Purification of a heterodimeric Reelin construct to investigate binding stoichiometry.

Reelin is a secreted glycoprotein that is integral in neocortex development and synaptic function. Reelin exists as a homodimer with two chains linked by a disulfide bond at cysteine 2101, a feature that is vital to the protein's function. This is highlighted by the fact that only dimeric Reelin can elicit efficient, canonical signaling, even though a mutated (C2101A) monomeric construct of Reelin retains the capacity to bind to its receptors. Receptor clustering has been shown to be important in the signaling pathway, however direct evidence regarding the stoichiometry of Reelin-receptor binding interaction is lacking. Here we describe the construction and purification of a heterodimeric Reelin construct to investigate the stoichiometry of Reelin-receptor binding and how it affects Reelin pathway signaling. We have devised different strategies and have finalized a protocol to produce a heterodimer of Reelin's central fragment using differential tagging and tandem affinity chromatography, such that chain A is wild type in amino acid sequence whereas chain B includes a receptor binding site mutation (K2467A). We also validate that the heterodimer is capable of binding to the extracellular domain of one of Reelin's known receptors, calculating the KD of the interaction. This heterodimeric construct will enable us to understand in greater detail the mechanism by which Reelin interacts with its known receptors and initiates pathway signaling.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the extracellular olfactomedin domain of gliomedin.

Gliomedin (GLDN) is one of the essential proteins in the development of the nodes of Ranvier in the vertebrate peripheral nervous system. An olfactomedin (OLF) domain is located at the GLDN extracellular C-terminus and is involved in the accumulation of neuronal plasma membrane voltage-gated sodium channels in the nodes by interacting with neurofascin and NrCAM. No structures of OLF domains have previously been reported. Here, the crystallization of the rat GLDN OLF domain, which was expressed in an insect-cell system, is reported. The crystal diffracted to 1.55 Šresolution and belonged to space group P21, with unit-cell parameters a=37.5, b=141.7, c=46.0 Å, ß=110.6°, and had two molecules in the asymmetric unit.

Functional expression of rat neuroligin-1 extracellular fragment by a bi-cistronic baculovirus expression vector.

The interaction between the synaptic adhesion molecules neuroligins and neurexins is essential for connecting the pre- and post-synaptic neurons, modulating neuronal signal transmission, and facilitating neuronal axogenesis. Here, we describe the simultaneous expression of the extracellular domain of rat neuroligin-1 (NL1) proteins along with the enhanced green fluorescent protein (EGFP) using the bi-cistronic baculovirus expression vector system (bi-BEVS). Recombinant rat NL1 protein, fused with signal sequence derived from human Azurocidin gene (AzSP), was secreted into the culture medium and the optimum harvest time for NL1 protein before the lysis of infected cells was determined through the release of cytosolic EGFP. The NL1 protein (0.129±0.013 mg/8×10(7) High Five cells; ~96% purity by metal affinity chromatography) was obtained from the supernatant of the recombinant virus-infected insect cells. A novel chip was employed to address whether the recombinant NL1 is functional in axogenesis. The purified rat NL1 promoted and enhanced the growth rate (137.07±9.74 µm/day) of the axon on NL1/PLL (poly-L-lysine)-coated fine lines on the chip compared to those lines that were coated with PLL alone (105.53±4.53 µm/day). These results were confirmed by fluorescence immunocytochemistry and demonstrated that the recombinant protein can be purified by a one-step process using IMAC combined with monitoring of cell lysis by bi-BEVS. This technique along with our novel chip offers a simple, cost-effective and useful platform for understanding the roles of NL1 protein in neuronal regeneration and synaptic formation studies.

Paragonimus westermani: identification and characterization of the fasciclin I domain-containing protein.

