RESUMEN

Dystroglycan (DG) serves as an adhesion complex linking the actin cytoskeleton to the extracellular matrix. DG is encoded by a single gene as a precursor, which is constitutively cleaved to form the α- and ß-DG subunits. α-DG is a peripheral protein characterized by an extensive glycosylation that is essential to bind laminin and other extracellular matrix proteins, while ß-DG binds the cytoskeleton proteins. The functional properties of DG depend on the correct glycosylation of α-DG and on the cross-talk between the two subunits. A reduction of α-DG glycosylation has been observed in muscular dystrophy and cancer while the inhibition of the interaction between α- and ß-DG is associated to aberrant post-translational processing of the complex. Here we used confocal microscopy based techniques to get insights into the influence of α-DG glycosylation on the functional properties of the ß-DG, and its effects on cell migration. We used epithelial cells transfected with wild-type and with a mutated DG harboring the mutation T190M that has been recently associated to dystroglycanopathy. We found that α-DG hypoglycosylation, together with an increased protein instability, reduces the membrane dynamics of the ß-subunit and its clustering within the actin-rich domains, influencing cell migration and spontaneous cell movement. These results contribute to give novel insights into the involvement of aberrant glycosylation of DG in the developing of muscular dystrophy and tumor metastasis.

Asunto(s)

Movimiento Celular , Distroglicanos/metabolismo , Seudópodos/metabolismo , Animales , Línea Celular , Distroglicanos/genética , Glicosilación , Ratones , Microscopía Confocal , Estabilidad Proteica , Seudópodos/genética

RESUMEN

A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl(2) solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC(50) values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein.

Asunto(s)

Antifúngicos/metabolismo , Cassia/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular

RESUMEN

PURPOSE: To report a new family belonging to a previously non-investigated geographic are a with a rare form of lattice corneal dystrophy (LCD). METHODS: Detailed ophthalmologic analysis was carried out on a Bulgarian woman, enrolled for perforating keratoplasty. In order to obtain a final diagnosis both histology and genetic analysis were performed. RESULTS: Upon transplantation, histologic analysis of the dystrophic cornea revealed the typical staining pattern and amyloid deposits of lattice corneal dystrophies. Genetic analysis of the subject and her daughter confirmed the presence of an autosomal dominant R124C mutation within exon 4 of the BIGH3 gene, encoding for keratoepithelin, while showing no abnormalities in her son. CONCLUSIONS: The identification of this mutation allows the unambiguous classification of this corneal dystrophy as LCD type I. A first case of LCD I in a family from Eastern Europe could help to better clarify the molecular epidemiology of the disease.

Asunto(s)

Distrofias Hereditarias de la Córnea/genética , Proteínas de la Matriz Extracelular/genética , Factor de Crecimiento Transformador beta/genética , Adolescente , Adulto , Amiloide/metabolismo , Bulgaria/epidemiología , Distrofias Hereditarias de la Córnea/etnología , Distrofias Hereditarias de la Córnea/metabolismo , Análisis Mutacional de ADN , Exones/genética , Femenino , Genes Dominantes , Humanos , Masculino , Epidemiología Molecular , Linaje , Mutación Puntual

RESUMEN

Inositol-containing molecules are involved in important cellular functions, including signalling, membrane transport and secretion. Our interest is in lysophosphatidylinositol and the glycerophosphoinositols, which modulate cell proliferation and G-protein-dependent activities such as adenylyl cyclase and phospholipase A(2). To investigate the role of glycerophosphoinositol (GroPIns) in the modulation of Ras-dependent pathways and its correlation to Ras transformation, we employed a novel liquid chromatography-tandem mass spectrometry technique to directly measure GroPIns in cell extracts. The cellular levels of GroPIns in selected parental and Ras-transformed cells, and in some carcinoma cells, ranged from 44 to 925 microM, with no consistent correlation to Ras transformation across all cell lines. Moreover, the derived cellular inositol concentrations revealed a wide range ( approximately 150 microM to approximately 100 mM) under standard [(3)H]-inositol-loading, suggesting a complex relationship between the inositol pool and the phosphoinositides and their derivatives. We have investigated these pools under specific loading conditions, designing a further HPLC analysis for GroPIns, combined with mass determinations of cellular phosphatidylinositol 4,5-bisphosphate. The data demonstrate that limiting inositol conditions identify a preferred pathway of inositol incorporation and retention into the polyphosphoinositides pool. Thus, under conditions of increased metabolic activity, such as receptor stimulation or cellular transformation, the polyphosphoinositide levels will be maintained at the expense of phosphatidylinositol and the turnover of its aqueous derivatives.

