Purification, molecular cloning, and application of a novel sphingomyelin-binding protein (clamlysin) from the brackishwater clam, Corbicula japonica.

A novel sphingomyelin-binding protein (clamlysin) was purified from the foot muscle of a brackishwater clam, Corbicula japonica. The purified 24.8-kDa protein lysed sheep, horse and rabbit erythrocytes and the hemolytic activity was inhibited by sphingomyelin, but not other phospholipids or glycosphingolipids. The open reading frame of the clamlysin gene encoded a putative 26.9-kDa protein (clamlysin B) which showed high sequence similarity with the actinoporin family. A surface plasmon resonance assay confirmed that clamlysin B specifically bound to sphingomyelin. Furthermore, two cDNA variants of clamlysin, encoding putative 31.4 kDa (clamlysin A) and 11 kDa (clamlysin C) proteins, were isolated. Only the 31.4-kDa variant was found to exhibit sphingomyelin-binding activity. Clamlysin A and B, but not C, shared a sequence (domain II) conserved in all known sphingomyelin-binding proteins. Domain II fused with a glutathione S-transferase bound to sphingomyelin. Horse erythrocytes, mouse melanoma B16 and GM95 cells, and Chinese hamster ovary CHO-K1 cells, but not the same cells treated with bacterial sphingomyelinase, were immunostained with clamlysin B. These results indicate that clamlysin B binds to the sphingomyelin of living cells and thus would be useful as a molecular probe to detect sphingomyelin.

PolyADP-ribosylation is required for pronuclear fusion during postfertilization in mice.

BACKGROUND: During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives.

A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides.

Enzymes capable of hydrolyzing the beta-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 3.2.1.123) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (beta/alpha)8 barrel structure in which Glu234 at the end of beta-strand 4 and Glu341 at the end of beta-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the beta-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lactoseries glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1'-beta-galactohydrolase" or tentatively designated "endogalactosylceramidase."

Identification of nitrated proteins in the normal rat brain using a proteomics approach.

BACKGROUND: The nitration of tyrosine has been suggested to play a role in the pathogenesis of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and Alzheimer's disease (AD). METHODS: In the present study, we identified four targets of protein nitration, T-complex polypeptide 1 alpha subunit (TCP-1), neurofilament L (NFL), glial fibrillary acidic protein (GFAP) and clathrin heavy chain (CHC), in the normal rat cortex using a proteomics approach. CONCLUSIONS: There have been no reports on these proteins being identified by proteomics as nitrated forms in the brain. For further study, we have to investigate alterations in these nitrated proteins during aging and in neurodegenerative disorders.

Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp.

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.

Identification of a novel Cochlin isoform in the perilymph: insights to Cochlin function and the pathogenesis of DFNA9.

The COCH gene mutated in DFNA9, an autosomal dominant hereditary sensorineural hearing loss and vestibular disorder, encodes Cochlin. Previously, we reported three bovine Cochlin isoforms, p63s, p44s, and p40s, which exhibit significant molecular heterogeneity in vivo. Here we have characterized Cochlin isoforms by generating four isoform-specific anti-Cochlin antibodies. The same three Cochlin isoforms, p63s, p44s, and p40s, were detected in human and cow inner ear tissue; however, p44s and p40s were not detected in perilymph. We identified a novel short 16kDa isoform in human perilymph and a 18-23kDa isoform in cow perilymph, named Cochlin-tomoprotein (CTP), corresponding to the N-terminus of full-length Cochlin (p63s) and the LCCL domain. Notably, CTP contains all of the known mutation sites associated with DFNA9. The pathogenesis of DFNA9 is not fully clarified as yet, and this novel perilymph-associated CTP isoform might provide mechanistic clues to how mutations in the COCH gene damage the inner ear function.

Organization and development of corticocortical associative neurons expressing the orphan nuclear receptor Nurr1.

The developmental mechanism that contributes to the highly organized axonal connections within the cerebral cortex is not well understood. This is partly due to the lack of molecular markers specifically expressed in corticocortical associative neurons during the period of circuit formation. We have shown previously that latexin, a carboxypeptidase A inhibitor, is expressed in intrahemispheric corticocortical neurons from the second postnatal week in the rat (Arimatsu et al. [1999] Cereb. Cortex 9:569-576). In the present study, we first demonstrate in the adult rat that the orphan nuclear receptor Nurr1 is coexpressed in latexin-expressing neurons located in layer V, sublayer VIa, and the white matter of the lateral sector of the neocortex, and also in latexin-negative early born neurons in sublayer VIb of the entire neocortex. Virtually all Nurr1-expressing neurons exhibit immunoreactivity for phosphate-activated glutaminase but not for gamma-aminobutyric acid, suggesting that they are glutamatergic-excitatory neurons. By combining Nurr1 immunohistochemistry and 5-bromo-2'-deoxyuridine-birthdating, we then show that Nurr1 is expressed in (early born) subplate neurons and (later born) presumptive latexin-expressing neurons from embryonic day 18 onward. Finally, by combination of Nurr1 immunohistochemistry and retrograde tracing, we show that Nurr1-expressing neurons, including those in sublayer VIb, contribute predominantly to long-range intrahemispheric corticocortical projections. These results raise the possibility that Nurr1 plays a role in the establishment and maintenance of normal corticocortical circuitry and function.