Paragonimus westermani is a trematode parasite that causes inflammatory lung disease as well as systemic infections in carnivorous mammals. The interaction of the parasite with host cells and paired worms is initiated by adhesion and plays an important role in parasite proliferation and differentiation. In this study, we isolated a cDNA encoding a P. westermani fasciclin I domain-containing protein (Pwfas-I). The fasiclin-I domain is suggested to be involved in cell adhesion, migration, and differentiation. Immunohistochemical analysis of P. westermani adult worms with polyclonal anti-Pwfas-I serum revealed immunoreactivity in the egg shells and the cells lining the sub-tegumental layer of adult worm throughout the contact regions of the cyst wall and paired worms. Using cell adhesion and spreading assays, we showed that Pwfas-I supports cell adhesion and spreading. Furthermore, we determined that the alphanubeta5 integrin was a functional receptor for the Pwfas-I. Taken together, these results suggest that Pwfas-I may be functional for the modulation of cell adhesion via binding with alphanubeta5 integrin in the extracellular matrix of Paragonimus.

Amalgam, an axon guidance Drosophila adhesion protein belonging to the immunoglobulin superfamily: over-expression, purification and biophysical characterization.

Amalgam, a multi-domain member of the immunoglobulin superfamily, possesses homophilic and heterophilic cell adhesion properties. It is required for axon guidance during Drosophila development in which it interacts with the extracellular domain of the transmembrane protein, neurotactin, to promote adhesion. Amalgam was heterologously expressed in Pichia pastoris, and the secreted protein product, bearing an NH(2)-terminal His(6)Tag, was purified from the growth medium by metal affinity chromatography. Size exclusion chromatography separated the purified protein into two fractions: a major, multimeric fraction and a minor, dimeric one. Two protocols to reduce the percentage of multimers were tested. In one, protein induction was performed in the presence of the zwitterionic detergent CHAPS, yielding primarily the dimeric form of amalgam. In a second protocol, agitation was gradually reduced during the course of the induction and antifoam was added daily to reduce the air/liquid interfacial foam area. This latter protocol lowered the percentage of multimer 2-fold, compared to constant agitation. Circular dichroism measurements showed that the dimeric fraction had a high beta-sheet content, as expected for a protein with an immunoglobulin fold. Dynamic light scattering and sedimentation velocity measurements showed that the multimeric fraction displays a monodisperse distribution, with R(H)=16 nm. When co-expressed together with amalgam the ectodomain of neurotactin copurified with it. Furthermore, both purified fractions of amalgam were shown to interact with Torpedo californica acetylcholinesterase, a structural homolog of neurotactin.

Novel affinity tag system using structurally defined antibody-tag interaction: application to single-step protein purification.

Biologically important human proteins often require mammalian cell expression for structural studies, presenting technical and economical problems in the production/purification processes. We introduce a novel affinity peptide tagging system that uses a low affinity anti-peptide monoclonal antibody. Concatenation of the short recognition sequence enabled the successful engineering of an 18-residue affinity tag with ideal solution binding kinetics, providing a low-cost purification means when combined with nondenaturing elution by water-miscible organic solvents. Three-dimensional information provides a firm structural basis for the antibody-peptide interaction, opening opportunities for further improvements/modifications.

The ligand-binding profile of HARE: hyaluronan and chondroitin sulfates A, C, and D bind to overlapping sites distinct from the sites for heparin, acetylated low-density lipoprotein, dermatan sulfate, and CS-E.

The hyaluronic acid receptor for endocytosis (HARE)/ Stabilin-2 is the primary systemic scavenger receptor for hyaluronan (HA), the chondroitin sulfates (CS), dermatan sulfate (DS), and nonglycosaminoglycan (GAG) ligands such as acetylated low-density lipoprotein (AcLDL), pro-collagen propeptides, and advanced glycation end products. We recently discovered that HARE is also a systemic scavenger receptor for heparin (Hep) (Harris EN, Weigel JA, Weigel PH. 2008. The human hyaluronan receptor for endocytosis [HARE/Stabilin-2] is a systemic clearance receptor for heparin. J Biol Chem. 283:17341-17350). Our goal was to map the binding sites of eight different ligands within HARE. We used biotinylated GAGs and radio-iodinated streptavidin or AcLDL to assess the binding activities of ligands directly or indirectly (by competition with unlabeled ligands) in endocytosis assays using stable cell lines expressing the 315 or 190 kDa HA receptor for endocytosis (315- or 190-HARE) isoforms, and ELISA-like assays, with purified recombinant soluble 190-HARE ecto-domain. For example, Hep binding to HARE was competed by DS, CS-E, AcLDL, and dextran sulfate, but not by other CS types, HA, dextran, or heparosan. (125)I-AcLDL binding to HARE was partially competed by Hep and dextran sulfate, but not competed by HA. Two ligands, DS and CS-E, competed with both Hep and HA to some degree. Hep and HA binding or endocytosis is mutually inclusive; binding of these two GAGs occurs with functionally separate, noncompetitive, and apparently noninteracting domains. Thus, HARE binds to HA and Hep simultaneously. Although the domain(s) responsible for Hep binding remains unknown, the Link domain was required for HARE binding to HA, CS-A, CS-C, and CS-D. These results enable us to outline, for the first time, a binding activity map for multiple ligands of HARE.

