Identification of a New Coiled Body Component

Coiled bodies are small, round nuclear inclusions that have been identified in many somatic cell types. Equivalent structures are found in the germinal vesicles of amphibian and insect oocytes, known respectively as sphere organelles and Binnenkörper. Their functions are not known, but their molecular composition is being brought to light. In addition to the nucleolar protein, fibrillarin, coiled bodies contain DNA topoisomerase I and an array of RNA processing molecules characteristic of spliceosomes. One coiled body protein absent from nucleoli and spliceosomes, known as p80-coilin, has also been described. We have now identified pigpen, a new member of the EWS family of proteins, as a second protein enriched in coiled bodies. In an earlier report we found that pigpen's structure and expression pattern were suggestive of a role in endothelial cell proliferation and differentiation. In this brief report we characterize pigpen's nuclear compartment and describe its reorganization during mitosis.     

Plasmodium falciparum: cloned and expressed CIDR domains of PfEMP1 bind to chondroitin sulfate A

Adherence of erythrocytes infected with mature asexual Plasmodium falciparum parasites (iRBC) to microvascular endothelial cells contributes to the pathology of P. falciparum malaria. It has been shown that the variant P. falciparum erythrocyte membrane protein 1 (PfEMP1) confers adhesion to a wide range of cell surface receptors. Previously, the cysteine-rich interdomain region (CIDR) of PfEMP1 has been identified as binding site to CD36. We provide evidence that the same region can also mediate binding to chondroitin sulfate A (CSA). CIDR domains of two different parasite strains were expressed in Escherichia coli as a 6xHis-tagged protein. Purified recombinant protein bound to Chinese hamster ovary (CHO) cells which naturally express chondroitin sulfate A. Treatment of wild-type CHO cells with chondroitinase ABC reduced binding up to 94.4%. Competitive binding using soluble CSA inhibited binding to CHO cells by up to 100% at 2 mg/ml and by 62.4% at 0.5 mg/ml, whereas 1 mg/ml heparan sulfate had only a little effect (18.1%). In contrast, a recombinant 6xHis-tagged DBL1 domain showed no binding to wild-type CHO cells. Such an approach of analyzing various domains of PfEMP1 as recombinant proteins may elucidate their functions and may lead to novel anti-adherence therapeutics, especially for maternal malaria infections.     

Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. selective targeting of endothelial regulatory protein pigpen

Tumor microvessels differ in structure and metabolic function from normal vasculature, and neoangiogenesis is associated with quantitative and qualitative changes in expression of endothelial proteins. Such molecules could serve as molecular addresses differentiating the tumor vasculature from those of the normal brain. We have applied Systematic Evolution of Ligands by EXponential enrichment (SELEX) against transformed endothelial cells as a complex target to select single-stranded DNA-ligands (aptamers) that function as histological markers to detect microvessels of rat experimental glioma, a fatal brain tumor that is highly vascularized. Both the SELEX selection procedure as well as subsequent deconvolution-SELEX were analyzed by fluorescence based methods (flow cytometry and fluorescence microscopy). Of 25 aptamers analyzed, one aptamer was selected that selectively bound microvessels of rat brain glioblastoma but not the vasculature of the normal rat brain including peritumoral areas. The molecular target protein of aptamer III.1 was isolated from endothelial cells by ligand-mediated magnetic DNA affinity purification. This protein was identified by mass spectrometry as rat homologue of mouse pigpen, a not widely known endothelial protein the expression of which parallels the transition from quiescent to angiogenic phenotypes in vitro. Because neoangiogenesis, the formation of new blood vessels, is a key feature of tumor development, the presented aptamer can be used as a probe to analyze pathological angiogenesis of glioblastoma. The presented data show that pigpen is highly expressed in tumor microvessels of experimental rat brain glioblastoma and may play an important role in warranting blood supply, thus growth of brain tumors.     