Identification of S1 proteins B2, C1 and D1 as AUF1 isoforms and their major role as heterogeneous nuclear ribonucleoprotein proteins.

AUF1 (A+U-rich RNA binding factor) participates in the rapid decay of mRNAs in the cytoplasm. It is sometimes called heterogeneous nuclear ribonucleoprotein (hnRNP) D0; however, evidence for its characterization as an hnRNP protein has been scarce. S1 proteins A-D are those selectively extracted at pH 4.9 from isolated nuclei pretreated with either RNase A or DNase I. In the present study we identified S1 ('first supernatant') proteins B2, C1 and D1 with p45, p40 and p37 AUF1s respectively, by microsequencing and product analysis of transfected cDNAs. We found, further, that more than 96% of the S1 proteins occurred in the nucleus, and localized largely in RNase-sensitive structures. B2 was confined in the nucleus and C1 directly bound to heterogeneous nuclear RNAs (hnRNAs). These B2 and C1 proteins formed hnRNP structures responsible for the 33 S, and, to lesser extent, the 40 S particles, which were liberated upon mild nucleolytic cleavage. On the other hand, D1 and the remainder of C1 were associated with nuclease-hypersensitive sites of hnRNAs, and comprised the major cytoplasmic AUF1s that may be involved in mRNA decay. Two-dimensional immunoblotting resolved each S1 isoform into up to six spots or more, and suggested that the previous uncertain relationship of hnRNP D0 and hnRNP D is resolved in terms of charge differences and differential splicing arising from one gene. The present results thus indicate that S1 proteins B2, C1 and D1 are identical with AUF1 proteins, but largely occur as hnRNP proteins in the nucleus. That hnRNP D0 is indeed an hnRNP protein was verified.

Establishment of latrunculin-A resistance in HeLa cells by expression of R183A D184A mutant beta-actin.

Actin plays central roles in cell motility through formation of the actin cytoskeleton. Recently, the very intriguing possibility that actin also contributes to processes in the cell nucleus has been emerging. To dissect its dynamics and functions, several actin-disrupting drugs have been widely and effectively employed. Among them, latrunculin-A has proved particularly useful, supplanting the classical drug cytochalasin-D. One reason is that latrunculin-A appears to bind only to actin monomers impairing the nucleotide exchange, the mode being simpler than with cytochalasin. This property may be especially crucial when studying actin functions as a monomer, as suggested for nuclear actin. Very importantly, actin mutations that cause cells to become resistant to the effects of latrunculin-A have been identified in budding yeast. However, it remains controversial as to whether all of the various phenotypes observed with latrunculin in mammalian cells more complicated than yeast are truly the consequence of its specific actions against actin. Here, we show that the expression of R183A D184A mutant beta-actin specifically confers resistance to the effects of latrunculin-A on actin cytoskeleton formation and cell growth in HeLa cells. The established system provides a strong tool to address the various functions of actin in mammalian cells.

Gerbils of a seizure-sensitive strain have a mitochondrial inner membrane protein with different isoelectric points from those of a seizure-resistant strain.

The distribution of proteins in the cerebral cortex of a seizure-sensitive (SS) strain of gerbil and its seizure-resistant (SR) counterpart was profiled using two-dimensional gel electrophoresis. A series of proteins of similar molecular weight (around 83 kDa) showed small but consistent differences in their isoelectric point (pI) with indistinguishable profiles of distribution between the two strains. Amino acid sequences of peptides produced by limited proteolysis of each protein in the spots from the strains were identical or highly homologous to those of mitofilin, a mitochondrial inner membrane protein (IMMT) in humans. Analysis of cDNA sequences revealed the proteins of these spots to be gerbil mitofilin-like proteins (gIMMT), with a few base substitutions between SS and SR strains, in particular within a region near a putative transmembrane domain that is highly conserved in humans and gerbils. The amino acid at the site was acidic, Glu in humans and Asp in the strain SR of gerbil and a neutral, Asn in strain SS. In addition to these base substitutions, production of multiple species of mRNA for gIMMT by alternative splicing was observed.