The adhesion molecule Necl-3/SynCAM-2 localizes to myelinated axons, binds to oligodendrocytes and promotes cell adhesion.

BACKGROUND: Cell adhesion molecules are plasma membrane proteins specialized in cell-cell recognition and adhesion. Two related adhesion molecules, Necl-1 and Necl-2/SynCAM, were recently described and shown to fulfill important functions in the central nervous system. The purpose of the work was to investigate the distribution, and the properties of Necl-3/SynCAM-2, a previously uncharacterized member of the Necl family with which it shares a conserved modular organization and extensive sequence homology. RESULTS: We show that Necl-3/SynCAM-2 is a plasma membrane protein that accumulates in several tissues, including those of the central and peripheral nervous system. There, Necl-3/SynCAM-2 is expressed in ependymal cells and in myelinated axons, and sits at the interface between the axon shaft and the myelin sheath. Several independent assays demonstrate that Necl-3/SynCAM-2 functionally and selectively interacts with oligodendrocytes. We finally prove that Necl-3/SynCAM-2 is a bona fide adhesion molecule that engages in homo- and heterophilic interactions with the other Necl family members, leading to cell aggregation. CONCLUSION: Collectively, our manuscripts and the works on Necl-1 and SynCAM/Necl-2 reveal a complex set of interactions engaged in by the Necl proteins in the nervous system. Our work also support the notion that the family of Necl proteins fulfils key adhesion and recognition functions in the nervous system, in particular between different cell types.

The crystal structure of the ligand-binding module of human TAG-1 suggests a new mode of homophilic interaction.

Human TAG-1 is a neural cell adhesion molecule that is crucial for the development of the nervous system during embryogenesis. It consists of six immunoglobulin-like and four fibronectin III-like domains and is anchored to the membrane by glycosylphosphatidylinositol. Herein we present the crystal structure of the four N-terminal immunoglobulin-like domains of TAG-1 (TAG-1(Ig1-4)), known to be important in heterophilic and homophilic macromolecular interactions. The contacts of neighboring molecules within the crystal were investigated. A comparison with the structure of the chicken ortholog resulted in an alternative mode for the molecular mechanism of homophilic TAG-1 interaction. This mode of TAG-1 homophilic interaction is based on dimer formation rather than formation of a molecular zipper as proposed for the chicken ortholog.

Stabilin-2 is involved in lymphocyte adhesion to the hepatic sinusoidal endothelium via the interaction with alphaMbeta2 integrin.

Although lymphocyte recirculation to the endothelium plays a critical role in the movement of immune cells from the blood into tissues and sites of inflammation, the mechanisms involved in lymphocyte trafficking via the hepatic circulation have yet to be elucidated fully. In this study, we investigated the role of stabilin-2, which is expressed specifically in the sinusoidal endothelium, in the adhesion of lymphocytes to the hepatic endothelium. Stabilin-2-expressing cells mediate the adhesion of PBLs. This interaction was attributed specifically to the interaction of stabilin-2 with alphaMbeta2 integrin. Using mutant stabilin-2 molecules with deletions in the extracellular domain, we mapped the binding site for alphaMbeta2 integrin to the fasciclin 1 (FAS1) domains of stabilin-2. The specificity of the interaction between alphaMbeta2 integrin and the FAS1 domain was confirmed further by binding assays using neutralizing antibodies. More physiologically, we showed that the down-regulation of stabilin-2 results in the defective binding of lymphocytes to hepatic sinusoidal endothelial cells under conditions of static and physiological flow. Together, these data show that stabilin-2 can reconstitute the lymphocyte-endothelial adhesion cascade under physiological shear stress. We propose a critical role for stabilin-2 in lymphocyte adhesion to specialized endothelia, such as that of the hepatic sinusoid.