Soluble lactose-binding vertebrate lectins: a growing family

Extracts of rat intestine contain nine soluble lactose-binding lectins with subunit molecular weights ranging from 14,500 to 19,000 that were purified by affinity chromatography and ion-exchange chromatography. Two of them are either identical with or closely related to other known rat lectins. A third appears to be the isolated carbohydrate-binding C-terminal domain of a known lectin but lacks the N-terminal domain presumed to mediate a different function. The others have not been described previously. Among them, the major rat intestinal lectin, RI-H, and a related protein, RI-G, have N-terminal amino acid sequences with similarities to sequences found in other known rat lectins. Therefore, these results introduce new members of a growing family of these structurally homologous soluble lactose-binding proteins.     

Structure determination of Discoidin II from Dictyostelium discoideum and carbohydrate binding properties of the lectin domain

The social amoeba Dictyostelium discoideum adopts a cohesive stage upon starvation and then produces Discoidin I and II, two proteins able to bind galactose and N-acetyl-galactosamine. The N-terminal domain or discoidin domain (DS) is widely distributed in eukaryotes where it plays a role in extracellular matrix binding while the C-terminal domain displays sequence similarities to invertebrate lectins. We present the first X-ray structures of the wild-type and recombinant Discoidin II in unliganded state and in complex with monosaccharides. The protein forms a homotrimer which presents two binding surfaces situated on the opposite boundaries of the structure. The binding sites of the N-terminal domain contain PEG molecules that could mimics binding of natural ligand. The C-terminal lectin domain interactions with N-acetyl-D-galactosamine and methyl-beta-galactoside are described. The carbohydrate binding sites are located at the interface between monomers. Specificity for galacto configuration can be rationalized since the axial O4 hydroxyl group is involved in several hydrogen bonds with protein side chains. Titration microcalorimetry allowed characterization of affinity and demonstrated the enthalpy-driven character of the interaction. Those results highlight the structural differentiation of the DS domain involved in many cell-adhesion processes from the lectin activity of Dictyostelium discoidins.     

Biophysical investigation of human heparan sulfate D-glucosaminyl 3-O-sulfotransferase-3A: a mutual effect of enzyme oligomerisation and glycosaminoglycan ligand binding

3-O-sulfation of heparan sulfate (HS) is the rarest modification within heparan sulfate biosynthesis resulting in unique biological activities. Heparan sulfate d-glucosaminyl 3-O-sulfotransferase-3A (3-OST-3A) (EC 2.8.2.23) generates a binding site for the envelope glycoprotein D (gD) of herpes simplex virus 1. We have expressed the sulfotransferase domain of the human heparan sulfate 3-OST-3A isoform in Escherichia coli and subsequently purified the active enzyme which was found to be present as an oligomer under nonreducing conditions. The activity of the enzyme was tested by a novel gD-dependent gel mobility assay. A biophysical characterisation of 3-OST-3A was performed to study ligand binding and ligand-induced structural changes. Interestingly, the natural substrate HS did not cause a secondary structural change in the enzyme, whereas heparin and chondroitin sulfate did, both of which also exhibited similar high affinity binding to 3-OST-3A compared to HS as detected by isothermal fluorescence titrations. In cross-link assays, only HS was found to induce high molecular aggregates of 3-OST-3A whereas other GAG ligands did not or even inhibited enzyme oligomerisation like the K5 polysaccharide, which was nevertheless found to bind to the enzyme. We therefore conclude that since 3-OST-3A is able to bind also non-substrate GAG ligands with high affinity, discrimination among ligands is triggered by protein oligomerisation.     

Neuroglycan C,A Brain-Specific Chondroitin Sulfate Proteoglycan,Interacts with Pleiotrophin,A Heparin-Binding Growth Factor

Neuroglycan C (NGC) is a transmembrane-type chondroitin sulfate proteoglycan that promotes neurite outgrowth. To identify the ligand of NGC, we applied a detergent-solubilized membrane fraction of fetal rat brains to an NGC-immobilized affinity column. Several proteins were eluted from the column including an 18 kDa-band protein recognized by an anti-pleiotrophin antibody. The binding of pleiotrophin (PTN) to NGC was confirmed by a quartz crystal microbalance method and had a Kd of 8.7 nM. PTN bound to the acidic amino acid cluster of the NGC extracellular domain. In addition, PTN bound to both chondroitin sulfate-bearing NGC and chondroitinase-treated NGC prepared from the neonatal rat brain. These results suggest that NGC interacts with PTN.     

