its antitumour action, consisting in a cytotoxic activity selective for malignant cells. This cytotoxic action is exerted when the protein reaches the cytosol of the affected cells, where it degrades ribosomal RNA, thus blocking protein synthesis and leading cells to death. The current model proposed for the mechanism of antitumour action of BS-RNase is based on the ability of the protein to resist the neutralizing action of the cytosolic RNase inhibitor, a resistance due to the dimeric structure of the enzyme. Monomeric RNases, and monomeric derivatives of BS-RNase, are strongly bound by the inhibitor and inactive as antitumor agents. Here we report on monomeric derivatives of BS-RNase that, although strongly inhibited by the cytosolic RNase inhibitor, are cytotoxic towards malignant cells. These monomers are produced by reductive cleavage of the intersubunit disulfides of the native, dimeric protein followed by linking the exposed sulfhydryls to small thiols through formation of mixed disulfides. We found that sulfhydryls from cell monolayers and cell membranes can attack these mixed disulfides in the monomeric derivatives, and reconstitute, through sulfhydryl-disulfide interchange reactions, the native dimeric protein, which is internalized as such, and displays its antitumour action.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

Characterization of monomeric and dimeric B domain of Staphylococcal protein A.

Both monomeric and dimeric constructs of the B domain of protein A from Staphylococcus aureus have been characterized by NMR, CD and fluorescence spectroscopy. The monomeric form of the protein was synthesized using a novel method incorporating the use of a recombinant, folded, chimeric protein. A comparison of the recombinant monomeric form with the commercially available dimeric form indicates that, although the dimer retains the integrity of the three-helix bundle structure present in the monomer, there are interdomain contacts in the dimeric form. A single long-lived water molecule in the hydrophobic core of the three-helix bundle of monomeric protein A may represent an important stabilizing factor for the three-helix bundle topology.     

The domain-swapped dimer of cyanovirin-N is in a metastable folded state: reconciliation of X-ray and NMR structures

The structure of the potent HIV-inactivating protein cyanovirin-N was previously found by NMR to be a monomer in solution and a domain-swapped dimer by X-ray crystallography. Here we demonstrate that, in solution, CV-N can exist both in monomeric and in domain-swapped dimeric form. The dimer is a metastable, kinetically trapped structure at neutral pH and room temperature. Based on orientational NMR constraints, we show that the domain-swapped solution dimer is similar to structures in two different crystal forms, exhibiting solely a small reorientation around the hinge region. Mutation of the single proline residue in the hinge to glycine significantly stabilizes the protein in both its monomeric and dimeric forms. By contrast, mutation of the neighboring serine to proline results in an exclusively dimeric protein, caused by a drastic destabilization of the monomer.     

Calcitonin receptor and Odz4 are differently expressed in Pax7-positive cells during skeletal muscle regeneration

Satellite cells, muscle-specific stem cells, are anatomically identified as the mononuclear cells residing external to the myofiber plasma membrane and beneath the basal lamina. Skeletal muscle has great regenerative potential, and the regeneration process depends absolutely on satellite cells. In uninjured muscle, satellite cells are maintained in a quiescent state, and some genes are expressed in a quiescent-specific manner. Here we show that Odz4/Ten-m4, a mouse homolog of the Drosophila pair-rule gene odd Oz (odz or Ten-m), is expressed in quiescent satellite cells on the protein level, but not in activated/proliferating myoblasts. Intriguingly, the timing of the reappearance of Odz4 and calcitonin receptor (another quiescence molecule) on Pax7-positive cells was different during the regeneration process. In addition, almost all neonatal satellite cells express Odz4, but only some of them express calcitonin receptor. These results indicate that Odz4 may be useful as a new marker of satellite cells and that quiescence molecules are differently expressed in regenerating and neonatal muscle.     

Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein

We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22(+) tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V(L)36(Leu-->Tyr)) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 (K(D)=0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I (rapLRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22(+) tumor cell lines with high efficacy (IC(50)=3-20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.