Asunto(s)

Transformación Celular Neoplásica/genética , Genes ras , Fosfatos de Inositol/fisiología , Inositol/fisiología , Lisofosfolípidos/fisiología , Cromatografía Liquida/métodos , Humanos , Líquido Intracelular/química , Espectrometría de Masas/métodos , Células Tumorales Cultivadas

RESUMEN

Dystroglycan is a receptor for extracellular matrix proteins that plays a crucial role during embryogenesis in addition to adult tissue stabilization. A precursor product of a single gene is post-translationally cleaved to form two different subunits, alpha and beta. The extracellular alpha-dystroglycan is a membrane-associated, highly glycosylated protein that binds to various extracellular matrix molecules, whereas the transmembrane beta-dystroglycan binds, via its cytosolic domain, to dystrophin and many other proteins. alpha- and beta-Dystroglycan interact tightly but noncovalently. We have previously shown that the N-terminal region of beta-dystroglycan, beta-DG(654-750), binds to the C-terminal region of murine alpha-dystroglycan independently from glycosylation. Preparing a series of deleted recombinant fragments and using solid-phase binding assays, the C-terminal sequence of alpha-dystroglycan containing the binding epitope for beta-dystroglycan has been defined more precisely. We found that a region of 36 amino acids, from position 550-585, is required for binding the extracellular region, amino acids 654-750 of beta-dystroglycan. Recently, a dystroglycan-like gene was identified in Drosophila that showed a moderate degree of conservation with vertebrate dystroglycan (31% identity, 48% similarity). Surprisingly, the Drosophila sequence contains a region showing a higher degree of identity and conservation (45% and 66%) that coincides with the 550-585 sequence of vertebrate alpha-dystroglycan. We have expressed this Drosophila dystroglycan fragment and measured its binding to the extracellular region of vertebrate (murine) beta-dystroglycan (Kd = 6 +/- 1 microM). These data confirm the proper identification of the beta-dystroglycan binding epitope and stress the importance of this region during evolution. This finding might help the rational design of dystroglycan-specific binding drugs, that could have important biomedical applications.

Asunto(s)

Proteínas del Citoesqueleto/química , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas del Citoesqueleto/metabolismo , Distroglicanos , Epítopos , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

We have probed the binding of a synthetic peptide corresponding to the region 550-585 of the alpha subunit of dystroglycan with a recombinant protein fragment corresponding to the N-terminal extracellular region of beta-dystroglycan (654-750), using NMR in solution. In a 30:1 molar ratio, the peptide binds to the recombinant protein fragment in the fast/intermediate exchange regime. By monitoring the peptide intra-residue HN-Halpha peak volumes of the 2D TOCSY NMR spectra, both in the absence and in the presence of the recombinant fragment, we determined the differential binding affinities of each amino acid. We found that the residues in the region 550-565 (SWVQFNSNSQLMYGLP) are more influenced by the presence of the protein, whereas the C-terminal portion is marginally involved. These NMR results have been confirmed by solid-phase binding assays.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Distroglicanos , Escherichia coli , Espectroscopía de Resonancia Magnética/métodos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/metabolismo

RESUMEN

Dystroglycan, originally identified in muscle as a component of the dystrophin-associated glycoprotein complex, is a ubiquitously expressed cell-surface receptor that forms a transmembrane link between the extracellular matrix and the cytoskeleton. It contains two subunits, alpha and beta, formed by proteolytic cleavage of a common precursor. In the brain, different neuronal subtypes and glial cells may express dystroglycan in complex with distinct cytoplasmic proteins such as dystrophin, utrophin and their truncated forms. To examine the distribution of dystroglycan in adult mouse brain, we raised antibodies against the recombinant amino- and carboxyl-terminal domains of alpha-dystroglycan. On western blot, the antibodies recognized specifically alpha-dystroglycan in cerebellar extracts. Using light microscopy, alpha-dystroglycan was found in neurons of the cerebral cortex, hippocampus, olfactory bulb, basal ganglia, thalamus, hypothalamus, brainstem and cerebellum, where dystrophin and its truncated isoforms are also known to be present. Electron microscopy revealed that alpha-dystroglycan immunoreactivity was preferentially associated with the postsynaptic specializations. Dystroglycan immunostaining was also detected in perivascular astrocytes and in those facing the pia mater, where utrophin and dystrophin truncated isoforms are present. The cell body and endfeet of astrocytes around blood vessels and the endothelial cells at the blood-brain barrier also expressed dystroglycan. From these data, we suggest that dystroglycan, by bridging the extracellular matrix and the cytoskeleton, may play an important functional role at specialized intercellular contacts, synapses and the blood-brain barrier, whose structural and functional organization strictly depend on the integrity of the extracellular matrix-cytoskeleton linkage.