Basis for low affinity binding of a lysosomal cysteine protease to the cation-independent mannose 6-phosphate receptor

In cultured mouse fibroblasts, secretion of the lysosomal cysteine protease, MEP (major excreted protein) is regulated by growth factors and viral transformation. The ability of this protein to be regulated has been attributed to its intrinsic low affinity for the cation-independent mannose 6-phosphate (Man-6-P) receptor (Dong, J., Prence, E. M., and Sahagian, G. G. (1989) J. Biol. Chem. 264, 7377-7383). In this study, the basis for this low affinity was examined. Chromatography on a cation-independent Man-6-P receptor affinity matrix was used to assess relative affinities of Man-6-P-containing oligosaccharides and proteins, and the state of phosphorylation of the oligosaccharides was determined by ion exchange chromatography on QAE-Sephadex. MEP proteins synthesized by normal NIH 3T3 cells or NIH cells transformed with Kirsten sarcoma virus displayed a similar low affinity for the receptor and were found to possess oligosaccharide species with two phosphomonoester moieties. The affinity of these oligosaccharides for the receptor was the same as intact MEP protein and as great as phosphorylated oligosaccharides obtained from lysosomal proteins with the usual high affinity for the receptor. These results indicate that the polypeptide portion of MEP has no effect on binding of the protein to the receptor and that the difference in affinity of MEP and lysosomal proteins with high affinity cannot be attributed to differences in oligosaccharide structure. To investigate this further, we examined the binding characteristics of MEP made by CHO cells. In contrast to mouse MEP, CHO MEP bound to the receptor with high affinity. Partial endoglycosidase H treatment indicated that CHO MEP has two phosphorylated oligosaccharides, whereas the mouse protein has only one. Both oligosaccharides of the CHO cell protein contained two phosphomonoester moieties and displayed an affinity for the receptor that was indistinguishable from that of oligosaccharides of the mouse protein. Conversion of CHO MEP to a one-oligosaccharide species by partial endoglycosidase H treatment produced a protein that displayed low affinity binding similar to that of mouse MEP. A substantial portion of the pool of CHO cell lysosomal protein was also converted to a low affinity ligand by this treatment. Taken together, these results suggest that high affinity binding to the cation-independent receptor involves a divalent interaction with lysosomal proteins that contain two or more phosphorylated oligosaccharides, and that the low affinity of MEP results from an inability to form this multivalent interaction.     

A novel C-type lectin secreted by a tissue-dwelling parasitic nematode.

Many parasitic nematodes live for surprisingly long periods in the tissues of their hosts, implying sophisticated mechanisms for evading the host immune system. The nematode Toxocara canis survives for years in mammalian tissues, and when cultivated in vitro, secretes antigens such as TES-32. From the peptide sequence, we cloned TES-32 cDNA, which encodes a 219 amino-acid protein that has a domain characteristic of host calcium-dependent (C-type) lectins, a family of proteins associated with immune defence. Homology modelling predicted that TES-32 bears remarkable structural similarity to mammalian immune-system lectins. Native TES-32 acted as a functional lectin in affinity chromatography. Unusually, it bound both mannose- and galactose-type monosaccharides, a pattern precluded in mammalian lectins by a constraining loop adjacent to the carbohydrate-binding site. In TES-32, this loop appeared to be less obtrusive, permitting a broader range of ligand binding. The similarity of TES-32 to host immune cell receptors suggests a hitherto unsuspected strategy for parasite immune evasion.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Structural and energetic aspects of Grb2-SH2 domain-swapping

