RESUMO
Two trials were conducted to assess the fate of the Cry3Bb1 protein from YieldGard rootworm corn (MON 863) when fed to laying hens. In the first trial, 2 diets, 1 formulated with MON 863 and 1 with conventional corn, were fed to laying hens (12 replicate cages with 4 hens/cage per treatment) for 8 wk. Daily feed intake (FI), egg production (EP), and BW were measured. Prestudy fecal samples, wk 4 and 8 egg and fecal samples, and hepatic and pectoralis tissue samples were collected from 12 killed hens and were tested for the Cry3Bb1 protein. Corn source had no significant effects on FI, EP, or BW. Feces from hens fed diets containing MON 863 were positive for the Cry3Bb1 protein or proteolytic fragments (1.5 to 4.0 ppm fecal dry matter). The Cry3Bb1 protein could not be determined in eggs due to the presence of an interfering substance in all test and control eggs. No Cry3Bb1 protein was detected in hepatic and pectoralis tissue. In the second trial, the same test and control diets were fed to 12 hens each. Six hens/treatment were sampled after 7 and 28 d. Samples included blood, feces, and digesta (crop, small and large intestine, and ceca). The Cry3Bb1 protein could not be determined in blood due to the presence of an interfering substance in all test and control blood samples. The Cry3Bb1 protein or partially digested fragments, or both, were found in the digesta sampled from all sections of the digestive tract. About 98 to >99% of the dietary Cry3Bb1 protein was digested. Overall, MON 863, when fed to laying hens, had no significant effects on FI, EP, or BW. The Cry3Bb1 protein was extensively digested, similar to that of other dietary proteins, and was not detected in hepatic or muscle tissue.
Assuntos
Ração Animal , Endotoxinas/farmacocinética , Zea mays , Animais , Peso Corporal , Galinhas/crescimento & desenvolvimento , Galinhas/fisiologia , Digestão , Endotoxinas/análise , Ingestão de Energia , Fezes/química , Feminino , Abrigo para Animais , Fígado/fisiologia , Músculo Esquelético/fisiologia , OviposiçãoRESUMO
Distribution and antithrombotic activity of orally administered unfractionated porcine heparin were studied. [14C]Heparin was prepared by de-N-acetylation of porcine mucosal heparin followed by re-N-acetylation, using [14C]acetic anhydride. [14C]Heparin and (or) cold heparin (60 mg/kg) were administered by stomach tube to male Wistar rats. Blood, all levels of gut and gut contents, liver, lung, spleen, kidney, and aortic and vena caval endothelium were collected under deep anesthesia at 3, 6, 15, 30, and 60 min and 4 and 24 h (6 rats/group) after administration. Urine and feces were collected at 24 h, using metabolic cages. In three additional rats, drugs were administered in gelatin capsules. Tissues listed above and tongue, esophagus, trachea, brain, heart, thymus, bile ducts, vena caval and aortic walls, ureters, bladder, samples of muscle, skin, hair, and bone marrow were collected at 24 h. Radioactivity and chemical heparin, measured by agarose gel electrophoresis, were observed in all tissues examined as well as gut washes, plasma, urine, and feces. Radiolabel recovered was confirmed to be heparin by autoradiograms of gradient polyacrylamide electrophoretic gels. [14C]Heparin and chemical heparin in gut tissue suggest a transit time of 4 h. Porcine or bovine heparin (7.5 mg/kg), administered by stomach tube, decreased the incidence of thrombosis induced by applying 10% formalin in 65% methanol to the exposed jugular vein of rats. Heparin isolation from non-gut tissue, endothelium, urine, and plasma and the observed antithrombotic effect are consistent with oral bioavailability.
Assuntos
Fibrinolíticos/farmacologia , Fibrinolíticos/farmacocinética , Heparina/farmacologia , Heparina/farmacocinética , Mucosa Intestinal/química , Administração Oral , Animais , Bovinos , Endotélio/metabolismo , Fibrinolíticos/análise , Heparina/análise , Marcação por Isótopo , Masculino , Ratos , Ratos Wistar , Suínos , Trombose/sangue , Trombose/prevenção & controle , Distribuição TecidualRESUMO
A beta-cyclodextrin sulfate mixture has been fractionated using discontinuous gradient polyacrylamide gel electrophoresis. Semidry electrotransfer of the sample onto a positively charged nylon membrane and visualization of a portion of this membrane with Alcian blue stain showed multiple bands. The bands were cut from the remaining portion of the membrane and after washing with 8 M urea, the beta-cyclodextrin sulfate fractions were eluted with 2 M sodium chloride and dialyzed. Analysis of each fraction using high resolution analytical gradient polyacrylamide gel electrophoresis as well as capillary electrophoresis, using indirect detection, showed some of the fractions to be pure while others were mixtures. Each beta-cyclodextrin sulfate fraction was complexed with a basic synthetic peptide and analyzed by electrospray ionization mass spectrometry to define the mass of the components in each mixture and thereby to determine the purity of each sample.
