RESUMEN
Dscam, a novel cell-adhesion molecule belonging to the Ig-superfamily mediates homophilic intercellular adhesion and is expressed abundantly in the nervous system during development. To gain better understanding on the role of Dscam in neuronal differentiation, we raised an antibody and characterized its protein product. Anti-Dscam antibody detected an approximately 200-kDa protein band in human and mouse brain lysates. Immunohistochemical studies showed that during embryonic development of mice, mouse Dscam is expressed throughout the neuronal tissues and also in nonneuronal tissues such as lung, liver, and limb buds. In adult brain Dscam expression is predominant in the cerebellum, hippocampus, and olfactory bulb. Immunofluorescence double labeling of hippocampal and cerebellar primary cultures revealed that Dscam is associated with axonal and dendritic processes. In view of its cellular localization and spatiotemporal expression pattern, we suggest that Dscam is involved in cell-cell interactions during axonal-dendritic development, and maintenance of functional neuronal networks.
Asunto(s)
Axones/metabolismo , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Sistema Nervioso Central/embriología , Dendritas/metabolismo , Red Nerviosa/embriología , Proteínas/química , Envejecimiento/metabolismo , Animales , Axones/ultraestructura , Tipificación del Cuerpo/fisiología , Moléculas de Adhesión Celular , Células Cultivadas/metabolismo , Células Cultivadas/ultraestructura , Sistema Nervioso Central/crecimiento & desarrollo , Sistema Nervioso Central/ultraestructura , Cerebelo/embriología , Cerebelo/crecimiento & desarrollo , Cerebelo/ultraestructura , Dendritas/ultraestructura , Femenino , Feto , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica/fisiología , Hipocampo/embriología , Hipocampo/crecimiento & desarrollo , Hipocampo/ultraestructura , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Microscopía Electrónica , Peso Molecular , Red Nerviosa/citología , Red Nerviosa/crecimiento & desarrollo , Estructura Terciaria de Proteína/fisiología , Proteínas/aislamiento & purificaciónRESUMEN
Juvenile myoclonic epilepsy (JME) is the most frequent and, hence, most important form of hereditary grand mal epilepsy. Genetic linkage, haplotype, and recombination analyses have indicated that 6p11-12 (EJM1) is one of the candidate regions harboring a gene responsible for JME. In efforts to identify a gene responsible for JME, we identified several expressed sequences in the EJM1 critical region. Here we report the identification and characterization of a gene, named C6orf33, in the EJM1 region. Northern blot analysis showed that C6orf33 is predominantly expressed in brain but in mice, testis shows additional transcripts. C6orf33 is predicted to encode a novel approximately 40-kDa membrane protein, LMPB1, that defines a novel protein family by having highly conserved orthologs in eukaryotes and three putative paralogs in human. Biochemical and immunocytochemical studies revealed that LMPB1 is indeed an integral membrane protein that targets to lysosomal structures. LMPB1 may be involved in specialized lysosomal functions that are unique to brain and testis, and the C6orf33 gene is of interest as a candidate for EJM1.
Asunto(s)
Encéfalo/metabolismo , Cromosomas Humanos Par 6 , Proteínas de la Membrana/genética , Epilepsia Mioclónica Juvenil/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario/análisis , Células HeLa , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Filogenia , Proteínas/genética , Proteínas/metabolismo , Receptores de Progesterona , Homología de Secuencia de Aminoácido , Fracciones SubcelularesRESUMEN
DSCAM, a conserved gene involved in neuronal differentiation, is a member of the Ig superfamily of cell adhesion molecules. Herein, we report the functional characterization of a human DSCAM (Down syndrome cell adhesion molecule) paralogue, DSCAML1, located on chromosome 11q23. The deduced DSCAML1 protein contains 10 Ig domains, six fibronectin-III domains, and an intracellular domain, all of which are structurally identical to DSCAM. When compared to DSCAM, DSCAML1 protein showed 64% identity to the extracellular domain and 45% identity to the cytoplasmic domain. In the mouse brain, DSCAML1 is predominantly expressed in Purkinje cells of the cerebellum, granule cells of the dentate gyrus, and in neurons of the cerebral cortex and olfactory bulb. Biochemical and immunofluorescence analyses indicated that DSCAML1 is a cell surface molecule that targets axonal features in differentiated PC12 cells. DSCAML1 exhibits homophilic binding activity that does not require divalent cations. Based on its structural and functional properties and similarities to DSCAM, we suggest that DSCAML1 may be involved in formation and maintenance of neural networks. The chromosomal locus for DSCAML1 makes it an ideal candidate for neuronal disorders (such as Gilles de la Tourette and Jacobsen syndromes) that have been mapped on 11q23.