The SH2 domain of growth factor receptor-bound protein 2 (Grb2) has been the focus of numerous studies, primarily because of the important roles it plays in signal transduction. More recently, it has emerged as a useful protein to study the consequences of ligand preorganization upon energetics and structure in protein-ligand interactions. The Grb2-SH2 domain is known to form a domain-swapped dimer, and as part of our investigations toward correlating structure and energetics in biological systems, we examined the effects that domain-swapping dimerization of the Grb2-SH2 domain had upon ligand binding affinities. Isothermal titration calorimetry was performed using Grb2-SH2 in both its monomeric and domain-swapped dimeric forms and a phosphorylated tripeptide AcNH-pTyr-Val-Asn-NH(2) that is similar to the Shc sequence recognized by Grb2-SH2 in vivo. The two binding sites of domain-swapped dimer exhibited a 4- and a 13-fold reduction in ligand affinity compared to monomer. Crystal structures of peptide-bound and uncomplexed forms of Grb2-SH2 domain-swapped dimer were obtained and reveal that the orientation of residues V122, V123, and R142 may influence the conformation of W121, an amino acid that is believed to play an important role in Grb2-SH2 ligand sequence specificity. These findings suggest that domain-swapping of Grb2-SH2 not only results in a lower affinity for a Shc-derived ligand, but it may also affect ligand specificity.     

The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression

The RNA recognition motif (RRM), also known as RNA-binding domain (RBD) or ribonucleoprotein domain (RNP) is one of the most abundant protein domains in eukaryotes. Based on the comparison of more than 40 structures including 15 complexes (RRM-RNA or RRM-protein), we reviewed the structure-function relationships of this domain. We identified and classified the different structural elements of the RRM that are important for binding a multitude of RNA sequences and proteins. Common structural aspects were extracted that allowed us to define a structural leitmotif of the RRM-nucleic acid interface with its variations. Outside of the two conserved RNP motifs that lie in the center of the RRM beta-sheet, the two external beta-strands, the loops, the C- and N-termini, or even a second RRM domain allow high RNA-binding affinity and specific recognition. Protein-RRM interactions that have been found in several structures reinforce the notion of an extreme structural versatility of this domain supporting the numerous biological functions of the RRM-containing proteins.     

Coiled body heterogeneity induced by G(1) arrest with amiloride + bumetanide

Ki67 is a nuclear protein expressed in proliferating cells, but not in quiescent or G(0)-arrested cells. Similar to the proliferating cell nuclear antigen and several other well-characterized molecules, Ki67 exhibits a repeating pattern of regulated expression and redistribution during the cell cycle, making it a useful marker for cell cycle phase. In addition to other structures labeled, concentrated foci may be observed in the nucleus and sometimes the cytoplasm. We observed that these Ki67 foci can be found at any stage of the endothelial cell cycle. They are not coincident with coiled bodies (CB), as determined in double-label immunofluorescence experiments with anti-Ki67 and antibodies to the CB marker protein pigpen. However, arrest of BPA47 endothelial cells in G(1) with amiloride + bumetanide induces colocalization of pigpen and Ki67 in 40% of cells exhibiting Ki67 foci. We conducted a series of experiments to examine the possibilities that pigpen was exported from CB and into unique, Ki67-containing foci or that Ki67 was imported into pigpen-containing CB. Our results showed us that although CB typically contain both coilin and pigpen, amiloride + bumetanide-induced G(1) arrest reconfigured the CB compartment into three populations of foci: one containing pigpen without coilin, the second containing coilin without pigpen, and a third containing both pigpen and coilin together. Furthermore, G(1) arrest resulted in Ki67 redistribution into both coilin- and pigpen-containing foci. The results suggest that under certain conditions, "resident" CB proteins can be differentially redistributed, and proteins not previously recognized as resident in CB can be driven into that compartment. Our observations underscore the fluid nature of CB. They demonstrate that previously reported heterogeneity in the CB compartment can be amplified by a specific experimental manipulation. This may be useful in future analyses of protein trafficking within the CB compartment and between CB and other cellular compartments. Finally, the redistribution of Ki67 into CB represents a new finding for a widely expressed but poorly understood molecule, one that may be useful in elucidating function.     

