Biomedical subjects

J J Calvete

Publications and source records attributed to J J Calvete.

At least 19 recordsLinked to original sources

PubMed2006

Isolation and characterization of a new agglutinin from the red marine alga Hypnea cervicornis J. Agardh.

The biochemical characterization of a new lectin (Hypnea cervicornis agglutinin or HCA) isolated from the Brazilian red alga H. cervicornis is reported. The haemagglutinating activity of the lectin was only inhibited by the glycoprotein porcine stomach mucin at a minimum inhibitory concentration of 19 microg x mL(-1). No haemagglutination inhibition was detected after the addition of simple sugars. The MALDI-TOF molecular masses of native and reduced and carbamidomethylated HCA were, respectively, 9196.6 Da and 9988.2 Da, indicating that the primary structure of the protein is crosslinked by 7 disulfide bonds. This unusual structural feature among lectins, along with its N-terminal sequence and amino-acid composition, clearly shows that HCA belongs to a protein family distinct from the isolectins Hypnin A1 and A2 isolated from the related Japanese alga Hypnea japonica. On the other hand, HCA displayed a high degree of similarity to the agglutinin from the Brazilian species Hypnea musciformis. Our data indicate the occurrence of structural diversity among lectins of closely related species living in distant ecosystems, i.e., the Pacific coast of Japan and the Atlantic coast of Brazil, and support the hypothesis that the lectin content (lectinome) might serve as a biomarker for taxonomical purposes.

Agglutinins↗

PubMed2005

Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.

The present study contributes to clarify the mechanism underlying the high efficacy of hepatocyte gene transfer mediated by hydrodynamic injection. Gene transfer experiments were performed employing the hAAT gene, and the efficacy and differential identification in mouse plasma of human transgene versus mouse gene was assessed by ELISA and proteomic procedures, respectively. By applying different experimental strategies such as cumulative dose-response efficacy, hemodynamic changes reflected by venous pressures, intravital microscopy, and morphological changes established by transmission electron microscopy, we found that: (a) cumulative multiple doses of transgene by hydrodynamic injection are efficient and well tolerated, resulting in therapeutic plasma levels of hAAT; (b) hydrodynamic injection mediates a transient inversion of intrahepatic blood flow, with circulatory stasis for a few minutes mainly in pericentral vein sinusoids; (c) transmission electron microscopy shows hydrodynamic injection to promote massive megafluid endocytic vesicles among hepatocytes around the central vein but not in hepatocytes around the periportal vein. We suggest that the mechanism of hydrodynamic liver gene transfer involves transient inversion of intrahepatic flow, sinusoidal blood stasis, and massive fluid endocytic vesicles in pericentral vein hepatocytes.

Animals↗

PubMed2002

Purification and characterization of a new lectin from the red marine alga Hypnea musciformis.

A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF/MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.

Animals↗

PubMed2002

Purification and partial characterization of a lectin from Canavalia grandiflora benth. seeds.

A D-glucose/D-mannose specific lectin from seeds of Canavalia grandiflora (ConGF) was purified by affinity chromatography on Sephadex G-50. By SDS-PAGE ConGF yielded three protein bands with apparent molecular masses of 29-30 kDa (alpha chain), 16-18 kDa (beta fragment) and 12-13 kDa (gamma fragment), like other related lectins from the genus Canavalia (Leguminosae). ConGF strongly agglutinates rabbit erythrocytes, has a high content of ASP and SER, and its N-terminal sequence (30 residues) is highly similar to the sequences of other related lectins from subtribe Diocleinae.

Amino Acids↗

PubMed2001

Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasma.

The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 A resolution were collected at the ESRF synchrotron-radiation source.

Amino Acid Sequence↗

PubMed2001

Crystal structure of native and Cd/Cd-substituted Dioclea guianensis seed lectin. A novel manganese-binding site and structural basis of dimer-tetramer association.