Asunto(s)
Moléculas de Adhesión Celular/genética , Proteínas de Drosophila , Proteínas/genética , Animales , Axones/metabolismo , Northern Blotting , Encéfalo/metabolismo , Adhesión Celular/genética , Moléculas de Adhesión Celular/biosíntesis , Línea Celular , Cromosomas Humanos Par 11/genética , Clonación Molecular , Secuencia Conservada , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Drosophila , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Proteínas de la Membrana , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Mapeo Físico de Cromosoma , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Lafora's disease, an autosomal recessive progressive myoclonus epilepsy, is caused by mutations in the EPM2A gene encoding a dual-specificity phosphatase (DSP) named laforin. Here, we analyzed the developmental and regional expression of murine Epm2a and discussed its evolutionary conservation. A phylogenetic analysis indicated that laforin is evolutionarily distant from other DSPs. Southern zoo blot analysis suggested that conservation of Epm2a gene is limited to mammals. Laforin orthologs (human, mouse, and rat) display more than 94% similarity. All missense mutations known in Lafora disease patients affect conserved residues, suggesting that they may be essential for laforin's function. Epm2a is expressed widely in various organs but not homogeneously in brain. The levels of Epm2a transcripts in mice brains increase postnatally, attaining its highest level in adults. The most intense signal was detected in the cerebellum, hippocampus, cerebral cortex, and the olfactory bulb. Our results suggest that Epm2a is functionally conserved in mammals and is involved in growth and maturation of neural networks.
Asunto(s)
Encéfalo/metabolismo , Evolución Molecular , Regulación del Desarrollo de la Expresión Génica , Filogenia , Proteínas Tirosina Fosfatasas/química , Proteínas Tirosina Fosfatasas/genética , Envejecimiento , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Corteza Cerebral/metabolismo , Secuencia Conservada , Fosfatasas de Especificidad Dual , Desarrollo Embrionario y Fetal , Biblioteca de Genes , Humanos , Hibridación Fluorescente in Situ , Masculino , Ratones , Datos de Secuencia Molecular , Mutación Missense , Especificidad de Órganos , Proteínas Tirosina Fosfatasas no Receptoras , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , VertebradosRESUMEN
Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in beta1 and alpha(I)-subunit genes of voltage-gated Na(+) channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R188W) [corrected] in the gene encoding the alpha-subunit of neuronal voltage-gated Na(+) channel type II (Na(v)1.2) in a patient with FS associated with afebrile seizures. The mutation R188W [corrected] occurring on Arg(187), a highly conserved residue among voltage-gated Na(+) channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNa(v)1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na(+) channel conductance was not affected. Prolonged residence in the open state of the R188W [corrected] mutant channel may augment Na(+) influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Na(v)1.2 in a human disease and propose the R188W [corrected] mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.
Asunto(s)
Mutación Missense , Proteínas del Tejido Nervioso/genética , Convulsiones Febriles/genética , Convulsiones/genética , Canales de Sodio/genética , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Niño , ADN Complementario , Electrofisiología , Humanos , Masculino , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.1 , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas del Tejido Nervioso/fisiología , Ratas , Canales de Sodio/fisiologíaRESUMEN
Down Syndrome Cell Adhesion molecule (DSCAM) is a member of the immunoglobulin superfamily, and represents a novel class of neuronal cell adhesion molecules. In order to understand the cellular functions of DSCAM, we isolated full-length mouse and human cDNA clones, and analysed its expression during mouse development and differentiation. Sequence analysis of the human DSCAM cDNA predicted at least 33 exons that are distributed over 840 kb. When compared to human DSCAM, the mouse homologue showed 90 and 98% identity at the nucleotide and amino acid levels, respectively. In mouse, DSCAM is located on 16C, the syntenic region for human chromosome band 21q22 and also the region duplicated in mouse DS models. DSCAM gene is predicted to encode an approximately 220-kDa protein, and its expression shows dynamic changes that correlate with neuronal differentiation during mouse development. Our results suggest that DSCAM may play critical roles in the formation and maintenance of specific neuronal networks in brain.