Ligand specificity in the CRAL-TRIO protein family

Intracellular trafficking of hydrophobic ligands is often mediated by specific binding proteins. The CRAL-TRIO motif is common to several lipid binding proteins including the cellular retinaldehyde binding protein (CRALBP), the alpha-tocopherol transfer protein (alpha-TTP), yeast phosphatidylinositol transfer protein (Sec14p), and supernatant protein factor (SPF). To examine the ligand specificity of these proteins, we measured their affinity toward a variety of hydrophobic ligands using a competitive [(3)H]-RRR-alpha-tocopherol binding assay. Alpha-TTP preferentially bound RRR-alpha-tocopherol over all other tocols assayed, exhibiting a K(d) of 25 nM. Binding affinities of other tocols for alphaTTP closely paralleled their ability to inhibit in vitro intermembrane transfer and their potency in biological assays. All other homologous proteins studied bound alpha-tocopherol but with pronouncedly weaker (> 10-fold) affinities than alpha-TTP. Sec14p demonstrated a K(d) of 373 nM for alpha-tocopherol, similar to that for its native ligand, phosphatidylinositol (381 nM). Human SPF had the highest affinity for phosphatidylinositol (216 nM) and gamma-tocopherol (268 nM) and significantly weaker affinity for alpha-tocopherol (K(d) 615 nM). SPF bound [(3)H]-squalene more weakly (879 nM) than the other ligands. Our data suggest that of all known CRAL-TRIO proteins, only alphaTTP is likely to serve as the physiological mediator of alpha-tocopherol's biological activity. Further, ligand promiscuity observed within this family suggests that caution should be exercised when suggesting protein function(s) from measurements utilizing a single ligand.     

Nuclear injection of anti-pigpen antibodies inhibits endothelial cell division

Endothelial cell proliferation is required for angiogenesis in both embryonic and adult tissues. In rat brain tumors, it has recently been shown that the nuclear protein pigpen is expressed selectively in endothelial cells of developing microvasculature but not in the established peritumoral vessels (Blank, M., Weinschenk, T., Priemer, M., and Schluesener, H. (2001) J. Biol. Chem. 276, 16464-16468). This finding suggests that pigpen may be important for promoting the undifferentiated, or "angiogenic" endothelial cell phenotype. Our studies show that pigpen protein and mRNA are expressed in actively dividing endothelial cells and down-regulated as they become confluent. Protein distribution is regulated in a cell cycle-dependent manner. We conclude that this expression pattern is important for and not simply ancillary to proliferation because nuclear microinjection of anti-pigpen Fab fragments inhibited endothelial cell division. Moreover, expression of the proliferating cell marker Ki67 was inhibited in antibody-injected cells. The absence of Ki67 suggests exit from rather than arrest within (for example, at the G(1)/S interface) the cell cycle. Together with earlier observations on the structure and expression of this molecule, our data support the hypothesis that pigpen helps regulate endothelial cell differentiation state.     

Animal lectins: a historical introduction and overview

Some proteins we now regard as animal lectins were discovered before plant lectins, though many were not recognised as carbohydrate-binding proteins for many years after first being reported. As recently as 1988, most animal lectins were thought to belong to one of two primary structural families, the C-type and S-type (presently known as galectins) lectins. However, it is now clear that animal lectin activity is found in association with an astonishing diversity of primary structures. At least 12 structural families are known to exist, while many other lectins have structures apparently unique amongst carbohydrate-binding proteins, although some of those "orphans" belong to recognised protein families that are otherwise not associated with sugar recognition. Furthermore, many animal lectins also bind structures other than carbohydrates via protein-protein, protein-lipid or protein-nucleic acid interactions. While animal lectins undoubtedly fulfil a variety of functions, many could be considered in general terms to be recognition molecules within the immune system. More specifically, lectins have been implicated in direct first-line defence against pathogens, cell trafficking, immune regulation and prevention of autoimmunity.