Identification of mRNA/protein (mRNP) complexes containing Puralpha, mStaufen, fragile X protein, and myosin Va and their association with rough endoplasmic reticulum equipped with a kinesin motor.

Puralpha, which is involved in diverse aspects of cellular functions, is strongly expressed in neuronal cytoplasm. Previously, we have reported that this protein controls BC1 RNA expression and its subsequent distribution within dendrites and that Puralpha is associated with polyribosomes. Here, we report that, following treatment with EDTA, Puralpha was released from polyribosomes in mRNA/protein complexes (mRNPs), which also contained mStaufen, Fragile X Mental Retardation Protein (FMRP), myosin Va, and other proteins with unknown functions. As the coimmunoprecipitation of these proteins by an anti-Puralpha antibody was abolished by RNase treatment, Puralpha may assist mRNP assembly in an RNA-dependent manner and be involved in targeting mRNPs to polyribosomes in cooperation with other RNA-binding proteins. The immunoprecipitation of mStaufen- and FMRP-containing mRNPs provided additional evidence that the anti-Puralpha detected structurally or functionally related mRNA subsets, which are distributed in the somatodendritic compartment. Furthermore, mRNPs appear to reside on rough endoplasmic reticulum equipped with a kinesin motor. Based on our present findings, we propose that this rough endoplasmic reticulum structure may form the molecular machinery that mediates and regulates multistep transport of polyribosomes along microtubules and actin filaments, as well as localized translation in the somatodendritic compartment.

An extra human chromosome 21 reduces mlc-2a expression in chimeric mice and Down syndrome.

An extra copy of human chromosome 21 (Chr 21) causes Down syndrome (DS), which is characterized by mental retardation and congenital heart disease (CHD). Chimeric mice containing Chr 21 also exhibit phenotypic traits of DS including CHD. In this study, to identify genes contributing to DS phenotypes, we compared the overall protein expression patterns in hearts of Chr 21 chimeras and wild type mice by two-dimensional electrophoresis. The endogenous mouse atrial specific isoform of myosin light chain-2 (mlc-2a) protein was remarkably downregulated in the hearts of chimeric mice. We also confirmed that the human MLC-2A protein level was significantly lower in a human DS neonate heart, as compared to that of a normal control. Since mouse mlc-2a is involved in heart morphogenesis, our data suggest that the downregulation of this gene plays a crucial role in the CHD observed in DS. The dosage imbalance of Chr 21 has a trans-acting effect which lowers the expression of other genes encoded elsewhere in the genome.

Study of expression of myelin basic proteins (MBPs) in developing rat brain using a novel antibody reacting with four major isoforms of MBP.

Myelin basic proteins (MBPs) are the major protein components of myelin. MBP isoforms are known to have different expression patterns. In order to distinguish the different expression patterns on myelination, we have developed a novel antibody reacting with the four major isoforms of MBPs with molecular masses of 21.5 kDa, 18.5 kDa, 17.0 kDa, and 14.0 kDa. These MBPs were initially separated by acid urea gel and sodium dodecyl sulfate polyacrylamide gel electrophoreses and detected with the luminol reaction. Then the antibody developed was used to determine the relative amounts of MBP isoforms. The MBPs of oligodendrocytes were detected by the enhanced luminol reaction using Renaissance (Dupont NEN, Boston, MA). From the immunological aspect, the MBP monoclonal antibody (Sires et al. [1981] Science 214:87-89) was revealed to recognize MBPs with molecular masses of 21.5 kDa and 18.5 kDa. Furthermore, we found that Ile-166 in the rat 18.5-kDa MBP isomers was replaced by methionine. The 14.0-kDa and 18.5-kDa isoforms of MBP are the most abundant MBP species and comprise more than 70% of the total MBPs in 3.5-and 24-month-old rats. MBPs are expressed during development and the compositions of MBPs in mature (3.5 months old) and aged (24 months old) rats were almost the same. The expression of the 14.0-kDa and 18.5-kDa MBPs occurred earlier in the cerebellum and the spinal cord than in the cerebrum by approximately 1 week. MBPs are also expressed upon oligodendrocyte maturation by interacting with astrocytes. The above results suggest that the regulation of MBP isoforms during development and oligodendrocyte differentiation may indicate the point of occurrence of both the unique patterns of isoform expression and the shift in intracellular localization of MBPs with the maturation of oligodendrocytes.

Purification, characterization, and cDNA cloning of alpha-N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera.

