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G Venkataraman

Publications and source records attributed to G Venkataraman.

At least 19 recordsLinked to original sources
PubMed2000

Sequencing of 3-O sulfate containing heparin decasaccharides with a partial antithrombin III binding site.

Heparin- and heparan sulfate-like glycosaminoglycans (HLGAGs) represent an important class of molecules that interact with and modulate the activity of growth factors, enzymes, and morphogens. Of the many biological functions for this class of molecules, one of its most important functions is its interaction with antithrombin III (AT-III). AT-III binding to a specific heparin pentasaccharide sequence, containing an unusual 3-O sulfate on a N-sulfated, 6-O sulfated glucosamine, increases 1,000-fold AT-III's ability to inhibit specific proteases in the coagulation cascade. In this manner, HLGAGs play an important biological and pharmacological role in the modulation of blood clotting. Recently, a sequencing methodology was developed to further structure-function relationships of this important class of molecules. This methodology combines a property-encoded nomenclature scheme to handle the large information content (properties) of HLGAGs, with matrix-assisted laser desorption ionization MS and enzymatic and chemical degradation as experimental constraints to rapidly sequence picomole quantities of HLGAG oligosaccharides. Using the above property-encoded nomenclature-matrix-assisted laser desorption ionization approach, we found that the sequence of the decasaccharide used in this study is DeltaU(2S)H(NS,6S)I(2S)H(NS, 6S)I(2S)H(NS,6S)IH(NAc,6S)GH(NS,3S,6S) (+/-DDD4-7). We confirmed our results by using integral glycan sequencing and one-dimensional proton NMR. Furthermore, we show that this approach is flexible and is able to derive sequence information on an oligosaccharide mixture. Thus, this methodology will make possible both the analysis of other unusual sequences in HLGAGs with important biological activity as well as provide the basis for the structural analysis of these pharamacologically important group of heparin/heparan sulfates.

Antithrombin III↗
PubMed2000

Cleavage of the antithrombin III binding site in heparin by heparinases and its implication in the generation of low molecular weight heparin.

Heparin has been used as a clinical anticoagulant for more than 50 years, making it one of the most effective pharmacological agents known. Much of heparin's activity can be traced to its ability to bind antithrombin III (AT-III). Low molecular weight heparin (LMWH), derived from heparin by its controlled breakdown, maintains much of the antithrombotic activity of heparin without many of the serious side effects. The clinical significance of LMWH has highlighted the need to understand and develop chemical or enzymatic means to generate it. The primary enzymatic tools used for the production of LMWH are the heparinases from Flavobacterium heparinum, specifically heparinases I and II. Using pentasaccharide and hexasaccharide model compounds, we show that heparinases I and II, but not heparinase III, cleave the AT-III binding site, leaving only a partially intact site. Furthermore, we show herein that glucosamine 3-O sulfation at the reducing end of a glycosidic linkage imparts resistance to heparinase I, II, and III cleavage. Finally, we examine the biological and pharmacological consequences of a heparin oligosaccharide that contains only a partial AT-III binding site. We show that such an oligosaccharide lacks some of the functional attributes of heparin- and heparan sulfate-like glycosaminoglycans containing an intact AT-III site.

Antithrombin III↗
PubMed2000

Heparin and heparan sulfate: biosynthesis, structure and function.

Heparin and heparan sulfate glycosaminoglycans are acidic complex polysaccharides found on the cell surface and in the extracellular matrix. Recent progress has uncovered a virtual explosion of important roles of these biopolymers in fundamental biological processes. Advances in the understanding of biosynthesis and structure and the development of novel analytical methods for composition and sequence analysis have provided remarkable insights into structure/function relationships of these complex and once elusive polysaccharides.

Animals↗
PubMed1999

Sequencing complex polysaccharides.

Although rapid sequencing of polynucleotides and polypeptides has become commonplace, it has not been possible to rapidly sequence femto- to picomole amounts of tissue-derived complex polysaccharides. Heparin-like glycosaminoglycans (HLGAGs) were readily sequenced by a combination of matrix-assisted laser desorption ionization mass spectrometry and a notation system for representation of polysaccharide sequences. This will enable identification of sequences that are critical to HLGAG biological activities in anticoagulation, cell growth, and differentiation.

