The cloning and preliminarily functional analysis of the human neurotrimin gene

~~The cloning and preliminarily functional analysis of the human neurotrimin gene~~     

Neurotractin, a novel neurite outgrowth-promoting Ig-like protein that interacts with CEPU-1 and LAMP.

The formation of axon tracts in nervous system histogenesis is the result of selective axon fasciculation and specific growth cone guidance in embryonic development. One group of proteins implicated in neurite outgrowth, fasciculation, and guidance is the neural members of the Ig superfamily (IgSF). In an attempt to identify and characterize new proteins of this superfamily in the developing nervous system, we used a PCR-based strategy with degenerated primers that represent conserved sequences around the characteristic cysteine residues of Ig-like domains. Using this approach, we identified a novel neural IgSF member, termed neurotractin. This GPI-linked cell surface glycoprotein is composed of three Ig-like domains and belongs to the IgLON subgroup of neural IgSF members. It is expressed in two isoforms with apparent molecular masses of 50 and 37 kD, termed L-form and S-form, respectively. Monoclonal antibodies were used to analyze its biochemical features and histological distribution. Neurotractin is restricted to subsets of developing commissural and longitudinal axon tracts in the chick central nervous system. Recombinant neurotractin promotes neurite outgrowth of telencephalic neurons and interacts with the IgSF members CEPU-1 (KD = 3 x 10(-8) M) and LAMP. Our data suggest that neurotractin participates in the regulation of neurite outgrowth in the developing brain.     

Structural analysis of glycosyl-phosphatidylinositol membrane anchor of the Toxoplasma gondii tachyzoite surface glycoprotein gp23

In this study we describe the biochemical features of the Toxoplasma gondii tachyzoite surface glycoprotein, gp23, demonstrating that it is attached to the parasite membrane by a glycosyl-phosphatidyl inositol anchor. Gp23 was metabolically labeled with tritiated palmitate, myristate, ethanolamine, inositol, glucosamine, mannose and galactose, as expected for a GPI-anchor structure. Gp23 was released from the surface of living parasites after treatment with phosphatidyl inositol-specific phospholipase C (PI-PLC) and the resulting water-soluble protein was immunoprecipitated with a monoclonal antibody specific for gp23. The GPIcore glycan was generated after aqueous-HF dephosphorylation followed by nitrous acid deamination and its carbohydrate structure was analyzed using selective exo- and endoglycosidase treatments. Finally, the phosphatidylinositol moiety of gp23 was characterized using PI-PLC and phospholipase A₂ (PLA₂) digestions. Our cumulative data suggest that gp23 of T gondii tachyzoites contains a modified GPI-backbone similar to the mammalian Thy-1 anchor, consisting of a conserved core structure (ethanolaminePO₄-6-Manαl-2-Manαl-6-Manαl-4-GIcNαl-6-PI) bearing β-linked N-acetylgalactosamine residue(s).     

Axonal Glycoproteins with Immunoglobulin- and Fibronectin Type III-Related Domains in Vertebrates: Structural Features, Binding Activities, and Signal Transduction

Abstract: The L1- and F11-like axonal glycoproteins, implicated in neurite outgrowth and fasciculation, are members of the Ig superfamily comprising multiple fibronectin type III-like domains. Their Ig-like and fibronectin type III-related domains are likely to be composed of seven β-strands arranged in two opposing β-sheets of highly similar topology. Whereas the F11-like molecules lack a transmembrane sequence and are anchored in the plasma membrane by a glycosylphosphatidylinositol, the L1 -like molecules comprise cytoplasmic domains with highly conserved sequence motifs. Most of the latter proteins occur in different isoforms generated by alternative pre-mRNA splicing, which has not been documented for molecules of the F11 subgroup. L1 -like proteins undergo heterophils as well as homophilic interactions, whereas only the former mode of binding was observed for F11 -like proteins. Evidence is accumulating that these Ig superfamily molecules with fibronectin type III-like domains are interacting in a complex manner with each other and molecules of the extracellular matrix. Investigations assigning structure to function reveal that their individual extracellular domains serve distinct binding activities. Recent studies also suggest that L1 and NCAM are implicated in the transduction of transmembrane signals.     

A direct interaction of axonin-1 with NgCAM-related cell adhesion molecule (NrCAM) results in guidance, but not growth of commissural axons

An interaction of growth cone axonin-1 with the floor-plate NgCAM-related cell adhesion molecule (NrCAM) was shown to play a crucial role in commissural axon guidance across the midline of the spinal cord. We now provide evidence that axonin-1 mediates a guidance signal without promoting axon elongation. In an in vitro assay, commissural axons grew preferentially on stripes coated with a mixture of NrCAM and NgCAM. This preference was abolished in the presence of anti-axonin-1 antibodies without a decrease in neurite length. Consistent with these findings, commissural axons in vivo only fail to extend along the longitudinal axis when both NrCAM and NgCAM interactions, but not when axonin-1 and NrCAM or axonin-1 and NgCAM interactions, are perturbed. Thus, we conclude that axonin-1 is involved in guidance of commissural axons without promoting their growth.     

