RESUMEN
We have cloned a new member of the interferon (IFN)-induced guanylate-binding protein (GBP) family of GTPases, murine GBP-2 (mGBP-2), from bone marrow-derived macrophages. mGBP-2 is located on murine chromosome 3, where it is linked to mGBP-1. With the identification of mGBP-2 there are now two human and two murine GBPs. Like other GBPs, mGBP-2 RNA and protein are induced by IFN-gamma. In addition, mGBP-2 shares with the other GBPs important structural features that distinguish this family from other GTPases. First, mGBP-2 contains only two of the three consensus sequences for nucleotide binding found within the classic GTP binding regions of other GTPases. A second amino acid motif found in mGBP-2 is a potential C-terminal site for isoprenoid modification, called a CaaX sequence. mGBP-2 is prenylated, as detected by [3H]mevalonate incorporation, when expressed in COS cells and preferentially incorporates the C-20 isoprenoid geranylgeraniol. Surprisingly, despite having a functional CaaX sequence, mGBP-2 is primarily cytosolic. GBP proteins are very abundant in IFN-exposed cells, but little is known about their function. mGBP-2 is expressed by IFN-gamma-treated cells from C57Bl/6 mice, whereas mGBP-1 is not. Thus, the identification of mGBP-2 makes possible the study of GBP function in the absence of a second family member.
Asunto(s)
GTP Fosfohidrolasas/aislamiento & purificación , Proteínas de Unión al GTP/genética , Interferón gamma/farmacología , Macrófagos/enzimología , Familia de Multigenes , Secuencia de Aminoácidos , Animales , Células COS , Células Cultivadas , Mapeo Cromosómico , Clonación Molecular , Inducción Enzimática , GTP Fosfohidrolasas/sangre , Proteínas de Unión al GTP/biosíntesis , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Prenilación de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Glypicans are a member of a family of glycosylphosphatidylinositol anchored heparan sulfate proteoglycans that are expressed in cell and development specific patterns. Rat GPC1 cDNA probes were used to screen rat genomic libraries. Three overlapping genomic clones that contained the entire rat GPC1 gene were isolated. The rat GPC1 gene is approximately 15kb in length and consists of eight exons interrupted by introns of varying lengths. Two of the introns are quite short, with lengths of 41 and 43 base pairs. Each exon-intron splice junction exhibited the consensus splice site sequence. Exon 1 encodes the putative signal peptide and the serine residue of the first putative heparan sulfate attachment site. The last exon encodes the cluster of three potential COOH-terminal heparan sulfate attachment sites, the putative GPI anchor and polypeptide cleavage site, and the 3'-untranslated region including the polyadenylation signal. One of the genomic clones extended approximately 2.8 kb 5' of the exon 1 coding sequence, and is thus likely to contain sequences that regulate GPC1 gene expression. Sequence analysis of the 5'-flanking sequence revealed a lack of consensus TATA and CAAT boxes. A search for potential transcription factor binding sites revealed a number of such motifs, including Sp1 (GC box), NF-kappaB, and MyoD (E-box). This region of the rat GPC1 gene shows significant sequence homology to the 5'-flanking region of the human GPC3 gene. Functional promoter activity of the rat GPC1 sequence was demonstrated by its ability to drive the expression of a luciferase reporter gene in several cell types.