Assuntos
Ciclodextrinas/análise , Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Espectrometria de Massas/métodos , beta-Ciclodextrinas , Sequência de Carboidratos , Dados de Sequência MolecularRESUMO
Brain natriuretic peptide (BNP) was examined as part of a continuing study of the interaction of proteins and peptides with the glycosaminoglycan heparin. BNP was tentatively identified as a heparin-binding protein on the basis of its cyclic structure and the high frequency of the basic amino acid residues, lysine and arginine. Thermodynamic analysis using isothermal titration calorimetry confirmed heparin binding to BNP with a micromolar Kd. Surprisingly, despite the high frequency (22%) of basic residues in BNP, only a small portion of the free energy of this interaction resulted from ionic contributions under physiologic conditions. The contribution of polar amino acids, representing 28% of BNP, was next examined in a variety of different buffers. These experiments demonstrated the transfer of five protons from buffer to BNP on heparin binding, suggesting that hydrogen bonding between the polar residues of BNP and heparin is a major factor contributing to the free energy of BNP binding to heparin. Hydrophobic forces apparently play only a small role in binding. Heparin contains few nonpolar functional groups, and a positive change in heat capacity (DeltaCp = 1 kcal/mol) demonstrates the loss of polar residues on BNP-heparin binding.
Assuntos
Heparina/química , Peptídeo Natriurético Encefálico/química , Peptídeos Cíclicos/química , Termodinâmica , Sequência de Aminoácidos , Soluções Tampão , Calorimetria , Heparina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peptídeo Natriurético Encefálico/metabolismo , Peptídeos Cíclicos/metabolismo , Polímeros/metabolismo , Ligação Proteica/efeitos dos fármacos , Cloreto de Sódio/farmacologia , TemperaturaRESUMO
Protease inhibition by secretory leukocyte protease inhibitor (SLPI) is accelerated by the sulfated polysaccharides. The nature of the SLPI-polysaccharide interaction, explored with affinity chromatography, indicated that this interaction was sensitive to the charge and type of polysaccharide. Dextran and chondroitin had the lowest affinity for SLPI, followed by dermatan, heparan, and dextran sulfates. While heparin bound SLPI tightly, the highest affinity heparin chains unexpectedly contained a lower level of sulfation than more weakly interacting chains. Heparin oligosaccharides, prepared using heparin lyase I were SLPI-affinity fractionated. Surprisingly, undersulfated heparin oligosaccharides bound SLPI with the highest affinity, suggesting the importance of free hydroxyl groups for high affinity interaction. Isothermal titration calorimetry was used to determine the thermodynamics of SLPI interaction with a low molecular weight heparin, an undersulfated decasaccharide and a tetrasaccharide. The studies showed 12-14 saccharide units, corresponding to molecular weight of approximately 4,800, were required for a 1:1 (SLPI:heparin) binding stoichiometry. Furthermore, an undersulfated decasaccharide was able to bind SLPI tightly (Kd approximately 13 nM), resulting in its activation and the inhibition of neutrophil elastase and pancreatic chymotrypsin. The in vitro assessment of heparin and the decasaccharide and tetrasaccharide using stopped-flow kinetics suggested that heparin was the optimal choice to study SLPI-based in vivo protease inhibition. SLPI and heparin were co-administered by inhalation in therapy against antigen-induced airway hyperresponsiveness in a sheep bronchoprovocation model. Heparin, in combination with SLPI demonstrated in vivo efficacy reducing early and late phase bronchoconstriction. Heparin also increased the therapeutic activity of SLPI against antigen-induced airway hyperresponsiveness.
Assuntos
Asma/enzimologia , Heparina/metabolismo , Proteínas/metabolismo , Inibidores de Serina Proteinase/metabolismo , Animais , Calorimetria/métodos , Sequência de Carboidratos , Bovinos , Cromatografia de Afinidade , Feminino , Heparina/isolamento & purificação , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Oligossacarídeos/metabolismo , Proteínas Secretadas Inibidoras de Proteinases , Proteínas/química , Inibidor Secretado de Peptidases Leucocitárias , Ovinos , TermodinâmicaRESUMO
Although interactions of proteins with glycosaminoglycans (GAGs), such as heparin and heparan sulphate, are of great biological importance, structural requirements for protein-GAG binding have not been well-characterised. Ionic interactions are important in promoting protein-GAG binding. Polyelectrolyte theory suggests that much of the free energy of binding comes from entropically favourable release of cations from GAG chains. Despite their identical charges, arginine residues bind more tightly to GAGs than lysine residues. The spacing of these residues may determine protein-GAG affinity and specificity. Consensus sequences such as XBBBXXBX, XBBXBX and a critical 20 A spacing of basic residues are found in some protein sites that bind GAG. A new consensus sequence TXXBXXTBXXXTBB is described, where turns bring basic interacting amino acid residues into proximity. Clearly, protein-GAG interactions play a prominent role in cell-cell interaction and cell growth. Pathogens including virus particles might target GAG-binding sites in envelope proteins leading to infection.
Assuntos
Glicosaminoglicanos/química , Proteínas/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Sequência de Carboidratos , Sequência Consenso/genética , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , TermodinâmicaRESUMO
A new capillary electrophoresis technique has been developed for the affinity resolution of synthetic heparin-binding peptides using an immobilized glycosaminoglycan. Heparin and heparan sulfate were immobilized onto fused silica capillaries using biotin-neutravidin conjugation. These capillaries exhibited markedly reduced electroosmotic flow and were able to distinguish peptides based on the heparin binding domain of acidic fibroblast growth factor (residues 125-144, GLKKNGSCKRGPRTHYGQKA) that differed only in the stereochemistry of the proline amino acid residue. The peptide based on the native sequence was retarded compared to the peptide having unnatural stereochemistry, consistent with its stronger interaction for immobilized glycosaminoglycan. Improved resolution is also obtained for additional arginine and lysine containing heparin-binding peptides.
Assuntos
Proteínas de Transporte/isolamento & purificação , Eletroforese Capilar/métodos , Heparina , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Fator 1 de Crescimento de Fibroblastos/química , Fator 1 de Crescimento de Fibroblastos/isolamento & purificação , Fator 1 de Crescimento de Fibroblastos/metabolismo , Glicosaminoglicanos , Ligantes , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismoRESUMO
Fibroblast growth factors (FGFs) are a family of angiogenic and mitogenic proteins that promote cell division. The binding of FGFs to the heparan sulfate of cell-surface-bound proteoglycans appears to be critical for their activity. The interaction of fibroblast growth factor-1 (FGF-1 or aFGF) using heparin lyase-derived oligosaccharides from heparan sulfate was investigated. FGF-1 was also shown to protect sequences in heparan sulfate from heparin lyase digestion and protected oligosaccharide products of octasaccharide and decasaccharide size were recovered by FGF-1 affinity chromatography, suggesting that the high-affinity binding of heparan sulfate to FGF-1 resides within an octasaccharide sequence. The FGF-1 binding affinity of heparan sulfate is reduced compared to heparin presumably due to the absence of 6-sulfate groups in heparan sulfate. Inspection of the FGF-1 heparan sulfate binding domain shows that the majority of interacting amino acids are contained within a 20-amino-acid sequence that folds back upon itself (because of three turns) forming a triangular shaped cup of positive charge. The importance of FGF-1 binding site topology was investigated using three synthetic peptide mimics of the FGF-1 glycosaminoglycan (GAG) binding site. Heparan sulfate affinity chromatography and isothermal titration calorimetry, used to measure binding thermodynamics, demonstrated that a synthetic peptide analogous to the GAG binding site in FGF-1 bound tightly to heparan sulfate. A peptide containing a D-proline in place of L-proline bound with considerably reduced affinity, presumably due to the altered structure of the second turn in the binding site. A cyclic peptide, expected to be topologically most similar to the triangular GAG binding site in FGF-1, bound with the highest affinity to heparan sulfate. These data suggest the triangular topology of the GAG binding site in FGF is critical for its interaction with heparan sulfate. Analysis of known GAG binding sites in 25 proteins using the Chou-Fasman algorithm show that these sites commonly contain turns.
Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Oligossacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calorimetria , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Mucosa Intestinal/metabolismo , Modelos Moleculares , Conformação Molecular , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Suínos , TermodinâmicaRESUMO
Dengue virus is a human pathogen that has reemerged as an increasingly important public health threat. We found that the cellular receptor utilized by dengue envelope protein to bind to target cells is a highly sulfated type of heparan sulfate. Heparin, highly sulfated heparan sulfate, and the polysulfonate pharmaceutical Suramin effectively prevented dengue virus infection of target cells, indicating that the envelope protein-target cell receptor interaction is a critical determinant of infectivity. The dengue envelope protein sequence includes two putative glycosaminoglycan-binding motifs at the carboxy terminus; the first could be structurally modeled and formed an unusual extended binding surface of basic amino acids. Similar motifs were also identified in the envelope proteins of other flaviviridae. Developing pharmaceuticals that inhibit target cell binding may be an effective strategy for treating flavivirus infections.
Assuntos
Vírus da Dengue/patogenicidade , Heparitina Sulfato/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Chlorocebus aethiops , Cricetinae , Vírus da Dengue/metabolismo , Glicosaminoglicanos/metabolismo , Heparina/farmacologia , Heparitina Sulfato/química , Heparitina Sulfato/farmacologia , Imuno-Histoquímica , Dados de Sequência Molecular , Ligação Proteica , Receptores Virais/metabolismo , Suramina/farmacologia , Células Vero , Virulência/efeitos dos fármacosRESUMO
Electrospray ionization mass spectrometry was used in the negative ion mode to analyze complexes of sucrose octasulfate, sucrose heptasulfate and sulfated alpha-, beta- and gamma-cyclodextrins with synthetically prepared basic peptides, the basic protein ubiquitin and polyamines. The spectra presented demonstrate that complexes with these basic molecules facilitate the analysis of these polysulfated oligosaccharides. Stable (1:1) complexes result from the ion pairing between the protonated basic arginine and lysine residues of the peptide and the anionic sulfate groups of the polysulfated oligosaccharides. Fragmentation of the polysulfated oligosaccharides resulting in the loss of SO3 could be suppressed by controlling the experimental conditions, such as the nozzle-skimmer voltage, used to obtain the spectra. In the absence of fragmentation, it was possible to obtain data on the purity of sucrose octasulfate and sucrose heptasulfate as well as the distribution of the sulfated cyclodextrins. The confounding presence of sodium counter-ions is also eliminated using this method. Complete chemical sulfation of oligosaccharides is difficult to achieve. Thus, data on sample purity are essential for the characterization of sulfated oligosaccharides used as pharmaceutical agents.
Assuntos
Carboidratos/análise , Oligossacarídeos/análise , Peptídeos/análise , Proteínas/análise , Sulfatos/análise , Ciclodextrinas/análise , Espectrometria de Massas , Peso Molecular , Sacarose/análogos & derivados , Sacarose/análiseRESUMO
Glycosaminoglycan (GAG)-protein interactions regulate a myriad of physiologic and pathologic processes, yet an understanding of how these molecules interact is lacking. The role of the pattern and spacing of basic amino acids (arginine (R) and lysine (K)) in heparin binding sites was investigated using peptide analogs as well as by examining known heparin binding sites. Peptides having the general structure R(n)W (n = 3-9, where tyrosine (W) was added for peptide detection) were synthesized and their interaction with heparin was determined by isothermal titration calorimetry. Binding affinity increased with increasing number of R residues. A 9-mer of R (R9W) bound as tightly to heparin as acidic fibroblast growth factor under physiologic conditions. Despite their high affinity for heparin, long stretches of basic amino acids are uncommon in heparin binding proteins. Known heparin binding sites most commonly contain single isolated basic amino acids separated by one nonbasic amino acid. Peptides having the structure, H3CCONH-GRRG(m)RRG(5-m)-CONH2 (denoted as the RRG(m)RR peptide series) and H3CCONH-GRRRG(m)RG(5-m)-CONH2 (denoted as the RRRG(m)R peptide series), where m = 0-5, were synthesized to test the hypothesis that the spacing of basic amino acids in heparin binding sites is optimally arranged to interact with different GAGs. The peptides, in both the -RRG(m)RR- and -RRRG(m)R- peptide series, when m = 0, bound most tightly with heparin, as measured by affinity chromatography. In contrast, the -RRG(m)RR-peptide series interacted most tightly with heparan sulfate when m = 0 or 1, whereas the -RRRG(m)R- peptide series bound tightest when m = 3. These results are consistent with our understanding of heparin and heparan sulfate structure. A highly sulfated GAG, such as heparin, interacts most tightly with peptides (or peptide sequences within proteins) containing a complementary binding site of high positive charge density. Heparan sulfate, having fewer and more highly spaced negatively charged groups, interacts most tightly with a complementary site on a peptide (or peptide sequences with proteins) that has more widely spaced cationic residues.
Assuntos
Aminoácidos/química , Heparina/química , Sequência de Aminoácidos , Sítios de Ligação , Calorimetria , Cromatografia de Afinidade , Heparina/metabolismo , Dados de Sequência Molecular , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1-->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity.
Assuntos
Fator 2 de Crescimento de Fibroblastos/farmacologia , Glicosaminoglicanos/farmacologia , Mitógenos/farmacologia , Animais , Sítios de Ligação , Calorimetria , Dimerização , Fator 2 de Crescimento de Fibroblastos/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Heparina/metabolismo , Heparina/farmacologia , Espectroscopia de Ressonância Magnética , Mitógenos/metabolismo , Estrutura Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Ligação Proteica , Caramujos/química , Timidina/metabolismoRESUMO
This study presents a comparison of heparan sulphate chains isolated from various porcine and bovine tissues. 1H-NMR spectroscopy (500 MHz) was applied for structural and compositional studies on intact heparan sulphate chains. After enzymic digestion of heparan sulphate using heparin lyase I (EC 4.2.2.7) II and III (EC 4.2.2.8), the compositions of unsaturated disaccharides obtained were determined by analytical capillary electrophoresis. Correlations between the N-sulphated glucosamine residues and O-sulphation and between iduronic acid content and total sulphation were discovered using the data obtained by NMR and disaccharide analysis. Heparan sulphate chains could be classified into two groups based on the sulphation degree and the iduronic acid content. Heparan sulphate chains with a high degree of sulphation possessed also a significant number of iduronic acid residues and were isolated exclusively from porcine brain, liver and kidney medulla. The presence and amount of N-unsubstituted glucosamine residues (GlcNp) was established in all of the heparan sulphates examined. The structural context in which this residue occurs was demonstrated to be: high sulphation domain --> 4)-beta-D-GlcAp-(1 --> 4)-alpha-D-GlcNp-(1 --> 4)-beta-D-GlcAp-(1 --> low sulphation domain (where GlcNp is 2-amino-2-deoxyglucopyranose, and GlcAp is glucopyranosyluronic acid), based on the isolation and characterization of a novel, heparin lyase III-derived, GlcNp containing tetrasaccharide and hexasaccharide. The results presented suggest that structural differences may play a role in important biological events controlled by heparan sulphate in different tissues.
Assuntos
Amino Açúcares/análise , Heparitina Sulfato/química , Animais , Sequência de Carboidratos , Bovinos , Dissacarídeos/química , Eletroforese Capilar , Glucosamina/análise , Heparina/química , Ácido Idurônico/análise , Espectroscopia de Ressonância Magnética , Masculino , Dados de Sequência Molecular , Peso Molecular , Oligossacarídeos/química , Especificidade da Espécie , Suínos , Distribuição TecidualRESUMO
Porcine intestinal mucosal heparan sulfate was exhaustively depolymerized on a large scale using heparin lyase II (heparinase II) or heparin lyase III (heparitinase, EC 4.2.2.8). The oligosaccharide mixtures formed with each enzyme were fractionated by low pressure gel permeation chromatography. Size-uniform mixtures of disaccharides, tetrasaccharides, and hexasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semipreparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 13 homogenous oligosaccharides. The purity of each oligosaccharide was demonstrated by the presence of a single peak on analytical strong-anion-exchange high-performance liquid chromatography and reversed polarity capillary electrophoresis. The structures of these oligosaccharides were established using 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy. Three of the thirteen structures that were solved were novel while the remaining 10 have been previously described. All of the structures obtained using heparin lyase III contained a delta UAp residue (where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid) at their nonreducing termini. Structures obtained using heparin lyase II contained both delta UAp and delta UAp2S (where S is sulfate) at their nonreducing termini. These results are consistent with the reported specificity of both enzymes.
Assuntos
Heparitina Sulfato/química , Oligossacarídeos/química , Animais , Sequência de Carboidratos , Cromatografia/métodos , Heparina Liase , Heparitina Sulfato/metabolismo , Hidrogênio/química , Mucosa Intestinal/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Polissacarídeo-Liases/metabolismo , Análise de Sequência , SuínosRESUMO
Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.
Assuntos
Fatores de Crescimento Endotelial/metabolismo , Heparina/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Animais , Células CHO , Bovinos , Células Cultivadas , Cromatografia de Afinidade/métodos , Cricetinae , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Heparina/isolamento & purificação , Heparina/farmacologia , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Oligossacarídeos/análise , Ligação Proteica , Receptores Proteína Tirosina Quinases/química , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão , Fator A de Crescimento do Endotélio Vascular , Fator C de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Two new oligosaccharides were prepared from heparin by its partial depolymerization using heparin lyase I (EC 4.2.2.7) in an attempt to prepare oligosaccharides having intact antithrombin III binding sites. The oligosaccharides were purified by chromatography on the basis of both size and charge and demonstrated a high level of purity by capillary electrophoresis. One- and two-dimensional 1H NMR spectroscopy at 500 MHz revealed the structure of each oligosaccharide. The octasaccharide and decasaccharide are DeltaUAp2S(1-->4)-alpha-DGlcNpS6S(1-->4)-alpha-L-IdoAp (1-->4)-alpha-D -GlcNpAc6S(1-->4)-betaD-GlcAp(1-->4)-alpha-D-GlcNpS 3S6S(1-->4)-alpha- L-IdoAp2S(1-->4)alpha-D-GlcNpS6S (where DeltaUAp is 4-deoxy-alpha-L-threo-hex-enopyranosyluronic acid, GlcNp is 2-amino-2-deoxy-glucopyranose, GlcAp is glucopyranosyluronic acid, S is sulfate and Ac is acetate) and DeltaUAp2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp++ +(1-->4)-alpha- D-GlcNpAc6S (1-->4)-beta-D-GlcAp(1-->4)-alpha-D-GlcNpS3S6S(1-->4)-alpha- L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S(1-->4)-alpha -D-GlcNpS 6S, respectively. A hexasaccharide containing a similar structural motif to that found in the antithrombin III binding site and having greatly reduced anticoagulant activity was also isolated. The structure of the hexasaccharide is DeltaUAp2S(1-->4)-alpha-D-GlcNpAc6S(1-->4)-beta-D-GlcAp++ +(1-->4)-alpha- D-GlcNpS3S6S(1-->4)-alpha-L-IdoAp(1-->4)-alpha-D-GlcNpS6S . The octasaccharide and decasaccharide correspond to the predominant structural motif found in porcine intestinal mucosal heparin. Sufficient quantities of the decasaccharide were obtained to examine its interaction with antithrombin III using microtitration calorimetry. This decasaccharide bound to antithrombin III with similar avidity as heparin and showed comparable anticoagulant activity, as determined using an antithrombin III dependent anti-factor Xa assay. Interestingly, while both decasaccharide and heparin bound to antithrombin with nanomolar affinity, very little heat of binding was observed.
Assuntos
Antitrombina III/metabolismo , Heparina/análogos & derivados , Oligossacarídeos/metabolismo , Animais , Sítios de Ligação , Cromatografia em Gel , Espectroscopia de Ressonância Magnética , SuínosRESUMO
Crystal structures of heparin-derived tetra- and hexasaccharides complexed with basic fibroblast growth factor (bFGF) were determined at resolutions of 1.9 and 2.2 angstroms, respectively. The heparin structure may be approximated as a helical polymer with a disaccharide rotation of 174 degrees and a translation of 8.6 angstroms along the helix axis. Both molecules bound similarly to a region of the bFGF surface containing residues asparagine-28, arginine-121, lysine-126, and glutamine-135, the hexasaccharide also interacted with an additional binding site formed by lysine-27, asparagine-102, and lysine-136. No significant conformational change in bFGF occurred upon heparin oligosaccharide binding, which suggests that heparin primarily serves to juxtapose components of the FGF signal transduction pathway.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Heparina/química , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Cristalização , Cristalografia por Raios X , Heparina/metabolismo , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Conformação ProteicaRESUMO
Although the interaction of proteins with glycosaminoglycans (GAGs) such as heparin are of great importance, the general structural requirements for protein- or peptide-GAG interaction have not been well characterized. Electrostatic interactions between sulfate and carboxylate groups on the GAG and basic residues in the protein or peptide dominate the interaction, but the thermodynamics of these electrostatic interactions have not been studied. Arginine residues occur frequently in the known heparin binding sites of proteins. Arginine is also more common than lysine in randomly synthesized 7-mer peptides that bind to immobilized heparin and heparan sulfate. We have used heparin affinity chromatography, equilibrium dialysis, and isothermal titration calorimetry techniques to further investigate these interactions. A 7-mer of arginine eluted from a heparin-affinity column at 0.82 M NaCl, whereas the analogous 7-mer of lysine eluted at 0.64 M. Similarly, the putative heparin binding site peptide (amino acid residues 110-130) from acidic fibroblast growth factor, which contained four lysine and two arginine residues, eluted at 0.50 M, whereas the analogous peptide with six lysine residues eluted at 0.41 M and one with six arginine residues eluted at 0.54 M. At 25 degrees C in 10 mM sodium phosphate, pH 7.4, carboxy and amino termini blocked arginine (blocked arginine) bound to heparin twice as tightly as blocked lysine as measured by equilibrium dialysis Similarly, at 30 degrees C in 10 mM sodium phosphate, pH 7.4, and in water, blocked arginine bound 2.5 times more tightly to anions in heparin than blocked lysine. Using titration calorimetry, the enthalpy of blocked arginine and lysine binding to heparin was 1.14 +/- 0.24 and 0.45 +/- 0.35 kJ/mol, respectively, under identical conditions. Our observations show that blocked arginine- and arginine-containing peptides bound more tightly to GAGs than the analogous lysine species and suggest that the difference was due to the intrinsic properties of the arginine and lysine side chains. The greater affinity of the guanidino cation for sulfate in GAGs is probably due to stronger hydrogen bonding and a more exothermic electrostatic interaction. This can be rationalized by soft acid, soft base concepts.
Assuntos
Arginina/química , Fator 1 de Crescimento de Fibroblastos/química , Heparina/química , Lisina/química , Sequência de Aminoácidos , Calorimetria , Sequência Consenso , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
A method for the preparation of heparan sulfate from peptidoglycan heparin is described. The objective of this research was to provide a basis for the development and validation of an industrial process to support the preclinical development of heparan sulfate and/or heparan sulfate derivatives. In the preparation of heparan sulfate, heparin was recovered by alcohol fractionation and dermatan sulfate was isolated by selective precipitation. The remaining crude heparan sulfate was fractionated by anion-exchange chromatography into five subfractions. The biological activities of these subfractions were examined by anticoagulant and amidolytic assays. Molecular weight and molecular size were determined using capillary viscometry and polyacrylamide gel electrophoresis. Charge density and degree of sulfation were determined by cellulose acetate electrophoresis and elemental analysis. Oligosaccharide and disaccharide analysis relied on enzymatic depolymerization using heparin lyases followed by polyacrylamide gel and capillary electrophoresis. 1H NMR analysis provided detailed structural information on each subfraction. Crude heparin sulfate and its subfractions showed significant differences in physical, structural and biological properties.
Assuntos
Heparina/química , Heparitina Sulfato/química , Heparitina Sulfato/isolamento & purificação , Mucosa Intestinal/química , Peptidoglicano/química , Animais , Sequência de Carboidratos , Fracionamento Químico , Heparitina Sulfato/metabolismo , Dados de Sequência Molecular , SuínosRESUMO
1. Dermatan sulfate is a linear, sulfated polysaccharide and is a glycosaminoglycan component of several important proteoglycans. This minireview discusses the biosynthesis, structure and biological function of dermatan sulfate proteoglycans. 2. Dermatan sulfate and its derivatives are being investigated as a new class of anticoagulant and antithrombotic agents. 3. The preparation, chemistry and structure-activity relationship of dermatan sulfate is described. 4. Dermatan sulfate, low molecular weight dermatan sulfate and glycosaminoglycan mixtures containing dermatan sulfate have been used clinically. 5. The future prospects of these agents and other new, potentially useful dermatan sulfate based therapeutics are discussed.