Carbohydrate Sequence↗
PubMed1999

Oligomeric self-association of basic fibroblast growth factor in the absence of heparin-like glycosaminoglycans.

Basic fibroblast growth factor (FGF-2) represents a class of heparin-binding growth factors that are stored in the extracellular matrix attached to heparin-like glycosaminoglycans (HLGAGs). It has been proposed that cell surface HLGAGs have a central role in the biological activity of FGF-2, presumably by inducing dimers or oligomers of FGF-2 and leading to the dimerization or oligomerization of FGF receptor and hence signal transduction. We have previously proposed that FGF-2 possesses a natural tendency to self-associate to form FGF-2 dimers and oligomers; HLGAGs would enhance FGF-2 self-association. Here, through a combination of spectroscopic, chemical cross-linking and spectrometric techniques, we provide direct evidence for the self-association of FGF-2 in the absence of HLGAGs, defying the notion that HLGAGs induce FGF-2 oligomerization. Further, the addition of HLGAGs seems to enhance significantly the FGF-2 oligomerization process without affecting the relative percentages of FGF-2 dimers, trimers or oligomers. FGF-2 self-association is consistent with FGF-2's possessing biological activity both in the presence and in the absence of HLGAGs; this leads us to propose that FGF-2 self-association enables FGF-2 to signal both in the presence and in the absence of HLGAGs.

Biopolymers↗
PubMed1999

Molecular characteristics of fibroblast growth factor-fibroblast growth factor receptor-heparin-like glycosaminoglycan complex.

Fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. Understanding of the molecular nature of interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF-FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, we determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. We propose that the FGF-FGFR interaction mediated by the 'conserved' primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the 'variable' secondary sites, on both FGF as well as FGFR mediates specificity of a given FGF to a given FGFR isoform. Furthermore, as the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, we find that the spatial orientation of the primary receptor-binding site of FGF-2 coincides structurally with the IL-1beta receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF-FGFR interactions. In the FGF-FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG.

Amino Acid Sequence↗
PubMed1999

Fibroblast growth factors 1 and 2 are distinct in oligomerization in the presence of heparin-like glycosaminoglycans.

Fibroblast growth factor (FGF) 1 and FGF-2 are prototypic members of the FGF family, which to date comprises at least 18 members. Surprisingly, even though FGF-1 and FGF-2 share more than 80% sequence similarity and an identical structural fold, these two growth factors are biologically very different. FGF-1 and FGF-2 differ in their ability to bind isoforms of the FGF receptor family as well as the heparin-like glycosaminoglycan (HLGAG) component of proteoglycans on the cell surface to initiate signaling in different cell types. Herein, we provide evidence for one mechanism by which these two proteins could differ biologically. Previously, it has been noted that FGF-1 and FGF-2 can oligomerize in the presence of HLGAGs. Therefore, we investigated whether FGF-1 and FGF-2 oligomerize by the same mechanism or by a different one. Through a combination of matrix-assisted laser desorption ionization mass spectrometry and chemical crosslinking, we show here that, under identical conditions, FGF-1 and FGF-2 differ in the degree and kind of oligomerization. Furthermore, an extensive analysis of FGF-1 and FGF-2 uncomplexed and HLGAG complexed crystal structures enables us to readily explain why FGF-2 forms sequential oligomers whereas FGF-1 forms only dimers. FGF-2, which possesses an interface capable of protein association, forms a translationally related oligomer, whereas FGF-1, which does not have this interface, forms only a symmetrically related dimer. Taken together, these data show that FGF-1 and FGF-2, despite their sequence homology, differ in their mechanism of oligomerization.

Binding Sites↗
PubMed1999

The calcium-binding sites of heparinase I from Flavobacterium heparinum are essential for enzymatic activity.