Asunto(s)
Encéfalo/metabolismo , Proteínas/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Encéfalo/embriología , Moléculas de Adhesión Celular , Mapeo Cromosómico , ADN Complementario , Genoma , Humanos , Hibridación in Situ , Proteínas de la Membrana , Ratones , Datos de Secuencia Molecular , Proteínas/química , ARN Mensajero/genética , Homología de Secuencia de AminoácidoRESUMEN
The progressive myoclonus epilepsy of Lafora type is an autosomal recessive disorder caused by mutations in the EPM2A gene. EPM2A is predicted to encode a putative tyrosine phosphatase protein, named laforin, whose full sequence has not yet been reported. In order to understand the function of the EPM2A gene, we isolated a full-length cDNA, raised an antibody and characterized its protein product. The full-length clone predicts a 38 kDa laforin that was very close to the size detected in transfected cells. Recombinant laforin was able to hydrolyze phosphotyrosine as well as phosphoserine/threonine substrates, demonstrating that laforin is an active dual-specificity phosphatase. Biochemical, immunofluorescence and electron microscopic studies on the full-length laforin expressed in HeLa cells revealed that laforin is a cytoplasmic protein associated with polyribosomes, possibly through a conformation-dependent protein-protein interaction. We analyzed the intracellular targeting of two laforin mutants with missense mutations. Expression of both mutants resulted in ubiquitin-positive perinuclear aggregates suggesting that they were misfolded proteins targeted for degradation. Our results suggest that laforin is involved in translational regulation and that protein misfolding may be one of the molecular bases of the Lafora disease phenotype caused by missense mutations in the EPM2A gene.
Asunto(s)
Enfermedad de Lafora/genética , Polirribosomas/metabolismo , Proteínas Tirosina Fosfatasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Fraccionamiento Celular , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Enfermedad de Lafora/metabolismo , Microscopía Confocal , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación Missense , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Ubiquitinas/metabolismoRESUMEN
Down Syndrome (DS) caused by trisomy 21 is the most common birth defect associated with mental retardation. Recently, a novel gene named, DSCAM, has been identified in the DS critical region. DSCAM is predicted to be a transmembrane protein with a very high structural and sequence homology to Ig superfamily of cell adhesion molecules and is expressed in the developing nervous system with the highest level in fetal brain. Diverse glycoproteins of cell surfaces and extracellular matrices operationally termed as 'adhesion molecule' are important in the specification of cell interactions during development, maintenance and regeneration of the nervous system. To understand the cellular function of DSCAM protein, we transfected human DSCAM cDNA into mouse fibroblast L cells and analysed its expression. On Western blot analysis, antibodies raised against recombinant DSCAM-Ig3 recognized a 198 kDa protein band in the membrane fraction of DSCAM transfected L cells. Stable transformants expressing DSCAM showed uniform surface expression. DSCAM-expressing transfectants exhibited enhanced adhesive properties, aggregating with faster kinetics and forming aggregates in a homophilic manner. Divalent cations are not required for this cell aggregation. These results demonstrate that DSCAM is a cell adhesion molecule that can mediate cation-independent homophilic binding activity between DSCAM expressing cells.
Asunto(s)
Proteínas/fisiología , Animales , Adhesión Celular , Moléculas de Adhesión Celular , Agregación Celular , ADN Complementario , Síndrome de Down , Humanos , Células L , Proteínas de la Membrana , Ratones , Proteínas/genética , TransfecciónRESUMEN
Hyperhomocysteinemia, a risk factor for vascular disease, injures endothelial cells through undefined mechanisms. We previously identified several homocysteine-responsive genes in cultured human vascular endothelial cells, including the endoplasmic reticulum (ER)-resident molecular chaperone GRP78/BiP. Here, we demonstrate that homocysteine induces the ER stress response and leads to the expression of a novel protein, Herp, containing a ubiquitin-like domain at the N terminus. mRNA expression of Herp was strongly up-regulated by inducers of ER stress, including mercaptoethanol, tunicamycin, A23187, and thapsigargin. The ER stress-dependent induction of Herp was also observed at the protein level. Immunochemical analyses using Herp-specific antibodies indicated that Herp is a 54-kDa, membrane-associated ER protein. Herp is the first integral membrane protein regulated by the ER stress response pathway. Both the N and C termini face the cytoplasmic side of the ER; this membrane topology makes it unlikely that Herp acts as a molecular chaperone for proteins in the ER, in contrast to GRP78 and other ER stress-responsive proteins. Herp may, therefore, play an unknown role in the cellular survival response to stress.
Asunto(s)
Retículo Endoplásmico/fisiología , Endotelio Vascular/fisiología , Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana/genética , Transcripción Genética , Ubiquitinas/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcimicina/farmacología , Células Cultivadas , Clonación Molecular , Retículo Endoplásmico/efectos de los fármacos , Chaperón BiP del Retículo Endoplásmico , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Membranas Intracelulares/fisiología , Membranas Intracelulares/ultraestructura , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Mercaptoetanol/farmacología , Ratones , Datos de Secuencia Molecular , Biosíntesis de Proteínas , Conformación Proteica , ARN Mensajero/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Tapsigargina/farmacología , Transcripción Genética/efectos de los fármacos , Tunicamicina/farmacología , Venas UmbilicalesRESUMEN
RTP, also called Drg1/Cap43/rit42/TDD5/Ndr1, was originally identified as a homocysteine-responsive gene product, and is now considered to be involved in stress responses, atherosclerosis, carcinogenesis, differentiation, androgen responses, hypoxia, and N-myc pathways. We raised an antiserum against a recombinant human RTP. Western blot analysis showed that RTP expression was induced in human umbilical vein endothelial cells under conditions causing endoplasmic reticulum stress. RTP was partially phosphorylated at seven or more sites. The phosphorylation was reversible, and was enhanced by an increased level of intracellular cAMP and inhibited by both a protein kinase A inhibitor and a calmodulin kinase inhibitor. Protein kinase A directly phosphorylated recombinant RTP in vitro. The phosphorylated forms were abundant in cells at the early log phase, and then decreased with increasing cell density. These data demonstrated that RTP is a phosphorylated stress-responsive protein, and its phosphorylation may be related to cell growth.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Citoplasma/química , Retículo Endoplásmico/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Recuento de Células , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/inmunología , Células Cultivadas , AMP Cíclico/análogos & derivados , AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cicloheximida/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Retículo Endoplásmico/química , Retículo Endoplásmico/efectos de los fármacos , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Glicosilación , Células HeLa , Homocisteína/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Peso Molecular , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Transfección , Tunicamicina/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
An elevated plasma level of homocysteine is associated with arteriosclerosis and thrombosis. The mechanisms by which homocysteine may promote vascular diseases have not yet been elucidated. In the present study, we have applied a modified nonradioactive differential display analysis to evaluate changes in gene expression induced by homocysteine treatment of cultured human umbilical vein endothelial cells (HUVEC). We identified six upregulated and one downregulated gene. One upregulated gene was GRP78/BiP, a stress protein, suggesting that unfolded proteins would accumulate in the endoplasmic reticulum because of redox potential changes caused by homocysteine. Another upregulated gene encoded a bifunctional enzyme with activities of methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase, which is involved in a homocysteine metabolism. A third upregulated gene encoded activating transcription factor 4. The remaining four were uncharacterized genes. We isolated a full-length cDNA of one of the upregulated genes from a HUVEC library. It encoded a novel protein with 394 amino acids, which was termed reducing-agents and tunicamycin-responsive protein (RTP). Northern blot analysis revealed that RTP gene expression was induced in HUVEC after 4 h of incubation with homocysteine. RTP mRNA was also observed in unstimulated cells and induced by not only homocysteine but also 2-mercaptoethanol and tunicamycin. The mRNA was ubiquitously expressed in human tissues. These observations indicate that homocysteine can alter the expressivity of multiple genes, including a stress protein and several novel genes. These responses may contribute to atherogenesis.
Asunto(s)
Antibacterianos/farmacología , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Endotelio Vascular/fisiología , Regulación de la Expresión Génica , Proteínas de Choque Térmico , Homocisteína/toxicidad , Chaperonas Moleculares/genética , Tunicamicina/farmacología , Secuencia de Aminoácidos , Proteínas Portadoras/biosíntesis , Células Cultivadas , Clonación Molecular , Chaperón BiP del Retículo Endoplásmico , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular , Chaperonas Moleculares/biosíntesis , Datos de Secuencia MolecularRESUMEN
Small granules of horseshoe crab hemocytes contain two known major antimicrobial substances, tachyplesin and big defensin (S5), and at least five protein components (S1 to S6), with unknown functions. In the present study, we examined the biological properties and primary structure of a small granular component S2, named tachycitin. This component was purified from the acid extract of hemocyte debris by two steps of chromatography. The purified tachycitin was a single chain protein with an apparent M(r) = 8,500 on Tricine-SDS-polyacrylamide gel electrophoresis. Ultracentrifugation analysis revealed tachycitin to be present in monomer form in solution. Tachycitin inhibited the growth of both Gram-negative and -positive bacteria, and fungi, with a bacterial agglutinating property. Moreover, tachycitin and big defensin acted synergistically in antimicrobial activities. The amino acid sequence and intrachain disulfide bonds of tachycitin were determined by amino acid and sequence analyses of peptides produced by enzymatic cleavages. The mature tachycitin consisted of 73 amino acid residues containing five disulfide bonds with no N-linked sugar. A cDNA coding for tachycitin was isolated from a hemocyte cDNA library. The open reading frame coded for an NH2-terminal signal sequence followed by the mature peptide and an extension sequence of -Gly-Arg-Lys at the COOH-terminus, which is a putative amidating signal. The COOH-terminal threonine amide released after digestion of tachycitin with lysylendopeptidase was identified. The NH2-terminal 28 residues of tachycitin shows sequence homology to a part of chitin-binding regions found in antifungal chitin-binding peptides, chitin-binding lectins, and chitinases, all of which have been isolated from plants. Tachycitin showed a specific binding to chitin but did not bind with the polysaccharides cellulose, mannan, xylan, and laminarin. Tachycitin may represent a new class of chitin-binding protein family in animals.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Portadoras/química , Quitina/metabolismo , Hemocitos/química , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Secuencia de Bases , Proteínas Sanguíneas/metabolismo , Proteínas Portadoras/metabolismo , Defensinas , Disulfuros/química , Precursores Enzimáticos/metabolismo , Cangrejos Herradura , Modelos Moleculares , Datos de Secuencia Molecular , Polisacáridos/metabolismo , Alineación de Secuencia , Serina Endopeptidasas/metabolismoRESUMEN
We reported that limulus intracellular coagulation inhibitor type-1 (LICI-1) (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J. Biol. Chem. 269, 542-547) and LICI type-2 (LICI-2) (Miura, Y., Kawabata, S. , Wakamiya, Y., Nakamura, T., and Iwanaga, S. (1995) J. Biol. Chem. 270, 558-565) found in the hemocyte lysate belong to the serpin family. The LICI-1 specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C (k1 = 2.5 x 10(6) M-1 s-1), whereas LICI-2 inhibits preferentially limulus clotting enzyme (k1 = 4.3 x 10(5) M-1 s-1). In our ongoing studies on limulus serpin, we found another inhibitor, named LICI type-3 (LICI-3), which strongly inhibits (1,3)-beta-D-glucan-sensitive serine protease, factor G (k1 = 3.9 x 10(5) M-1 s-1). Thus, the limulus hemolymph coagulation cascade is effectively regulated by at least the three endogenous serpins. LICI-3, newly identified in hemocytes, is a single chain glycoprotein with an apparent Mr = 53,000, the largest one among known limulus serpins. A cDNA sequence for LICI-3 coded a mature protein of 392 amino acids, of which 68 residues were confirmed by peptide sequencing. LICI-3 showed significant sequence similarity to LICI-1 (45.8% identity) and LICI-2 (33.7% identity). LICI-3 contained a putative reactive site, -Arg-Ser-, distinct from that of LICI-2 (-Lys-Ser-) but the same as that of LICI-1. Expression of LICI-3 mRNA was detected only in hemocytes, and not in heart, brain, stomach, intestine, coxal gland, and skeletal muscle. Immunoblotting of the hemocyte-derived large and small granules with antiserum against LICI-3 suggested that it is stored specifically in large granules, as in the case of LICI-1 and LICI-2, and is released in response to external stimuli.