Cloning of the cDNA encoding neural adhesion molecule F3 from bovine brain

We cloned a bovine cDNA encoding the neural adhesion molecule F3 and analyzed its nucleotide sequence. The coding region consisted of 3054 bp encoding 1018 amino acid (aa) residues. The M_r calculated from the deduced as sequence was 113 383. Bovine F3 had 93, 94 and 77% as identity with the mouse, human and chicken homologs, respectively. Bovine F3, similar to those of chicken and human, was devoid of two as residues (Ile-Thr) in the sixth immunoglobulin type C2-like domain, as compared with the mouse homolog. Parts of bovine F3 protein were overproduced in Escherichia coli. The antibodies raised against the recombinant proteins in rabbits reacted specifically with F3. F3 protein was detected in cerebellum, cerebrum and spinal cord in Western blot analysis.     

Myelin protein zero is one of the components of the detergent-resistant membrane microdomain fraction prepared from rat pituitary

Pituitary gland is a well-known endocrine tissue. The hypothalamo-neurohypophysial system, containing arginine vasopressin and oxytocin, shows a reversible morphological reorganization of both neurons and glial cells during chronic physiological stimulations. Since many signal transducing and cell adhesion molecules (CAMs) are recovered in membrane microdomain (MD) fractions, MDs are considered as signaling platforms of cells. In order to know the molecular background for these endocrine systems, we characterized MD-components derived from rat pituitary and found specific enrichment of several proteins in the fraction. One of them was identified as myelin protein zero (P0) with mass analysis and this result was further confirmed by a result that a specific antibody to this protein reacted to the authentic P0 protein in the myelin fraction of rat sciatic nerve. P0 is one of type-I transmembrane CAMs and a major structural component of mammalian peripheral nerve myelin. In mammals, expression of P0 has been considered to be restricted to peripheral nervous system. This result however indicates that P0 expresses more widely and its enrichment in the MD-fraction from rat pituitary suggests the participation in cell-cell communications.     

cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

A common protocadherin tail: Multiple protocadherins share the same sequence in their cytoplasmic domains and are expressed in different regions of brain

To study the diversity of protocadherins, a rat brain cDNA library was screened using a cDNA for the cytoplasmic domain of human protocadherin Pcdh2 as a probe. The resultant clones contained three different types. One type corresponds to rat Pcdh2; the other two types are distinct from Pcdh2 but contain the same sequence in their cytoplasmic domains and part of the 3′ flanking sequence. To clarify the structure of the proteins defined by the new clones, a putative entire coding sequence corresponding to one of the clones was determined. The overall structure is essentially the same as Pcdh2, indicating that the proteins defined by this clone, and probably by other clones, belong to the protocadherin family. Two PCR experiments and an RNase protection assay showed the existence of the corresponding mRNAs in rat brain preparations. Human and mouse cDNA clones with the same sequence properties were also isolated. Taken together, these results indicate that the clones are not cloning artifacts and that corresponding mRNAs are actually expressed in brains of various species. The results of in situ hybridization showed that the mRNAs corresponding to these clones were expressed in different regions in brain. Since protocadherins encoded by these mRNAs are likely to have different specificity in their interaction and share a common activity at their cytoplasmic domains, these protocadherins may provide a molecular basis, in part, to support the complex cell-cell interaction in brain.     

Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA

Chromogranin A (CGA), also referred to as secretory protein I, is an acidic protein that has been detected in all neuroendocrine cell types examined and is often present in large amounts relative to other secreted proteins. For example, CGA comprises at least 40% of the soluble protein of the adrenal chromaffin granule, and it appears to be the major secretory protein in the parathyroid secretory granules. CGA complementary DNAs (cDNAs) from bovine adrenal and pituitary have recently been cloned and sequenced and found to be nearly identical. A region of bovine CGA has a high degree of amino acid sequence identity to pancreastatin, a recently isolated porcine peptide that inhibits glucose-induced insulin secretion. This suggests that CGA may be a prohormone. We have cloned and sequenced a human cDNA encoding CGA. This human CGA cDNA has an overall 86% nucleic acid identity to the bovine cDNA. Like the bovine CGA cDNA, the human cDNA has little homology to pancreastatin at the 5' region of this peptide but significant amino acid homology to the carboxyl-terminal portion of pancreastatin where the biologic activity resides. There is an area within the pancreastatin region of human CGA and porcine pancreastatin with a 70% amino acid identity to the calcium-binding moiety of the E-F hand proteins such as parvalbumin and oncomodulin. These data suggest that CGA and pancreastatin may both be members of a larger family of calcium-binding proteins.     