In the accompanying paper (Shriver, Z., Liu, D., Hu, Y., and Sasisekharan, R. (1999) J. Biol. Chem. 274, 4082-4088), we have shown that calcium binds specifically to heparinase I and have identified two major calcium-binding sites (CB-1 and CB-2) that partly conform to the EF-hand calcium-binding motif. In this study, through systematic site-directed mutagenesis, we have confirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity. More specifically, we identified critical residues (viz. Asp210, Asp212, Gly213, and Thr216 in CB-1 and Asn375, Tyr379, and Glu381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Mutations in CB-1 resulted in a lower kcat, but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the kcat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides. Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I. Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I. These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism. Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.

Amino Acid Sequence↗
PubMed1999

FGF-2/fibroblast growth factor receptor/heparin-like glycosaminoglycan interactions: a compensation model for FGF-2 signaling.

Heparin-like glycosaminoglycans (HLGAGs) play a central role in the biological activity and signaling behavior of basic fibroblast growth factor (FGF-2). Recent studies, however, indicate that FGF-2 may be able to signal in the absence of HLGAG, raising the question of the nature of the role of HLGAG in FGF-2 signaling. In this study, we present a conceptual framework for FGF-2 signaling and derive a simple model from it that describes signaling via both HLGAG-independent and HLGAG-dependent pathways. The model is validated with F32 cell proliferation data using wild-type FGF-2, heparin binding mutants (K26A, K119A/R120A, K125A), and receptor binding mutants (Y103A, Y111A/W114A). In addition, this model can predict the cellular response of FGF-2 and its mutants as a function of FGF-2 and HLGAG concentration based on experimentally determined thermodynamic parameters. We show that FGF-2-mediated cellular response is a function of both FGF-2 and HLGAG concentrations and that a reduction of one of the components can be compensated for by an increase in the other to achieve the same measure of cellular response. Analysis of the mutant FGF-2 molecules show that reduction in heparin binding interactions and primary receptor site binding interactions can also be compensated for in the same manner. These results suggest a molecular mechanism that could be used by cells in physiological systems to modulate the FGF-2-mediated cellular response by controlling HLGAG expression.

Binding Sites↗
PubMed1997

Heparin-induced self-association of fibroblast growth factor-2. Evidence for two oligomerization processes.

Fibroblast growth factor-2 (FGF-2), a potent angiogenic factor, requires heparin for dimerization and activation of the FGF receptor tyrosine kinase. The binding of multiple fibroblast growth factors by heparin may be necessary for dimerization of the FGF receptor. Analytical ultracentrifugation of FGF-2 in the presence of heparin-derived saccharides shows that both an active heparin octasaccharide and an inactive heparin-like disaccharide induce fibroblast growth factor-2 self-association. Analysis of the data indicates that the heparin octasaccharide induces a monomer-dimer-tetramer assembly of FGF-2 while the disaccharide induces a monomer-dimer equilibrium. Evidence is presented indicating that the dimer conformation induced by the heparin octasaccharide is a side by side dimer with the FGF-2 molecules cis to the heparin, while the disaccharide-induced dimer is a head to head dimer in which FGF-2 molecules are trans to the ligand. These results, combined with previous studies, support the model that formation of a specific side by side heparin-induced FGF-2 dimer is required for activation of the FGF receptor.

Dimerization↗
PubMed1997

Single-stranded DNA and RNA targeted triplex-formation: UV, CD and molecular modeling studies of foldback triplexes containing different RNA, 2'-OMe-RNA and DNA strand combinations.

We studied the influence of different 2'-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D.D:D, R*.D:D, D.D:R*, R*.D:R*, D.R:D, R*.R:D, D.R:R*, and R*.R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2'-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2'-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2'-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D:D), while hetero-foldback triplex spectra suggest intermediate conformation to both A-type and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).

Circular Dichroism↗
PubMed1997

Somatostatin receptor subtype expression in human thyroid and thyroid carcinoma cell lines.

Somatostatin (SRIH) analogs can suppress the proliferation of human differentiated thyroid carcinoma cell lines that express SRIH receptors (SSTRs) demonstrated by radioligand binding analysis. Five distinct human SSTR subtypes (hSSTR1-5) that bind native SRIH exhibit diverse affinities to a wide range of SRIH analogs. Reverse transcriptase-PCR amplification of ribonucleic acids (RNAs) obtained from normal thyroid tissues and nine human thyroid carcinoma cell lines, grown as monolayer cultures and xenograft tumors in nude mice, were used to discriminate expression of SSTR subtype messenger RNAs (mRNAs). The cell lines were derived from a follicular adenoma (KAK-1), two follicular carcinomas (MRO-87 and WRO-82), two papillary carcinomas (NPA87 and KAT-10), and four anaplastic thyroid carcinomas (DRO-90, ARO-81, KAT-4, and KAT-18). Most thyroid cancer cell line monolayers and xenografts expressed SSTR3 and SSTR5 mRNAs. SSTR1 expression was more varied between monolayers and xenografts, whereas SSTR2 mRNA was only faintly detectable at the most extreme resolution. SSTR4 mRNA was faintly positive in only one anaplastic carcinoma xenograft. Normal thyroid also expressed SSTR3 and SSTR5 mRNAs, with only faint expression of SSTR1 and SSTR2 mRNAs (in one of five and three of five samples, respectively). SSTR mRNA expression was dependent upon in vitro culture conditions, as xenograft SSTR mRNA expression tended to decrease compared to that in each respective monolayer culture. Characterization of SSTR subtype expression in human thyroid carcinomas may permit targeting of specific SRIH analogs to inhibit proliferation of differentiated and anaplastic thyroid carcinomas in patients.

Animals↗
PubMed1996

Heparinase III from Flavobacterium heparinum: cloning and recombinant expression in Escherichia coli.

Heparinase III (E.C. 4.2.2.8), formerly heparinase I, produced by Flavobacterium heparinum is an enzyme that specifically cleaves heparan sulfate-rich regions of acidic polysaccharides. In this study, we report the cloning of the heparinase III gene using polymerase chain reaction (PCR). Two degenerate oligonucleotides, based on amino acid sequences derived from tryptic peptides of purified heparinase III were used to generate a approximately 1100-bp probe by PCR amplification using Flavobacterium genomic DNA as the template. The PCR-derived probe was used to screen a Flavobacterium genomic DNA library in lambda ZAP II. The open reading frame of the heparinase III gene is 1980 bp in length, encoding a precursor protein of 75,950 Da; 10 of the tryptic peptides mapped onto the open reading frame which corresponded to approximately 18% of the protein. Recombinant heparinase III was expressed in Escherichia coli using the T7 polymerase pET expression system. This is the first report of the cloning and recombinant expression of an enzyme primarily degrading heparan sulfate.

Amino Acid Sequence↗
PubMed1996

Expression in Escherichia coli, purification and characterization of heparinase I from Flavobacterium heparinum.

The use of heparin for extracorporeal therapies has been problematical due to haemorrhagic complications; as a consequence, heparinase I from Flavobacterium heparinum is used for the determination of plasma heparin and for elimination of heparin from circulation. Here we report the expression of recombinant heparinase I in Escherichia coli, purification to homogeneity and characterization of the purified enzyme. Heparinase I was expressed with an N-terminal histidine tag. The enzyme was insoluble and inactive, but could be refolded, and was purified to homogeneity by nickel-chelate chromatography. The cumulative yield was 43%, and the recovery of purified heparinase I was 14.4 mg/l of culture. The N-terminal sequence and the molecular mass as analysed by matrix-assisted laser desorption MS were consistent with predictions from the heparinase I gene structure. The reverse-phase HPLC profile of the tryptic digest, the Michaelis-Menten constant Km (47 micrograms/ml) and the specific activity (117 units/mg) of purified recombinant heparinase I were similar to those of the native enzyme. Degradation of heparin by heparinase I results in a characteristic product distribution, which is different from those obtained by degradation with heparinase II or III from F. heparinum. We developed a rapid anion-exchange HPLC method to separate the products of enzymic heparin degradation, using POROS perfusion chromatography media. Separation of characteristic di-, tetra- and hexa-saccharide products is performed in 10 min. These methods for the expression, purification and analysis of recombinant heparinase I may facilitate further development of heparinase I-based medical therapies as well as further investigation of the structures of heparin and heparan sulphate and their role in the extracellular matrix.

Amino Acid Sequence↗
PubMed1996

Heparinase I from Flavobacterium heparinum. Mapping and characterization of the heparin binding domain.

In this study we have identified the primary heparin binding site of heparinase I (EC 4.2.2.7). Chemical and proteolytic digests of heparinase I were used in direct binding and competition assays, to map the regions of heparinase I that interact specifically with heparin. We find the heparin binding site contains two Cardin-Weintraub heparin binding consensus sequences and a calcium co-ordination consensus motif. We show that heparin binding to heparinase I is independent of calcium (Kd of 60 nm) and that calcium is able to activate heparinase I catalytically. We find that sulfhydryl selective labeling of cysteine 135 of heparinase I protects the lysines of the heparin binding sequence from proteolytic cleavage, suggesting the close proximity of the heparin binding site to the active site. Site-directed mutagenesis of H203A (contained in the heparin binding site) inactivated heparinase I; however, a H203D mutant retained marginal activity, indicating a role for this residue in catalysis. The above results taken together suggest that histidine 203 (hence the heparin binding site) is immediately adjacent to the scissile bond. We propose that the heparin binding site and active site are in close proximity to each other and that the calcium coordination motif, contained in the heparin binding site, may bridge heparin to heparinase I through calcium in a ternary complex during catalysis.

Amino Acid Sequence↗
PubMed1996

Preferential self-association of basic fibroblast growth factor is stabilized by heparin during receptor dimerization and activation.

Central to signaling by fibroblast growth factors (FGFs) is the oligomeric interaction of the growth factor and its high-affinity cell surface receptor, which is mediated by heparin-like polysaccharides. It has been proposed that the binding of heparin-like polysaccharides to FGF induces a conformational change in FGF, resulting in the formation of FGF dimers or oligomers, and this biologically active form is 'presented' to the FGF receptor for signal transduction. In this study, we show that monomeric basic FGF (FGF-2) preferentially self-associates and forms FGF-2 dimers and higher-order oligomers. As a consequence, FGF-2 monomers are oriented for binding to heparin-like polysaccharides. We also show that heparin-like polysaccharides can readily bind to self-associated FGF-2 without causing a conformational change in FGF-2 or disrupting the FGF-2 self-association, but that the bound polysaccharides only additionally stabilize the FGF-2 self-association. The preferential self-association corresponds to FGF-2 translations along two of the unit cell axes of the FGF-2 crystal structures. These two axes represent the two possible heparin binding directions, whereas the receptor binding sites are oriented along the third axis. Thus, we propose that preferential FGF-2 self-association, further stabilized by heparin, like "beads on a string," mediates FGF-2-induced receptor dimerization and activation. The observed FGF-2 self-association, modulated by heparin, not only provides a mechanism of growth factor activation but also represents a regulatory mechanism governing FGF-2 biological activity.

Apoproteins↗
PubMed1995

Single strand targeted triplex formation: targeting purine-pyrimidine mixed sequences using abasic linkers.

Foldback triplex-forming oligonucleotides (FTFOs) that contain an abasic linker, [2-(4-aminobutyr-1-yl)-1,3-propanediol] (APD linker), in the Hoogsteen domain against pyrimidine bases of a C:G and a T:A base pair were studied for their relative stability and sequence specificity of triplex formation. In general, the APD linker has less destabilizing effect against a C:G base pair than a T:A base pair. Incorporation of three APD linker moieties resulted in decreased binding to the target, which was comparable to results observed with three imperfectly matched natural base triplets. The APD linker incorporation did not result in the loss of sequence specificity of FTFOs, unlike in the case of normal triplex-forming oligonucleotides (TFOs). The introduction of a positively charged abasic linker, however, resulted in decreased stability of the triplex, because of loss of hydrogen bonding and stacking interactions in the major groove. The results of a molecular modeling study show that APD linker can be readily incorporated without any change in the conformation of the natural sugar-phosphate backbone conserving overall triple helix geometry. Further, the modeling study suggests a hydrogen bond formation between the amino group of linker and N4 of cytosine mediated by a solvent molecule (water) in the floor of the base triplet in addition to a contribution from the positive charge on the APD linker amino group. Either a direct or water-mediated hydrogen bond between the amino group of the APD linker and the O4 of thymine is unlikely when the linker is placed against a T:A base pair.

Base Composition↗