Diocleinae legume lectins are a group of oligomeric proteins whose subunits display a high degree of primary structure and tertiary fold conservation but exhibit considerable diversity in their oligomerisation modes. To elucidate the structural determinants underlaying Diocleinae lectin oligomerisation, we have determined the crystal structures of native and cadmium-substituted Dioclea guianensis (Dguia) seed lectin. These structures have been solved by molecular replacement using concanavalin (ConA) coordinates as the starting model, and refined against data to 2.0 A resolution. In the native (Mn/Ca-Dguia) crystal form (P4(3)2(1)2), the asymmetric unit contains two monomers arranged into a canonical legume lectin dimer, and the tetramer is formed with a symmetry-related dimer. In the Cd/Cd-substituted form (I4(1)22), the asymmetric unit is occupied by a monomer. In both crystal forms, the tetrameric association is achieved by the corresponding symmetry operators. Like other legume lectins, native D. guianensis lectin contains manganese and calcium ions bound in the vicinity of the saccharide-combining site. The architecture of these metal-binding sites (S1 and S2) changed only slightly in the cadmium/cadmium-substituted form. A highly ordered calcium (native lectin) or cadmium (Cd/Cd-substituted lectin) ion is coordinated at the interface between dimers that are not tetrameric partners in a similar manner as the previously identified Cd(2+) in site S3 of a Cd/Ca-ConA. An additional Mn(2+) coordination site (called S5), whose presence has not been reported in crystal structures of any other homologous lectin, is present in both, the Mn/Ca and the Cd/Cd-substituted D. guianensis lectin forms. On the other hand, comparison of the primary and quaternary crystal structures of seed lectins from D. guianensis and Dioclea grandiflora (1DGL) indicates that the loop comprising residues 117-123 is ordered to make interdimer contacts in the D. grandiflora lectin structure, while this loop is disordered in the D. guianensis lectin structure. A single amino acid difference at position 131 (histidine in D. grandiflora and asparagine in D. guianensis) drastically reduces interdimer contacts, accounting for the disordered loop. Further, this amino acid change yields a conformation that may explain why a pH-dependent dimer-tetramer equilibrium exists for the D. guianensis lectin but not for the D. grandiflora lectin.

Binding Sites↗

PubMed2001

Characterization of the sugar-binding specificity of the toxic lectins isolated from Abrus pulchellus seeds.

The sugar-binding specificity of the toxic lectins from Abrus pulchellus seeds was investigated by combination of affinity chromatography of glycopeptides and oligosaccharides of well-defined structures on a lectin-Sepharose column and measurement of the kinetic interactions in real time towards immobilized glycoproteins. The lectins showed strong affinity for a series of bi- and triantennary N-acetyllactosamine type glycans. The related asialo-oligosaccharides interact more strongly with the lectins. The best recognized structures were asialo-glycopeptides from fetuin. Accordingly, the kinetic interaction with immobilized asialofetuin was by far the most pronounced. Human and bovine lactotransferrins and human serotransferrin interacted to a lesser extent. The interaction with asialofetuin was inhibited by galactose in a dose dependent manner. Lactose, N-acetyllactosamine and lacto-N-biose exhibited similar degree of inhibition while N-acetylgalactosamine was a poor inhibitor. These results suggested that the carbohydrate-binding site of the Abrus pulchellus lectins was specific for galactose and possess a remarkable affinity for the sequences lactose [beta-D-Gal-(1-->4)-D-Glc], N-acetyllactosamine [beta-D-Gal-(1-->4)-D-GlcNAc] and lacto-N-biose [beta-D-Gal-(1-->3)-D-GlcNAc].

Abrus↗

PubMed2001

The amino-acid sequence of the glucose/mannose-specific lectin isolated from Parkia platycephala seeds reveals three tandemly arranged jacalin-related domains.

A mannose/glucose-specific lectin was isolated from seeds of Parkia platycephala, the most primitive subfamily of Leguminosae plants. The molecular mass of the purified lectin determined by mass spectrometry was 47 946 +/- 6 Da (by electrospray ionization) and 47 951 +/- 9 Da (by matrix-assisted laser-desoption ionization). The apparent molecular mass of the lectin in solutions of pH in the range 4.5-8.5 determined by analytical ultracentrifugation equilibrium sedimentation was 94 +/- 3 kDa, showing that the protein behaved as a non-pH-dependent dimer. The amino-acid sequence of the Parkia lectin was determined by Edman degradation of overlapping peptides. This is the first report of the primary structure of a Mimosoideae lectin. The protein contained a blocked N-terminus and a single, nonglycosylated polypeptide chain composed of three tandemly arranged homologous domains. Each of these domains shares sequence similarity with jacalin-related lectin monomers from Asteraceae, Convolvulaceae, Moraceae, Musaceae, Gramineae, and Fagaceae plant families. Based on this homology, we predict that each Parkia lectin repeat may display a beta prism fold similar to that observed in the crystal structure of the lectin from Helianthus tuberosus. The P. platycephala lectin also shows sequence similarity with stress- and pathogen-upregulated defence genes of a number of different plants, suggesting a common ancestry for jacalin-related lectins and inducible defence proteins.

Amino Acid Sequence↗

PubMed2000

Inhibitory effects of MLDG-containing heterodimeric disintegrins reveal distinct structural requirements for interaction of the integrin alpha 9beta 1 with VCAM-1, tenascin-C, and osteopontin.

The integrin alpha9beta1 is expressed on epithelial cells, smooth muscle cells, skeletal muscle, and neutrophils and recognizes at least three distinct ligands: vascular cell adhesion molecule 1 (VCAM-1), tenascin-C, and osteopontin. The alpha9 subunit is structurally similar to the integrin alpha4 subunit, and alpha9beta1 and alpha4beta1 both recognize VCAM-1 as a ligand. We therefore examined whether the disintegrin EC3, which we have recently shown specifically inhibits the binding of alpha4 integrins to ligands, would also be a functional inhibitor of alpha9beta1. EC3 and a novel heterodimeric disintegrin that we identified, EC6, both were potent inhibitors of alpha9beta1-mediated adhesion to VCAM-1 and of neutrophil migration across tumor necrosis factor-activated endothelial cells. A peptide containing a novel MLDG motif shared by both of these disintegrins also inhibited alpha9beta1- and alpha4beta1-mediated adhesion to VCAM-1. Surprisingly though, concentrations of EC3 that completely inhibited adhesion of alpha9-transfected cells to VCAM-1 had little or no effect on adhesion to either of the other alpha9beta1 ligands, osteopontin and tenascin-C. Furthermore, peptides AEIDGIEL and SVVYGLR, which we have previously shown inhibit binding of alpha9beta1-expressing cells to tenascin-C and osteopontin, respectively, had no effect on adhesion to VCAM-1. These data suggest that there are structurally distinct requirements for interactions of the alpha9beta1 integrin with VCAM-1 and the extracellular matrix ligands osteopontin and tenascin-C.

Amino Acid Motifs↗

PubMed2000

Biophysical study of the perturbation of model membrane structure caused by seminal plasma protein PDC-109.

PDC-109, the major heparin-binding protein of bull seminal plasma, binds specifically to sperm choline lipids at ejaculation and mediates capacitation by stimulating cholesterol and phospholipid efflux. We carried out a biophysical study to investigate the membrane perturbation effect caused by PDC-109. Binding of PDC-109 to phosphatidylcholine model membranes was maximal at a 12:1 phosphatidylcholine to protein molar ratio. The process was independent of the membrane structure and involved a slight conformational change of the protein, compatible with an increased exposure to the solvent. PDC-109 binding to dimyristoylphosphatidylcholine prevented lipid molecules from participating in the gel-to-liquid phase transition, due to enhancement of both acyl chain disorder and interfacial hydration. Visualization of the lipid-protein complexes by electron microscopy showed surface irregularities and the presence of 10-nm particles. Permeability assays confirmed the PDC-109-induced disruption of the vesicles. This effect was not modified by heparin. However, presence of cholesterol inhibited the process in a concentration-dependent manner.

Animals↗

PubMed2000

Disulphide-bond pattern and molecular modelling of the dimeric disintegrin EMF-10, a potent and selective integrin alpha5beta1 antagonist from Eristocophis macmahoni venom.

The disulphide-bond pattern of the heterodimeric disintegrin EMF-10, a potent and selective integrin alpha(5)beta(1) antagonist from Eristocophis macmahoni venom, was established by combination of amino-acid analysis, N-terminal sequencing and collision-induced dissociation by nanoelectrospray ionization quadrupole ion-trap MS of fragments isolated by reversed-phase HPLC after degradation of EMF-10 with oxalic acid. Each EMF-10 subunit contains four intrachain disulphide bonds. Two interchain cystine residues join the EMF-10 polypeptides. The intrachain linkages are conserved in monomeric disintegrins. A molecular model of EMF-10 was built using averaged NMR co-ordinates of flavoridin as a template. The active hairpin loops of the EMF-10 subunits occupy opposite locations at the ends of an elongated disulphide-bond ladder. In the EMF-10 model the N-terminal polypeptide of EMF-10B is close to the RGD-loop of the EMF-10A subunit, suggesting that the N-terminal region of the B-subunit could potentially influence the biological activity of the A-subunit.

Amino Acid Sequence↗

PubMed2000

The amino acid sequence of the agglutinin isolated from the red marine alga Bryothamnion triquetrum defines a novel lectin structure.

The primary structure of a lectin isolated from the red alga Bryothamnion triquetrum was established by combination of Edman degradation of sets of overlapping peptides and mass spectrometry. It contains 91 amino acids and two disulphide bonds. The primary structure of the B. triquetrum lectin does not show amino acid sequence similarity with known plant and animal lectin structures. Hence, this protein may be the paradigm of a novel lectin family.

Algal Proteins↗

PubMed2000

Biochemical and functional characterization of the Tn-specific lectin from Salvia sclarea seeds.

SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAcalpha-O-Ser/Thr), a specific marker of many human carcinomas. Two-dimensional electrophoresis, amino-acid and amino-sugar analysis, and MALDI-TOF MS showed that SSL is an acidic (pI 5.5), 60-61-kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was 59 +/- 9 kDa. This value did not change in the pH range 2.5-8.5, indicating that SSL does not associate into higher order structures. Tandem mass spectrometry and methylation analysis of N-glycans released from SSL by hydrazinolysis indicated that SSL possesses 2-3 glycosylation sites occupied with the typical plant glycans Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalp ha1-3)GlcNAc and [(Manalpha1-3/6)(Xylbeta1-2)]Manbeta1-4-GlcNAcbeta1 -4(Fucalpha1-3)Glc NAc. The influence of adjacent Tn structures on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4 only reacted with glycopeptides displaying two or three consecutive Tn structures.

Antibodies, Monoclonal↗

PubMed2000

Purification, chemical, and immunochemical properties of a new lectin from Mimosoideae (Parkia discolor).

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P. discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.

Amino Acids↗

PubMed2000

The disulfide bond pattern of catrocollastatin C, a disintegrin-like/cysteine-rich protein isolated from Crotalus atrox venom.

The disulfide bond pattern of catrocollastatin-C was determined by N-terminal sequencing and mass spectrometry. The N-terminal disintegrin-like domain is a compact structure including eight disulfide bonds, seven of them in the same pattern as the disintegrin bitistatin. The protein has two extra cysteine residues (XIII and XVI) that form an additional disulfide bond that is characteristically found in the disintegrin-like domains of cellular metalloproteinases (ADAMs) and PIII snake venom Zn-metalloproteinases (SVMPs). The C-terminal cysteine-rich domain of catrocollastatin-C contains five disulfide bonds between nearest-neighbor cysteines and a long range disulfide bridge between CysV and CysX. These results provide structural evidence for a redefinition of the disintegrin-like and cysteine-rich domain boundaries. An evolutionary pathway for ADAMs, PIII, and PII SVMPs based on disulfide bond engineering is also proposed.

Amino Acid Motifs↗

PubMed1999

Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins.

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegrin selectively inhibiting alpha(IIb)beta(3). In order to identify echistatin motifs required for selective recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 15 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to express ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). Replacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu selectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT exhibited new activity with alpha(5)beta(1), which was 10-20-fold higher than that of wild type eristostatin. A hypothesis is proposed that the C terminus of echistatin interacts with separate sites on beta(1) and beta(3) integrin molecules.

Amino Acid Sequence↗

PubMed1999

Structural and functional characterization of EMF10, a heterodimeric disintegrin from Eristocophis macmahoni venom that selectively inhibits alpha 5 beta 1 integrin.

Alpha5beta1, a major fibronectin receptor, is a widely distributed integrin that is essential for cell growth and organ development. Here, we describe a novel heterodimeric disintegrin named EMF10, isolated from the Eristocophis macmahoni venom, that is an extremely potent and selective inhibitor of alpha5beta1. EMF10 inhibited adhesion of cells expressing alpha5beta1 to fibronectin (IC(50) = 1-4 nM) and caused expression of a ligand-induced binding site (LIBS) on the beta1 subunit of alpha5beta1 integrin. It partially inhibited adhesion of cells expressing alphaIIbbeta3, alphavbeta3, and alpha4beta1 to appropriate ligands only at concentration higher than 500 nM. Guinea pig megakaryocytes expressing alpha5beta1 adhered to immobilized EMF10 and showed extensive spreading and cytoskeletal mobilization. As determined by electrospray mass spectrometry, EMF10 is composed of two species with molecular masses of 14 575 and 14 949 Da, respectively. EMF10 is a heterodimer containing two subunits: EMF10A (Mr 7544 Da) and EMF10B (Mr 7405 and 7032 Da) linked covalently by S-S bonds. Subunit B showed heterogeneity and may be present as EMF10B1 (Mr 7032) and EMF10B2 (Mr 7405). In putative hairpin loops, EMF10A and EMF10B contained CKKGRGDNLNDYC and CWPAMGDWNDDYC motifs, respectively. The reduced and alkylated subunit B of EMF10 inhibited adhesion of K562 cells to fibronectin in a dose-dependent, saturable manner with IC(50) of 3 microM. The synthetic, cyclic CKKGRGDNLNDYC and CWPAMGDWNDDYC peptides expressed their inhibitory activity in the same system with IC(50) of 100 microM. We propose that alpha5beta1 recognition of EMF10 is associated with the MGDW motif located in a putative hairpin loop of the B subunit and that the expression of activity may also depend on the RGDN motif in the subunit A and on the C-termini of both subunits.

Amino Acid Sequence↗

PubMed1999

EC3, a novel heterodimeric disintegrin from Echis carinatus venom, inhibits alpha4 and alpha5 integrins in an RGD-independent manner.

EC3, a heterodimeric disintegrin (Mr = 14,762) isolated from Echis carinatus venom is a potent antagonist of alpha4 integrins. Two subunits called EC3A and EC3B were isolated from reduced and alkylated EC3 by reverse-phase high performance liquid chromatography. Each subunit contained 67 residues, including 10 cysteines, and displayed a high degree of homology to each other and to other disintegrins. EC3 inhibited adhesion of cells expressing alpha4beta1 and alpha4beta7 integrins to natural ligands vascular cell adhesion molecule 1 (VCAM-1) and mucosal addressin cell adhesion molecule 1 (MadCAM-1) with IC50 = 6-30 nM, adhesion of K562 cells (alpha5beta1) to fibronectin with IC50 = 150 nM, and adhesion of alphaIIbbeta3 Chinese hamster ovary cells to fibrinogen with IC50 = 500 nM; it did not inhibit adhesion of alphavbeta3 Chinese hamster ovary cells to vitronectin. Ethylpyridylethylated EC3B inhibited adhesion of Jurkat cells to immobilized VCAM-1 (IC50 = 6 microM), whereas EC3A was inactive in this system. The MLDG motif appeared to be essential for activity of EC3B. Linear MLDG peptide inhibited the adhesion of Jurkat to VCAM-1 in a dose-dependent manner (IC50 = 4 mM), whereas RGDS peptide was not active at the same concentration. MLDG partially inhibited adhesion of K562 cells to fibronectin (5-10 mM) in contrast to RGDS peptide (IC50 = 3 mM), inhibiting completely at 10 mM.

Amino Acid Sequence↗