Asunto(s)

Astrocitos/metabolismo , Encéfalo/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Neuronas/metabolismo , Animales , Especificidad de Anticuerpos , Astrocitos/ultraestructura , Encéfalo/ultraestructura , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Proteínas del Citoesqueleto/inmunología , Citoesqueleto/metabolismo , Citoesqueleto/ultraestructura , Distroglicanos , Distrofina/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/ultraestructura , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Inmunohistoquímica , Masculino , Glicoproteínas de Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Microscopía Electrónica , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patología , Distrofia Muscular de Duchenne/fisiopatología , Neuronas/ultraestructura , Sinapsis/metabolismo , Sinapsis/ultraestructura

RESUMEN

The secondary structure content of the N-terminal extracellular domain of beta-dystroglycan (a recombinant fragment extending from positions 654 to 750) has been quantitatively determined by means of CD and FTIR spectroscopies. The elements of secondary structure, namely an 8-10 residue long alpha-helix (10%) and two beta-strands (24%) have been assigned to specific amino acid sequences by means of a GOR constrained prediction method. The remaining 66% of the whole sequence is classified as turns or unordered. The temperature dependence of CD and FTIR spectra has been investigated in detail. A reversible, non-cooperative thermal transition is observed with both CD and FTIR spectroscopies up to 95 degrees C. The profile of the transition is typical of the unfolding of isolated peptides and corresponds to the progressive loss of the secondary structure elements of the protein with no evidence for collapsing phenomena, typical of globular proteins, upon heating.

Asunto(s)

Proteínas del Citoesqueleto/química , Distrofina/química , Glicoproteínas de Membrana/química , Secuencia de Aminoácidos , Dicroismo Circular , Distroglicanos , Matriz Extracelular/química , Modelos Químicos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Espectroscopía Infrarroja por Transformada de Fourier

RESUMEN

Dystroglycan is a receptor responsible for crucial interactions between extracellular matrix and cytoplasmic space. We provide the first evidence that dystroglycan is truncated. In HC11 normal murine and the 184B5 non-tumorigenic mammary human cell lines, the expected beta-dystroglycan 43 kDa band was found but human breast T47D, BT549, MCF7, colon HT29, HCT116, SW620, prostate DU145 and cervical HeLa cancer cells expressed an anomalous approximately 31 kDa beta-dystroglycan band. alpha-Dystroglycan was udetectable in most of the cell lines in which beta-dystroglycan was found as a approximately 31 kDa species. An anomalous approximately 31 kDa beta-dystroglycan band was also observed in N-methyl-N-nitrosurea-induced primary rat mammary tumours. Reverse transcriptase polymerase chain reaction experiments confirmed the absence of alternative splicing events and/or expression of eventual dystroglycan isoforms. Using protein extraction procedures at low- and high-ionic strength, we demonstrated that both the 43 kDa and approximately 31 kDa beta-dystroglycan bands harbour their transmembrane segment.

Asunto(s)

Proteínas del Citoesqueleto/genética , Glicoproteínas de Membrana/genética , Animales , Mama/citología , Mama/metabolismo , Neoplasias de la Mama , Línea Celular , Neoplasias del Colon , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/química , Distroglicanos , Células Epiteliales/citología , Células Epiteliales/metabolismo , Femenino , Células HeLa , Humanos , Masculino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Neoplasias Mamarias Experimentales , Glicoproteínas de Membrana/análisis , Glicoproteínas de Membrana/química , Ratones , Neoplasias de la Próstata , Conejos , Ratas , Receptores de Laminina/análisis , Receptores de Laminina/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas

RESUMEN

A novel thrombin-like enzyme (named contortrixobin) has been purified to homogeneity from the venom of Agkistrodon contortrix contortrix by affinity chromatography on arginine-Sepharose, anionic exchange chromatography, and HPLC. The complete amino acid sequence has been determined by Edman degradation and by mass spectral analysis of peptides generated by enzymatic cleavage. A microheterogeneity at the level of residue 234 has been detected, as demonstrated by peptides differing for the occurrence of Pro234 ( approximately 85%) or Asp234 ( approximately 15%). Contortrixobin (i) has six disulfide bonds whose sequence positions have been determined by mass spectrometry and (ii) does not contain carbohydrates in its structure. As expected, the 234 residue sequence of contortrixobin exhibits strong homology with snake venom serine proteases acting on either fibrinogen or other blood coagulation components. The interaction of contortrixobin with chromogenic substrates indicates a higher specificity for arginine over lysine in the primary subsite and a faster attack to ester than amides. The hydrolytic activity of contortrixobin is strongly inhibited by diisopropyl fluorophosphate and to a less extent by phenylmethylsulfonyl fluoride, benzamidine, and 4', 6-diamidino-2-phenylindole; hirudin (a specific alpha-thrombin inhibitor) as well as basic pancreatic trypsin inhibitor has a small effect on contortrixobin's catalytic properties. Contortrixobin (i) preferentially releases fibrinopeptide B from human fibrinogen, (ii) activates blood coagulation Factors V and XIII with a rate 250-500-fold lower than human alpha-thrombin, and (iii) does not induce thrombocyte aggregation, intracytoplasmatic calcium ion increase in platelets, and activation of Factor VIII. Evidence for biorecognition properties different from thrombin is also reported.

Asunto(s)

Agkistrodon , Serina Endopeptidasas/metabolismo , Venenos de Víboras/enzimología , Secuencia de Aminoácidos , Animales , Disulfuros , Endopeptidasas/metabolismo , Fibrinógeno/metabolismo , Fibrinopéptido A/metabolismo , Fibrinopéptido B/metabolismo , Humanos , Hidrólisis , Datos de Secuencia Molecular , Inhibidores de Proteasas/farmacología , Análisis de Secuencia de Proteína , Serina Endopeptidasas/química , Venenos de Serpiente/enzimología , Especificidad por Sustrato , Trombina

RESUMEN

The beta-dystroglycan/Grb2 interaction was investigated and a proline-rich region within beta-dystroglycan that binds Grb2-src homology 3 domains identified. We used surface plasmon resonance (SPR), fluorescence analysis, and solid-phase binding assay to measure the affinity constants between Grb2 and the beta-dystroglycan cytoplasmic tail. Analysis of the data obtained from SPR reveals a high-affinity interaction (K(D) approximately 240 nM) between Grb2 and the last 20 amino acids of the beta-dystroglycan carboxyl-terminus, which also contains a dystrophin-binding site. A similar K(D) value (K(D) approximately 280 nM) was obtained by solid-phase binding assay and in solution by fluorescence. Both Grb2-SH3 domains bind beta-dystroglycan but the N-terminal SH3 domain binds with an affinity approximately fourfold higher than that of the C-terminal SH3 domain. The Grb2-beta-dystroglycan interaction was inhibited by dystrophin in a range of concentration of 160-400 nM. These data suggest a highly regulated and dynamic dystrophin/dystroglycan complex formation and that this complex is involved in cell signaling.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas/metabolismo , Animales , Sitios de Unión , Proteínas del Citoesqueleto/química , Distroglicanos , Distrofina/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteína Adaptadora GRB10 , Glutatión Transferasa/metabolismo , Cinética , Ligandos , Glicoproteínas de Membrana/química , Prolina/química , Unión Proteica , Estructura Terciaria de Proteína , Proteínas/química , Conejos , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Fluorescencia , Resonancia por Plasmón de Superficie , Factores de Tiempo , Dominios Homologos src

RESUMEN

New efforts in cancer therapy are being focused at various levels of signaling pathways. With phosphoinositide 3-kinase (PI3-K) potentially being necessary for a range of cancer-related functions, we have investigated the influence of selected inositol tris- to hexakisphosphates on cell growth and tumorigenicity. We show that micromolar concentrations of inositol 1,3,4,5,6-pentakisphosphate and inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P(4)] inhibit IGF-1-induced [(3)H]-thymidine incorporation in human breast cancer (MCF-7) cells and the ability to grow in liquid medium and form colonies in agarose semisolid medium by small cell lung cancer (SCLC) cells, a human cancer cell line containing a constitutively active PI3-K. In an ovarian cancer cell line that also contains a constitutively active PI3-K (SKOV-3 cells), Ins(1,4,5,6)P(4) again inhibited liquid medium growth. Furthermore, when applied extracellularly, inositol 1,3,4,5-tetrakisphosphate was shown indeed to enter SCLC cells. These effects appeared specifically related to PH domains known to bind to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)] and phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)], indicating involvement of the PI3-K downstream target protein kinase B (PKB/Akt). This was further supported by inhibition of PKB/Akt PH domain membrane targeting in COS-7 cells by Ins(1,4,5,6)P(4). Thus, we propose that specific inositol polyphosphates inhibit PI3-K by competing with PtdIns(3,4, 5)P(3)-binding PH domains and that this occurs mainly at the level of the downstream PI3-K target, PKB/Akt.