Molecular cloning and structural analysis of human norepinephrine transporter gene（NETHG）

A cDNA molecule encoding a major part of the human Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene.A positive clone harbouring the whole gene was obtained from a human lymphocyte genomic library through utilizing the “genomic walking” technique.The clone,designated as phNET,harbours a DNA fragment of about 59 kd in length inserted into BamH I site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein.A single exon encodes a distinct transmembrane domain,except for transmembrane domain 10 and 11,which are encoded by part of two exons respectively,and exon 12,which encodes part of domain 11 and all of domain 12.These results imply that there is a close relationship between exon splicing of a gene and structureal domains of the protein,as is the case for the human γ－aminobutyric acid transporter(hGAT) and a number of other membrane proteins.     

cDNA cloning,expression pattern and RNA binding analysis of human selenocysteine insertion sequence (SECIS) binding protein 2

Selenocysteine and selenoprotein synthesis require a complex molecular machinery in mammals. Among the key players is the RNA-protein complex formed by the selenocysteine insertion sequence (SECIS) binding protein (SBP2) and the SECIS element, an RNA hairpin in the 3' untranslated regions of selenoprotein messenger RNAs (mRNAs). We have isolated the DNA complementary to mRNA of the human SBP2, enabling us to establish that it differs from a previously reported human SBP2-like protein. Examination of the expression pattern revealed that the human SBP2 protein is encoded by a 4 kb long mRNA that is over-expressed in testis. Compared to the rat SBP2 sequence, the human SBP2 protein displays two highly conserved domains with 92 and 95% amino acid identity, the latter one containing the RNA binding domain. The inter-domain section carries 55% sequence identity, the remainder of the SBP2 sequences showing about 65% identity, values lower than expected for two mammalian proteins. Interestingly, we could show that the binding of human SBP2 to the SECIS RNA is stimulated by the selenoprotein-specialized elongation translation factor mSelB/eEFsec.     

Structure-function analysis of protein complexes involved in the molecular architecture of juxtaparanodal regions of myelinated fibers

Demyelinating disorders, including multiple sclerosis (MS), are common causes of neurological disability. One critical step towards the management and therapy of demyelinating diseases is to understand the basic functions of myelinating glia and their relationship with axons. Axons and myelinating glia, oligodendrocytes in the central (CNS) and Schwann cells in the peripheral (PNS) nervous systems, reciprocally influence each other's development and trophism. These interactions are critical for the formation of distinct axonal domains in myelinated fibers that ensure the rapid propagation of action potentials. Macromolecular complexes mediating axo-glial interactions in these domains have been identified, consisting of members of the immunoglobulin superfamily (IgSF) of adhesion molecules and the neurexin/NCP superfamily as well as other proteins. We have investigated the molecular details of axo-glial interactions in the juxtaparanodal region of myelinated fibers by utilizing domain-specific GFP constructs and immunoprecipitation assays on transfected cells. We have shown that the immunoglobulin domains of the IgSF member TAG-1/Cnt-2 are necessary and sufficient for the direct, cis interaction of this protein with Caspr2 and potassium channels.     

Differential gene expression in the developing human macula: microarray analysis using rare tissue samples

The macula is a unique and important region in the primate retina that achieves high resolution and color vision in the central visual field. We recently reported data obtained from microarray analysis of gene expression in the macula of the human fetal retina (Kozulin et al., Mol Vis 15:45–59, 1). In this paper, we describe the preliminary analyses undertaken to visualize differences and verify comparability of the replicates used in that study, report the differential expression of other gene families obtained from the analysis, and show the reproducibility of our findings in several gene families by quantitative real-time PCR.     

Developmental and pathophysiological aspects of the myelin-associated glycoprotein

1. A glycoslylated sulfate-containing protein known as myelin-associated glycoprotein (MAG) appears to be unique to the central and peripheral nervous systems. This component has been characterized and cDNA clones have been isolated. 2. MAG is a member of the immunoglobulin superfamily. The principal form of MAG synthesized in brain during active myelination has an apparent molecular weight of 100,000. Alternate exon splicing leads to an additional 5000-dalton smaller form with a different C terminus. 3. In patients with multiple sclerosis, MAG is rapidly lost in areas of active disease. It is immunologically reactive in patients with benign monoclonal gammopathy associated with peripheral neuropathy. 4. The role of MAG in the formation of the myelin sheath and its participation in autoimmune neurological disorders are outlined.