We report here the purification, characterization, and cDNA cloning of a novel N-acetylgalactosamine-specific lectin from starfish, Asterina pectinifera. The purified lectin showed 19-kDa, 41-kDa, and 60-kDa protein bands on SDS-PAGE, possibly corresponding to a monomer, homodimer, and homotrimer. Interestingly, on 4-20% native PAGE the lectin showed at least nine protein bands, among which oligomers containing six to nine subunits had potent hemagglutination activity for sheep erythrocytes. The hemagglutination activity of the lectin was specifically inhibited by N-acetylgalactosamine, Tn antigen, and blood group A trisaccharide, but not by N-acetylglucosamine, galactose, galactosamine, or blood group B trisaccharide. The specificity of the lectin was further examined using various glycosphingolipids and biotin-labeled lectin. The lectin was found to bind to Gb5Cer, but not Gb4Cer, Gb3Cer, GM1a, GM2, or asialo-GM2, indicating that the lectin specifically binds to the terminal alpha-GalNAc at the nonreducing end. The hemagglutination activity of the lectin was completely abolished by chelation with EDTA or EGTA and completely restored by the addition of CaCl(2). cDNA cloning of the lectin showed that the protein is composed of 168 amino acids, including a signal sequence of 18 residues, and possesses the typical C-type lectin motif. These findings indicate that the protein is a C-type lectin. The recombinant lectin, produced in a soluble form by Escherichia coli, showed binding activity for asialomucin in the presence of Ca(2+) but no hemagglutination.

Interaction of elongation factor-1alpha and pleckstrin homology domain of phospholipase C-gamma 1 with activating its activity.

The pleckstrin homology (PH) domain is a small motif for membrane targeting in the signaling molecules. Phospholipase C (PLC)-gamma1 has two putative PH domains, an NH(2)-terminal and a split PH domain. Here we report studies on the interaction of the PH domain of PLC-gamma1 with translational elongation factor (EF)-1alpha, which has been shown to be a phosphatidylinositol 4-kinase activator. By pull-down of cell extract with the glutathione S-transferase (GST) fusion proteins with various domains of PLC-gamma1 followed by peptide sequence analysis, we identified EF-1alpha as a binding partner of a split PH domain of PLC-gamma1. Analysis by site-directed mutagenesis of the PH domain revealed that the beta2-sheet of a split PH domain is critical for the interaction with EF-1alpha. Moreover, Dot-blot assay shows that a split PH domain specifically binds to phosphoinositides including phosphatidylinositol 4-phosphate and phosphatidylinositol 4, 5-bisphosphate (PIP(2)). So the PH domain of PLC-gamma1 binds to both EF-1alpha and PIP(2). The binding affinity of EF-1alpha to the GST.PH domain fusion protein increased in the presence of PIP(2), although PIP(2) does not bind to EF-1alpha directly. This suggests that EF-1alpha may control the binding affinity between the PH domain and PIP(2). PLC-gamma1 is substantially activated in the presence of EF-1alpha with a bell-shaped curve in relation to the molar ratio between them, whereas a double point mutant PLC-gamma1 (Y509A/F510A) that lost its binding affinity to EF-1alpha shows basal level activity. Taken together, our data show that EF-1alpha plays a direct role in phosphoinositide metabolism of cellular signaling by regulating PLC-gamma1 activity via a split PH domain.

Molecular cloning and characterization of sphingolipid ceramide N-deacylase from a marine bacterium, Shewanella alga G8.

Recently, lyso-sphingolipids have been identified as ligands for several orphan G protein-coupled receptors, although the molecular mechanism for their generation has yet to be clarified. Here, we report the molecular cloning of the enzyme, which catalyzes the generation of lyso-sphingolipids from various sphingolipids (sphingolipid ceramide N-deacylase). The 75-kDa enzyme was purified from the marine bacterium, Shewanella alga G8, and its gene was cloned from a G8 genomic library using sequences of the purified enzyme. The cloned enzyme was composed of 992 amino acids, including a signal sequence of 35 residues, and its molecular weight was estimated to be 109,843. Significant sequence similarities were found with an unknown protein of Streptomyces fradiae Y59 and a Lumbricus terrestris lectin but not other known functional proteins. The 106-kDa recombinant enzyme expressed in Escherichia coli hydrolyzed various glycosphingolipids and sphingomyelin, although it seems to be much less active than the native 75-kDa enzyme. In vitro translation using wheat germ extract revealed the activity of a 75-kDa deletion mutant lacking a C terminus to be much stronger than that of the full-length enzyme, suggesting that C-terminal processing is necessary for full activity.