Asunto(s)

Inhibidores Enzimáticos/farmacología , Fosfatos de Inositol/farmacología , Neoplasias/enzimología , Neoplasias/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas , Animales , Sitios de Unión , Células COS , División Celular/efectos de los fármacos , Membrana Celular/enzimología , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/metabolismo , Transformación Celular Neoplásica/patología , ADN de Neoplasias/biosíntesis , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Fosfatos de Inositol/antagonistas & inhibidores , Fosfatos de Inositol/metabolismo , Fosfatos de Inositol/uso terapéutico , Factor I del Crecimiento Similar a la Insulina/antagonistas & inhibidores , Factor I del Crecimiento Similar a la Insulina/farmacología , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-akt , Células Tumorales Cultivadas

RESUMEN

Insulin evokes diverse biological effects through receptor-mediated tyrosine phosphorylation of the insulin receptor substrate (IRS) proteins. Here, we show that, in vitro, the IRS-1, -2 and -3 pleckstrin homology (PH) domains bind with different specificities to the 3-phosphorylated phosphoinositides. In fact, the IRS-1 PH domain binds preferentially to phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3), the IRS-2 PH domain to phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P2), and the IRS-3 PH domain to phosphatidylinositol 3-phosphate. When expressed in NIH-IR fibroblasts and L6 myocytes, the IRS-1 and -2 PH domains tagged with green fluorescent protein (GFP) are localized exclusively in the cytoplasm. Stimulation with insulin causes a translocation of the GFP-IRS-1 and -2 PH domains to the plasma membrane within 3-5 min. This translocation is blocked by the phosphatidylinositol 3-kinase (PI 3-K) inhibitors, wortmannin and LY294002, suggesting that this event is PI 3-K dependent. Interestingly, platelet-derived growth factor (PDGF) did not induce translocation of the IRS-1 and -2 PH domains to the plasma membrane, indicating the existence of specificity for insulin. In contrast, the GFP-IRS-3 PH domain is constitutively localized to the plasma membrane. These results reveal a differential regulation of the IRS PH domains and a novel positive feedback loop in which PI 3-K functions as both an upstream regulator and a downstream effector of IRS-1 and -2 signaling.

Asunto(s)

Proteínas Sanguíneas/química , Fosfatidilinositoles/metabolismo , Fosfoproteínas/análisis , Fosfoproteínas/química , Fracciones Subcelulares/química , Animales , Membrana Celular/química , Citoplasma/química , Fibroblastos/ultraestructura , Proteínas Fluorescentes Verdes , Humanos , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina , Péptidos y Proteínas de Señalización Intracelular , Proteínas Luminiscentes , Ratones , Músculos/ultraestructura , Mutagénesis Sitio-Dirigida , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , Homología de Secuencia

RESUMEN

Current studies involve an investigation of the role of the pleckstrin homology (PH) domain in membrane targeting and activation of phospholipase Cbeta(1) (PLCbeta(1)). Here we report studies on the membrane localization of the isolated PH domain from the amino terminus of PLCbeta(1) (PLCbeta(1)-PH) using fluorescence microscopy of a green fluorescent protein fusion protein. Whereas PLCbeta(1)-PH does not localize to the plasma membrane in serum-starved cells, it undergoes a rapid but transient migration to the plasma membrane upon stimulation of cells with serum or lysophosphatidic acid (LPA). Regulation of the plasma membrane localization of PLCbeta(1)-PH by phosphoinositides was also investigated. PLCbeta(1)-PH was found to bind phosphatidylinositol 3-phosphate most strongly, whereas other phosphoinositides were bound with lower affinity. The plasma membrane localization of PLCbeta(1)-PH induced by serum and LPA was blocked by wortmannin pretreatment and by LY294002. In parallel, activation of PLCbeta by LPA was inhibited by wortmannin, by LY294002, or by the overexpression of PLCbeta(1)-PH. Microinjection of betagamma subunits of G proteins in serum-starved cells induced the translocation of PLCbeta(1)-PH to the plasma membrane. These results demonstrate that a cooperative mechanism involving phosphatidylinositol 3-phosphate and the Gbetagamma subunit regulates the plasma membrane localization and activation of PLCbeta(1)-PH.