Nuclear translocation of CAM-associated protein activates transcription for long-term facilitation in Aplysia.

Repeated pulses of serotonin (5-HT) induce long-term facilitation (LTF) of the synapses between sensory and motor neurons of the gill-withdrawal reflex in Aplysia. To explore how apCAM downregulation at the plasma membrane and CREB-mediated transcription in the nucleus, both of which are required for the formation of LTF, might relate to each other, we cloned an apCAM-associated protein (CAMAP) by yeast two-hybrid screening. We found that 5-HT signaling at the synapse activates PKA which in turn phosphorylates CAMAP to induce the dissociation of CAMAP from apCAM and the subsequent translocation of CAMAP into the nucleus of sensory neurons. In the nucleus, CAMAP acts as a transcriptional coactivator for CREB1 and is essential for the activation of ApC/EBP required for the initiation of LTF. Combined, our data suggest that CAMAP is a retrograde signaling component that translocates from activated synapses to the nucleus during synapse-specific LTF.

Neuroglian on hemocyte surfaces is involved in homophilic and heterophilic interactions of the innate immune system of Manduca sexta.

Neuroglian, a member of the L1 family of cell adhesion molecules (L1-CAMs), is expressed on surfaces of granular cells and a subset of large plasmatocytes of Manduca sexta that act as foci for hemocyte aggregation during the innate immune response. Neuroglian expressed on surfaces of transfected Sf9 cells induced their homophilic aggregation, with the aggregation being abolished in the presence of recombinant immunoglobulin (Ig) domains of neuroglian. Neuroglian and its Ig domains also can interact with hemocyte-specific integrin (HS integrin) as demonstrated with an enzyme-linked immunoassay and a surface plasmon resonance (SPR) assay. Neuroglian double-stranded (ds) RNA not only depresses expression of neuroglian in hemocytes but also depresses the cell-mediated encapsulation response of these hemocytes to foreign surfaces. After injection of a monoclonal antibody (MAb 3B11) into M. sexta larvae that recognizes the Ig domains of neuroglian, the cell-mediated encapsulation response of hemocytes was likewise inhibited. The Ig domains of neuroglian are involved in both homophilic and heterophilic interactions, and subsets of these six different Ig domains may affect different functions of neuroglian.

Rat neurological disease creeping is caused by a mutation in the reelin gene.

Reelin (Reln) is an extracellular matrix protein secreted from distinct neuronal populations and controls neural cell positioning during brain development. Alterations of human RELN have been reported in two pedigrees with an autosomal recessive lissencephaly. Although several alleles of the mouse reeler mutation were identified as disruptions of Reln, there is no other animal model with a confirmed mutation in Reln. We recently established the Komeda Zucker creeping (KZC) rat strain with an autosomal recessive mutation creeping (cre), showing a reeler-like phenotype. We also found that creeping was located in the genomic segment on rat chromosome 4 containing Reln and that the expression level of Reln mRNA was markedly reduced in cre/cre homozygous mutant animals. Here we report positional candidate cloning of creeping, and identify a nucleotide insertion mutation in Reln. This mutation leads to a translational frameshift and results in truncation of the predicted protein in the fourth reelin-specific repeat, removing the C-terminal region required for secretion and function of the protein. We conclude that the mutation detected here is causative and is probably a null allele. The KZC rat is the first rat model with a confirmed Reln mutation and would therefore contribute to the understanding of the Reln function.

Purification and molecular identification of the human hyaluronan receptor for endocytosis.

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.

SynCAM, a synaptic adhesion molecule that drives synapse assembly.

Synapses, the junctions between nerve cells through which they communicate, are formed by the coordinated assembly and tight attachment of pre- and postsynaptic specializations. We now show that SynCAM is a brain-specific, immunoglobulin domain-containing protein that binds to intracellular PDZ-domain proteins and functions as a homophilic cell adhesion molecule at the synapse. Expression of the isolated cytoplasmic tail of SynCAM in neurons inhibited synapse assembly. Conversely, expression of full-length SynCAM in nonneuronal cells induced synapse formation by cocultured hippocampal neurons with normal release properties. Glutamatergic synaptic transmission was reconstituted in these nonneuronal cells by coexpressing glutamate receptors with SynCAM, which suggests that a single type of adhesion molecule and glutamate receptor are sufficient for a functional postsynaptic response.

Conserved and divergent patterns of Reelin expression in the zebrafish central nervous system.

The protein Reelin is suggested to function in cell-cell interactions and in mediating neuronal migrations in layered central nervous system structures. With the aim of shedding light on the development of the teleost telencephalon, which forms through the process of eversion and results in the formation of a nonlaminar pallium, we isolated a zebrafish ortholog of the reelin gene and studied its expression in developing and adult brain. The pattern of expression is highly dynamic during the first 24-72 hours of development. By 5 days postfertilization, high amounts of reelin mRNA are found in the dorsal telencephalon, thalamic and hypothalamic regions, pretectal nuclei, optic tectum, cerebellum, hindbrain, reticular formation, and spinal cord, primarily confined to postmitotic neurons. This pattern persists in 1- to 3-month-old zebrafish. This study, together with reports on reelin expression in other vertebrates, shows that reelin mRNA distribution is conserved in many regions of the vertebrate brain. A major exception is that reelin is expressed in the majority of the cells of the dorsal regions of the everted telencephalon in zebrafish embryos, whereas it is restricted to specific neuronal populations in the developing telencephalon of amniotes. To better understand the origin of these differences, we analyzed reelin expression in the telencephalon of an amphibian. Telencephalic reelin expression in Xenopus laevis shows more similarities with the sauropsidian than with the teleostean pattern. Thus, the differences in the telencephalic expression of reelin between teleosts and tetrapods are likely to be due to different roles for Reelin during eversion, a process that is specific for the teleost telencephalon.

Reelin is a serine protease of the extracellular matrix.

Reelin is an extracellular matrix protein that plays a pivotal role in development of the central nervous system. Reelin is also expressed in the adult brain, notably in the cerebral cortex, where it might play a role in synaptic plasticity. The mechanism of action of reelin at the molecular level has been the subject of several hypotheses. Here we show that reelin is a serine protease and that proteolytic activity is relevant to its function, since (i) Reelin expression in HEK 293T cells impairs their ability to adhere to fibronectin-coated surfaces, and adhesion to fibronectin is restored by micromolar concentrations of diisopropyl phosphorofluoridate, a serine hydrolase inhibitor; (ii) purified Reelin binds FP-Peg-biotin, a trap probe which irreversibly binds to serine residues located in active catalytic sites of serine hydrolases; (iii) purified Reelin rapidly degrades fibronectin and laminin, while collagen IV is degraded at a much slower rate; fibronectin degradation is inhibited by inhibitors of serine proteases, and by monoclonal antibody CR-50, an antibody known to block the function of Reelin both in vitro and in vivo. The proteolytic activity of Reelin on adhesion molecules of the extracellular matrix and/or receptors on neurons may explain how Reelin regulates neuronal migration and synaptic plasticity.

The adhesion molecule TAG-1 mediates the migration of cortical interneurons from the ganglionic eminence along the corticofugal fiber system.

Cortical nonpyramidal cells, the GABA-containing interneurons, originate mostly in the medial ganglionic eminence of the ventral telencephalon and follow tangential migratory routes to reach the dorsal telencephalon. Although several genes that play a role in this migration have been identified, the underlying cellular and molecular cues are not fully understood. We provide evidence that the neural cell adhesion molecule TAG-1 mediates the migration of cortical interneurons. We show that the migration of these neurons occurs along the TAG-1-expressing axons of the developing corticofugal system. The spatial and temporal pattern of expression of TAG-1 on corticofugal fibers coincides with the order of appearance of GABAergic cells in the developing cortex. Blocking the function of TAG-1, but not of L1, another adhesion molecule and binding partner of TAG-1, results in a marked reduction of GABAergic neurons in the cortex. These observations reveal a mechanism by which the adhesion molecule TAG-1, known to be involved in axonal pathfinding, also takes part in neuronal migration.