Asunto(s)
Proteoglicanos de Heparán Sulfato/genética , Regiones Promotoras Genéticas/genética , Animales , Secuencia de Bases , Células Cultivadas , Clonación Molecular , ADN Complementario/análisis , Exones , Fibroblastos , Genes , Proteoglicanos de Heparán Sulfato/química , Intrones , Datos de Secuencia Molecular , Ratas , Secuencias Reguladoras de Ácidos Nucleicos , Células de SchwannRESUMEN
The cytoplasmic domain of the syndecan family of heparan sulfate proteoglycans is punctuated by the presence of four regularly spaced tyrosine residues. In this report, we explore the possibility of whether the four tyrosine residues in the cytoplasmic domain of N-syndecan (Syndecan 3) are potential substrates for phosphorylation by a tyrosine kinase. Bacterially expressed elk kinase was used to phosphorylate a series of bacterially expressed N-syndecan fusion proteins. Our results clearly demonstrate that the tyrosine residues in the cytoplasmic domain of N-syndecan can be phosphorylated by a tyrosine-specific kinase, and that all four tyrosine residues are capable of being phosphorylated.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Proteoglicanos/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Clonación Molecular , Drosophila melanogaster/metabolismo , Glutatión Transferasa , Proteínas de Unión a Maltosa , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Reacción en Cadena de la Polimerasa , Proteoglicanos/biosíntesis , Proteoglicanos/química , Ratas , Proteínas Tirosina Quinasas Receptoras/biosíntesis , Receptor EphB4 , Receptores de la Familia Eph , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Sindecano-3RESUMEN
Heparan sulfate moieties of cell-surface proteoglycans modulate the biological responses to fibroblast growth factors (FGFs). We have reported previously that cell-associated heparan sulfates inhibit the binding of the keratinocyte growth factor (KGF), but enhance the binding of acidic FGF to the KGF receptor, both in keratinocytes, which naturally express this receptor, and in rat myoblasts, which ectopically express it (Reich-Slotky, R., Bonneh-Barkay, D., Shaoul, E., Berman, B., Svahn, C. M., and Ron, D. (1994) J. Biol. Chem. 269, 32279-32285). The proteoglycan bearing these modulatory heparan sulfates was purified to homogeneity from salt extracts of rat myoblasts by anion-exchange and FGF affinity chromatography and was identified as rat glypican. Affinity-purified glypican augmented the binding of acidic FGF and basic FGF to human FGF receptor-1 in a cell-free system. This effect was abolished following digestion of glypican by heparinase. Addition of purified soluble glypican effectively replaced heparin in supporting basic FGF-induced cellular proliferation of heparan sulfate-negative cells expressing recombinant FGF receptor-1. In keratinocytes, glypican strongly inhibited the mitogenic response to KGF while enhancing the response to acidic FGF. Taken together, these findings demonstrate that glypican plays an important role in regulating the biological activity of fibroblast growth factors and that, for different growth factors, glypican can either enhance or suppress cellular responsiveness.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Proteoglicanos/metabolismo , Receptores Virales/metabolismo , Animales , Línea Celular , Factor 10 de Crecimiento de Fibroblastos , Factor 7 de Crecimiento de Fibroblastos , Sustancias de Crecimiento/metabolismo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/química , Heparitina Sulfato/aislamiento & purificación , Humanos , Proteoglicanos/química , Proteoglicanos/aislamiento & purificación , RatasRESUMEN
The amino acid sequence of rat N-syndecan core protein was deduced from the cloned cDNA sequence. The sequence predicts a core protein of 442 amino acids with six structural domains: an NH2-terminal signal peptide, a membrane distal glycosaminoglycan attachment domain, a mucin homology domain, a membrane proximal glycosaminoglycan attachment domain, a single transmembrane domain, and a noncatalytic COOH-terminal cytoplasmic domain. Transfection of human 293 cells resulted in the expression of N-syndecan that was modified by heparan sulfate chain addition. Heparitinase digestion of the expressed proteoglycan produced a core protein that migrated on SDS-polyacrylamide gels at an apparent molecular weight of 120, 000, identical to N-syndecan synthesized by neonatal rat brain or Schwann cells. Rat genomic DNA coding for N-syndecan was isolated by hybridization screening. The rat N-syndecan gene is comprised of five exons. Each exon corresponds to a specific core protein structural domain, with the exception of the fifth exon, which contains the coding information for both the transmembrane and cytoplasmic domains as well as the 3'-untranslated region of the mRNA. The first intron is large, with a length of 22 kilobases. The expression of N-syndecan was investigated in late embryonic, neonatal, and adult rats by immunoblotting and Northern blotting analysis. Among the tissues and developmental stages studied, high levels of N-syndecan expression were restricted to the early postnatal nervous system. N-syndecan was expressed in all regions of the nervous system, including cortex, midbrain, spinal cord, and peripheral nerve. Immunohistochemical staining revealed high levels of N-syndecan expression in all brain regions and fiber tract areas.
Asunto(s)
Encéfalo/metabolismo , Glicoproteínas de Membrana/biosíntesis , Glicoproteínas de Membrana/genética , Proteoglicanos/biosíntesis , Proteoglicanos/genética , Médula Espinal/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Exones , Expresión Génica , Biblioteca Genómica , Humanos , Inmunohistoquímica , Intrones , Riñón , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/química , Señales de Clasificación de Proteína/metabolismo , Proteoglicanos/química , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Sindecano-3 , TransfecciónRESUMEN
The expression of cell surface heparan sulfate proteoglycans in rat heart was investigated by Northern blot analysis with specific cDNA probes. In adult heart syndecan-3 and glypican mRNAs were abundantly expressed. Lower levels of syndecan-2 mRNA and very low levels of syndecan-1 mRNA were also detected. Analysis of RNA isolated from hearts of rats of various ages revealed that syndecan-3 and glypican mRNAs levels increased dramatically at birth, and continued to be expressed at high levels in adult animals. To determine which of these proteoglycans was expressed in cardiomyocytes, primary cultures of cardiomyocytes and nonmyocytes isolated from neonatal rat hearts were analyzed for proteoglycan expression. Glypican mRNA was localized almost exclusively to cardiomyocytes. Syndecan-3 mRNA was not detected in myocytes, but was detected in the nonmyocyte cells. Biochemical characterization of cardiomyocyte glypican revealed that it was a phosphatidylinositol-anchored heparan sulfate proteoglycan. Results of immunofluorescent staining of rat hearts with anti-glypican antibodies were consistent with the Northern blot data, and localized glypican to the lateral regions of myocyte plasma membrane that contact the basement membrane, as well as sites of myocyte adhesion junctions. At the latter site glypican colocalized with vinculin. Visualization of basic fibroblast growth factor binding sites by means of a tissue slice overlay assay also revealed colocalization with glypican. These results demonstrate developmental and cell-type-specific expression of membrane heparan sulfate proteoglycans in the heart. They also show that glypican is a major heparan sulfate proteoglycan expressed on the cardiomyocyte plasma membrane.
Asunto(s)
Heparitina Sulfato/genética , Miocardio/metabolismo , Proteoglicanos/genética , Animales , Animales Recién Nacidos , Sitios de Unión , Membrana Celular/metabolismo , Células Cultivadas , Corazón Fetal/citología , Corazón Fetal/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Corazón/crecimiento & desarrollo , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/química , Glicoproteínas de Membrana/genética , Miocardio/citología , Proteoglicanos/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Sindecano-3RESUMEN
The guanylate binding proteins, GBPs, are a family of interferon-induced GTP-binding proteins that include the rat p67. We report here that rat p67, for which interferon regulation had not previously been demonstrated, is induced by IFN-gamma and also by LPS in both cultured bone marrow-derived macrophages and microglia. The basal level of rat p67 in macrophages is low but increases dramatically between 2 and 4 hours after treating cells with either IFN-gamma or LPS. It then remains elevated over the next 24 hours. Rat p67 is isoprenoid modified. The isoprenoid modification was detected in p67 isolated both from primary IFN-gamma-activated macrophages and when the gene for p67 was transfected into COS cells. This is the first demonstration of in vivo prenylation of a GBP. The interferon regulation and prenylation of rat p67 point toward this protein being significant in the functions of both activated macrophages and microglia.
Asunto(s)
Proteínas de Unión al GTP/biosíntesis , Interferón gamma/farmacología , Macrófagos/metabolismo , Ácido Mevalónico/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Células de la Médula Ósea , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al GTP/aislamiento & purificación , Immunoblotting , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa , Prenilación de Proteína , Ratas , Ratas Sprague-DawleyRESUMEN
We expressed domains of the core protein of the transmembrane heparan sulfate proteoglycan N-syndecan (syndecan-3) either individually or as maltose-binding protein fusion proteins. Biochemical characterization of the purified proteins revealed that some of them were capable of self-association and formed stable, noncovalent multimeric complexes. The formation of N-syndecan core protein complexes was also demonstrated in mammalian cells by in situ cross-linking. Identification of structural motifs in the core protein of N-syndecan responsible for the formation of these complexes was accomplished by analyzing a series of constructs comprising different regions of the protein as well as site-directed mutants. Self-association was assayed by SDS-polyacrylamide gel electrophoresis, glutaraldehyde cross-linking, and size-exclusion high pressure liquid chromatography. Our results indicated that (i) the transmembrane domain of the N-syndecan core protein was required but not sufficient for the formation of stable complexes; (ii) the minimal amino acid sequence that conferred the ability of the N-syndecan core protein to form multimeric complexes included the last four amino acids (ERKE) of the extracellular domain plus the transmembrane domain; (iii) point mutations that changed the basic residues in this sequence to alanine residues either partially or completely abolished the ability of the N-syndecan core protein to form complexes; and (iv) replacement of conserved glycine residues in the transmembrane domain with leucines abolished complex formation. This property is similar to the oligomerization activity of other transmembrane receptors and suggests that regulated self-association may be important for the biological activity of transmembrane proteoglycans.
Asunto(s)
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/química , Proteoglicanos/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Cromatografía Líquida de Alta Presión , Reactivos de Enlaces Cruzados , Electroforesis en Gel de Poliacrilamida , Factor Xa/metabolismo , Glutaral , Maltosa/metabolismo , Proteínas de Unión a Maltosa , Glicoproteínas de Membrana/aislamiento & purificación , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Proteoglicanos/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Sindecano-3RESUMEN
The cDNA coding for a 67 kDa protein (p67) was isolated from a rat Schwann cell library. A recombinant form of p67 expressed in bacteria was used to produce polyclonal anti-p67 antibodies. By immunoblot analysis p67 was found to be expressed in most tissues and cell lines examined. Inspection of the deduced amino acid sequence revealed a COOH-terminal consensus sequence for isoprenylation. Consistent with this finding, p67 was a substrate for isoprenylation in vitro by geranylgeranylpyrophosphate. p67 was associated predominantly with the particulate fraction of rat smooth muscle cells. The rat p67 sequence was highly homologous to a family of recently described human and mouse gamma-interferon inducible, guanine nucleotide binding proteins.
Asunto(s)
Proteínas Portadoras/genética , ADN Complementario/aislamiento & purificación , Proteínas de Unión al GTP/genética , Células de Schwann/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Prenilación de Proteína , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de AminoácidoRESUMEN
To investigate the biological functions of transmembrane proteoglycans we have produced clonal cell lines of rat Schwann cells that express the hybrid proteoglycan syndecan-1. This was done by transfection of newborn rat Schwann cells with a plasmid vector bearing the rat syndecan-1 cDNA sequence under transcriptional control of the constitutively active cytomegalovirus promoter, and a neomycin resistance gene. Stably expressing cells were selected by growth in G418. Expression of syndecan-1 was verified by Northern and immunoblot analysis and immunoprecipitation of 35SO4-labeled proteoglycans. The syndecan-1 expressing cells exhibited significantly enhanced spreading on several different substrata, including fibronectin and laminin, and an altered morphology. The enhanced spreading appeared to result from the presence of syndecan-1, based on the observation that anti-syndecan-1 antibodies inhibited the enhanced substratum spreading. There was also a reorganization of cytoskeletal structures and formation of focal adhesions, visualized by anti-vinculin staining, which were absent from control Schwann cells. There was no apparent stable association of cell surface syndecan-1 with focal contact sites, as determined by dual staining with anti-syndecan-1 and anti-vinculin antibodies. Colocalization of patches of cell surface syndecan-1 with actin was observed, but only during cell spreading. These findings provide evidence for a role of transmembrane proteoglycans in cellular morphogenesis, and suggest that transient association of syndecans with microfilaments may be an important aspect of their biological function.
Asunto(s)
Actinas/metabolismo , Adhesión Celular , Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/metabolismo , Células de Schwann/metabolismo , Animales , Animales Recién Nacidos , Compartimento Celular , Células Cultivadas , Citoesqueleto/ultraestructura , Técnica del Anticuerpo Fluorescente , Proteínas de Microfilamentos/metabolismo , Ratas , Sindecano-1 , Sindecanos , Transfección , Vinculina/metabolismoRESUMEN
We previously identified a phosphatidylinositol-specific phospholipase c (PI-PLC)-releasable heparan sulfate proteoglycan (HSPG) on the surface of rat Schwann cells (D. J. Carey and R. C. Stahl, J. Cell Biol. 111, 2053-2062, 1990). The present study was carried out to investigate the localization and processing of this proteoglycan. The HSPG was synthesized as a PI-PLC-releasable form that was shed into the culture medium with a T1/2 of 17 h. Degradation of the HSPG was negligible. The HSPG was present on the surface of Schwann cells on small (100-200 nm diameter) cylindrical membrane extensions that resembled filopodia. In neonatal peripheral nerve, brain, heart, and striated muscle the HSPG was found to be localized principally to regions of the cell surface that were in contact with basement membranes. Northern blot analysis with cDNA coding for rat glypican (a previously described human fibroblast HSPG) demonstrated abundant expression of glypican mRNA in Schwann cells. Antibodies made against recombinant rat glypican core protein immunoprecipitated the Schwann cell PI-PLC-releasable HSPG. These data demonstrate that the Schwann cell HSPG is rat glypican and support the hypothesis that this proteoglycan functions in cell-extracellular matrix interactions.
Asunto(s)
Heparitina Sulfato/metabolismo , Proteoglicanos/metabolismo , Células de Schwann/metabolismo , Animales , Secuencia de Bases , Compartimento Celular , Células Cultivadas , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Expresión Génica , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/genética , Técnicas In Vitro , Laminina/metabolismo , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos/química , Fosfatidilinositol Diacilglicerol-Liasa , Fosfoinositido Fosfolipasa C , Hidrolasas Diéster Fosfóricas/metabolismo , Proteoglicanos/genética , ARN Mensajero/genética , Ratas , Fosfolipasas de Tipo C/metabolismoRESUMEN
Two overlapping clones containing sequences homologous to bovine, human and chicken decorin have been recovered from poly A+ RNA isolated from rat vascular smooth muscle cells (VSMC) using cDNA cloning and reverse transcription-polymerase chain reaction (PCR) methodologies. Results of nucleotide sequence analysis performed on these clones demonstrated that they encode the complete mature rat decorin protein expressed by VSMC. Within the coding region, rat decorin exhibits 76% nucleotide sequence homology to human and bovine decorin, and 69% homologous to chicken decorin indicating a significant level of conservation among these species. This level of conservation among species was also maintained at the protein level with rat decorin being 77% homologous to its human, bovine and chicken homologues. As previously observed its human homologue, rat decorin, is made up of seven, tandem, leucine-rich repeat sequences. Furthermore, within the core of these repeats was the consensus protein sequence NKISK which has been proposed to be the fibronectin binding region of decorin (G. Schmidt et al., Biochem. J. 280, 411-414 (1991)). The vast majority of amino acid substitutions within rat decorin were of the conservative type. The highest frequency of amino acid substitutions were found to be localized within a hypervariable region located near the amino terminus of the decorin core protein. Unlike rat biglycan, rat decorin mRNA levels were found to increase significantly in density-arrested VSMC cultures. In contrast to rat biglycan gene expression, no quantitative differences in rat decorin mRNA levels were observed between proliferating VSMC and VSMC made quiescent through serum depletion. Finally, specific extracellular matrix (ECM) proteins were able to regulate the expression of decorin at the mRNA level in a slightly different manner than previously observed for biglycan.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas Musculares/química , Músculo Liso Vascular/química , Proteoglicanos/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Técnicas de Cultivo , Decorina , Proteínas de la Matriz Extracelular , Masculino , Datos de Secuencia Molecular , Proteínas Musculares/genética , Proteoglicanos/genética , Ratas , Ratas Sprague-Dawley , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
A cDNA clone coding for a membrane proteoglycan core protein was isolated from a neonatal rat Schwann cell cDNA library by screening with an oligonucleotide based on a conserved sequence in cDNAs coding for previously described proteoglycan core proteins. Primer extension and polymerase chain reaction amplification were used to obtain additional 5' protein coding sequences. The deduced amino acid sequence predicted a 353 amino acid polypeptide with a single membrane spanning segment and a 34 amino acid hydrophilic COOH-terminal cytoplasmic domain. The putative extracellular domain contains three potential glycosaminoglycan attachment sites, as well as a domain rich in Thr and Pro residues. Analysis of the cDNA and deduced amino acid sequences revealed a high degree of identity with the transmembrane and cytoplasmic domains of previously described proteoglycans but a unique extracellular domain sequence. On Northern blots the cDNA hybridized to a single 5.6-kb mRNA that was present in Schwann cells, neonatal rat brain, rat heart, and rat smooth muscle cells. A 16-kD protein fragment encoded by the cDNA was expressed in bacteria and used to immunize rabbits. The resulting antibodies reacted on immunoblots with the core protein of a detergent extracted heparan sulfate proteoglycan. The core protein had an apparent mass of 120 kD. When the anti-core protein antibodies were used to stain tissue sections immunoreactivity was present in peripheral nerve, newborn rat brain, heart, aorta, and other neonatal tissues. A ribonuclease protection assay was used to quantitate levels of the core protein mRNA. High levels were found in neonatal rat brain, heart, and Schwann cells. The mRNA was barely detectable in neonatal or adult liver, or adult brain.