Asunto(s)

Membrana Celular/enzimología , Isoenzimas/química , Isoenzimas/metabolismo , Fosfatidilinositoles/metabolismo , Fosfolipasas de Tipo C/química , Fosfolipasas de Tipo C/metabolismo , Células 3T3 , Androstadienos/farmacología , Animales , Células COS , Cromonas/farmacología , Medio de Cultivo Libre de Suero , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Proteínas de Unión al GTP/metabolismo , Glutatión Transferasa/análisis , Proteínas Fluorescentes Verdes , Sustancias de Crecimiento/farmacología , Células HeLa , Humanos , Proteínas Luminiscentes/análisis , Ratones , Microscopía Confocal , Microscopía Fluorescente , Morfolinas/farmacología , Fosfolipasa C beta , Reacción en Cadena de la Polimerasa , Ratas , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Wortmanina , Dominios Homologos src

RESUMEN

A protein fragment corresponding to the mouse beta-dystroglycan N-terminal extracellular region from position 654 to 750, beta-DG(654-750) was recombinantly expressed in BL21(DE3) Escherichia coli cells. Secondary structure prediction of the protein fragment reveals about 70% of random coil, as confirmed by circular dichroism analysis. Moreover, fluorescence analysis shows that the tryptophan residue in position 659 lays in a solvent-exposed fashion. These data suggest that the beta-DG(654-750) is likely to have a quite flexible structure and to be only partially folded. Interestingly, the protein still retains its biological function since using solid-phase assays we have detected binding of biotinylated beta-DG(654-750) both to native alpha-dystroglycan and to a recombinant fragment which spans the C-terminal region of alpha-dystroglycan.

Asunto(s)

Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Biotinilación , Dicroismo Circular , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/aislamiento & purificación , Disulfuros/química , Disulfuros/metabolismo , Distroglicanos , Escherichia coli/genética , Glicosilación , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Ratones , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Relación Estructura-Actividad , Triptófano/química , Triptófano/metabolismo

RESUMEN

The C-terminal perlecan domain V of about 90 kDa consists of laminin-type G domain modules (LG) (25 kDa) and epidermal growth factor-like modules (EG) (4 kDa) in the tandem arrangement LG1-EG1-EG2-LG2-EG3-EG4-LG3. Several shorter fragments have been prepared by recombinant production in mammalian cells and used to map the single glycosaminoglycan (GAG) substitution site and the binding of several carbohydrate and protein ligands. This identified a Ser3511 residue located in a short link region between EG4 and LG3 as being involved in GAG attachment. Electron microscopy provided evidence that the same substitution exists in tissue forms of perlecan. Heparan sulphate attached to this site was shown to bind to the alpha1LG4 module of laminin-1, indicating a role in basement membrane assembly and cell-matrix interactions. This site is also close to an Asn-Asp bond which is readily cleaved by an endogenous protease that depends on the presence of Asp and the LG2 module. A weak heparin binding site was shown to include the EG2 module, which contains five basic residues. Binding to sulphatides and the alpha-dystroglycan receptor was much stronger and required at least two LG modules. However, single LG modules appear to be sufficient for the interaction with the laminin-nidogen complex, while EG3-4 and some flanking regions are apparently involved in fibulin-2 binding. These observations indicate that a complex modular structure is required for domain V in order to provide a rich repertoire of potential biological functions.