Asunto(s)
Glicoproteínas/genética , Cangrejos Herradura/fisiología , Hormonas de Invertebrados/genética , Inhibidores de Serina Proteinasa/genética , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos , Secuencia de Bases , Factores de Coagulación Sanguínea/antagonistas & inhibidores , Proteínas Sanguíneas/química , Quimotripsina/química , ADN Complementario/genética , Defensinas , Expresión Génica , Genes , Glicoproteínas/fisiología , Cangrejos Herradura/genética , Hormonas de Invertebrados/fisiología , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Inhibidores de Serina Proteinasa/aislamiento & purificación , Serpinas , Distribución TisularRESUMEN
A cysteine protease inhibitor with an apparent Mr = 12,600, designated limulus (L)-cystatin, was isolated from hemocyte lysates of the Japanese horseshoe crab (Tachypleus tridentatus), using two steps of chromatography, including dextran sulfate-agarose, and carboxymethylated papain-agarose. L-cystatin inhibits amidolytic activity of papain by forming a noncovalent 1:1 complex with an equilibrium constant (Ki) of 0.08 nM. It also inhibits cathepsin L (Ki = 0.17 nM) and ficin (Ki = 0.52 nM), but not argingipain (a bacterial cysteine protease) and calpains. A cDNA for L-cystatin was isolated and the open reading frame coded for a mature protein of 114 amino acids, of which 99 residues were confirmed by peptide sequencing. L-cystatin shows significant sequence identities to members of the family 2 cystatin, such as bovine colostrum cystatin (33%) and human cystatin S (31%). Northern blotting revealed expression of the mRNA in hemocytes and slightly in heart but expression was negligible in hepatopancreas, intestine, stomach, and muscle. Immunoblotting revealed the localization to be in the large granules of hemocytes. Furthermore, L-cystatin has an antimicrobial activity against Gram-negative bacteria, which is much stronger than that of chicken egg white cystatin. These data suggest that the large granule-derived L-cystatin serves synergistically to accomplish an effective defense against invading microbes, together with other defense molecules that are released in response to external stimuli.
Asunto(s)
Cistatinas/química , Cistatinas/genética , Inhibidores de Cisteína Proteinasa/farmacología , Hemocitos/química , Cangrejos Herradura/química , Aglutininas/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Secuencia de Bases , Northern Blotting , Bovinos , Clonación Molecular , Cistatinas/farmacología , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/genética , ADN Complementario/química , ADN Complementario/genética , Hemocitos/ultraestructura , Cangrejos Herradura/citología , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Papaína/antagonistas & inhibidores , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Fracciones Subcelulares/químicaRESUMEN
O-Linked oligosaccharide chains were identified in the activation peptide (AP) of human blood coagulation factor IX. The peptide obtained from human factor IX was separated into three molecular species (AP alpha, AP beta, and AP gamma) by reversed-phase high-performance liquid chromatography. Amino acid analysis showed that AP alpha, but not AP beta and AP gamma, contained galactosamine in addition to glucosamine, thereby suggesting the presence of an O-linked sugar chain(s) in the molecule of AP alpha. A nonapeptide (AP alpha-D4, residues 157-165) and an undecapeptide (AP alpha-D5, 166-176) derived from AP alpha contained Thr-159 and Thr-169, neither of which could be identified using a gas-phase protein sequencer. All other serine and threonine residues present in AP alpha were identified by peptide sequencing. Component sugar and sialic acid analyses of AP alpha-D4 and AP alpha-D5 revealed that they contained 1 mol each of N-acetyl-D-galactosamine (GalNAc), D-galactose (Gal), and sialic acid. Fast atom bombardment tandem mass spectrometric analysis of AP alpha-D4 suggested the existence of Gal-GalNAc-Thr, NeuNAc-(Gal-)GalNAc-Thr, and NeuNAc-Gal-GalNAc-Thr structures. On the basis of amino acid analysis after the isolation of AP alpha, it accounted for approximately 35% of the total activation peptide obtained. From these results, it was concluded that a part of the activation peptide of human factor IX in circulating blood has tri- and tetrasaccharides O-glycosidically linked to the threonine residues at 159 and 169.