Asunto(s)

Glicosaminoglicanos/química , Proteoglicanos de Heparán Sulfato , Heparitina Sulfato/química , Proteoglicanos/química , Secuencia de Aminoácidos , Animales , Proteínas de Unión al Calcio/metabolismo , Conformación de Carbohidratos , Proteínas del Citoesqueleto/metabolismo , Distroglicanos , Factor de Crecimiento Epidérmico/química , Proteínas de la Matriz Extracelular/metabolismo , Fibroblastos/química , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Heparitina Sulfato/ultraestructura , Humanos , Laminina/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteoglicanos/metabolismo , Proteoglicanos/ultraestructura , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Sulfoglicoesfingolípidos/metabolismo , Resonancia por Plasmón de Superficie

RESUMEN

The carbohydrate chains of nine isoforms of chicken egg-white riboflavin-binding protein (RfBP) and six isoforms each of quail egg-white and yolk RfBP have been structurally characterized. The two N-glycosylation sites, Asn36 and Asn147, of the most abundant isoform of each of the three proteins were analyzed in further detail leading to the identification of different glycosylation patterns. In both chicken and quail egg-white RfBP the carbohydrates attached to position 36 had a lower degree of branching and, in the case of the quail protein, this site was only partially glycosylated. A very heterogeneous mixture of complex structures was characteristic of the other glycosylation site. Analysis of the two sites in quail yolk RfBP confirmed this result which agrees with what has been established for hen yolk RfBP. The presence in the three proteins of a highly heterogeneous mixture of differently branched glycans suggests that the differences in isoelectric points, which is a peculiarity of the different isoforms, are probably indeed due to differences in carbohydrate structure.

Asunto(s)

Proteínas Portadoras/química , Proteínas de Transporte de Membrana , Oligosacáridos/química , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Proteínas Portadoras/aislamiento & purificación , Pollos , Clara de Huevo/análisis , Yema de Huevo/química , Femenino , Cromatografía de Gases y Espectrometría de Masas , Datos de Secuencia Molecular , Oligosacáridos/aislamiento & purificación , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Codorniz , Riboflavina/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

Electron microscopy of ECADCOMP, a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 microM) stabilized the rod-like structure of E-cadherin. At medium Ca2+ concentration (500 microM), two adjacent ectodomains in a pentamer formed cis-dimers. At high Ca2+ concentration (>1 mM), two cis-dimers from different pentamers formed a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin revealed two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca2+-binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis-dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans- but not the cis-interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association.

Asunto(s)

Cadherinas/química , Cadherinas/ultraestructura , Secuencia de Bases , Sitios de Unión/genética , Cadherinas/genética , Calcio/metabolismo , Línea Celular , Cristalización , Cristalografía por Rayos X , Cartilla de ADN/genética , Humanos , Técnicas In Vitro , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/ultraestructura , Estereoisomerismo , Transfección

RESUMEN

The 395-residue proteolytic fragment E3, which comprises the two most C-terminal LG modules of the mouse laminin alpha1 chain, was previously shown to contain major binding sites for heparin, alpha-dystroglycan and sulfatides. The same fragment (alpha1LG4-5) and its individual alpha1LG4 and alpha1LG5 modules have now been obtained by recombinant production in mammalian cells. These fragments were apparently folded into a native form, as shown by circular dichroism, electron microscopy and immunological assays. Fragment alpha1LG4-5 bound about five- to tenfold better to heparin, alpha-dystroglycan and sulfatides than E3. These binding activities could be exclusively localized to the alpha1LG4 module. Side-chain modifications and proteolysis demonstrated that Lys and Arg residues in the C-terminal region of alpha1LG4 are essential for heparin binding. This was confirmed by 14 single to triple point mutations, which identified three non-contiguous basic regions (positions 2766-2770, 2791-2793, 2819-2820) as contributing to both heparin and sulfatide binding. Two of these regions were also recognized by monoclonal antibodies which have previously been shown to inhibit heparin binding. The same three regions and a few additional basic residues also make major contributions to the binding of the cellular receptor alpha-dystroglycan, indicating a larger binding epitope. The data are also consistent with previous findings that heparin competes for alpha-dystroglycan binding.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Heparina/metabolismo , Laminina/genética , Glicoproteínas de Membrana/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Unión Competitiva , Distroglicanos , Ensayo de Inmunoadsorción Enzimática , Laminina/química , Ratones , Microscopía Electrónica , Mutagénesis Sitio-Dirigida , Pepsina A/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Mutación Puntual/genética , Unión Proteica , Pliegue de Proteína , ARN Mensajero/genética , Proteínas Recombinantes/química

RESUMEN

The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Proteoglicanos de Heparán Sulfato , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Laminina/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteoglicanos/metabolismo , Sulfoglicoesfingolípidos/metabolismo , Animales , Sitios de Unión , Unión Competitiva , Proteínas de Unión al Calcio/metabolismo , Distroglicanos , Cinética , Ratones , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo