Characterization of the heparin-binding site of the mycobacterial heparin-binding hemagglutinin adhesin

The mycobacterial adhesin heparin-binding hemagglutinin (HBHA) contains several lysine-rich repeats at its carboxyl-terminal end. Using truncated recombinant HBHA forms and hybrid proteins containing HBHA repeats grafted onto the Escherichia coli maltose-binding protein (MBP), we found that these repeats are responsible for heparin binding. Immunofluorescence microscopy studies revealed that their deletion abrogates binding of HBHA to human pneumocytes. Conversely, when fused to MBP, the HBHA repeats confer pneumocyte adherence properties to the hybrid protein. Treatment of pneumocytes with glycosaminoglycan-degrading enzymes showed that HBHA binding depends on the presence of heparan sulfate chains on the cell surface. The epitope of a monoclonal antibody that inhibits mycobacterial adherence to epithelial cells was mapped within the lysine-rich repeats, confirming their involvement in mycobacterial adherence to epithelial cells. Surface plasmon resonance analyses showed that recombinant HBHA binds to immobilized heparin with fast association kinetics (k(a) = 5.62 (+/- 0.10) x 10(5) m(-1) s(-1)), whereas the dissociation kinetics were slower (k(d) = 0.015 (+/- 0.002) s(-1)), yielding a K(D) value of 26 nm. Similar analyses with grafted MBP indicated similar kinetic constants, indicating that the carboxyl-terminal repeats contain the entire heparin-binding site of HBHA. The molecular characterization of the interactions of HBHA with epithelial glycosaminoglycans should help to better understand mycobacterial adherence within the lungs and may ultimately lead to new approaches for therapy or immunoprophylaxis.     

Identification of the heparan sulfate binding sites in the cellular prion protein

Data from cell culture and animal models of prion disease support the separate involvement of both heparan sulfate proteoglycans and copper (II) ions in prion (PrP) metabolism. Though direct interactions between prion protein and heparin have been recorded, little is known of the structural features implicit in this interaction or of the involvement of copper (II) ions. Using biosensor and enzyme-linked immunosorbent assay methodology we report direct heparin and heparan sulfate-binding activity in recombinant cellular prion protein (PrP(c)). We also demonstrate that the interaction of recombinant PrP(c) with heparin is weakened in the presence of Cu(II) ions and is particularly sensitive to competition with dextran sulfate. Competitive inhibition experiments with chemically modified heparins also indicate that 2-O-sulfate groups (but not 6-O-sulfate groups) are essential for heparin recognition. We have also identified three regions of the prion protein capable of independent binding to heparin and heparan sulfate: residues 23-52, 53-93, and 110-128. Interestingly, the interaction of an octapeptide-spanning peptide motif amino acids 53-93 with heparin is enhanced by Cu(II) ions. Significantly, a peptide of this sequence is able to inhibit the binding of full-length prion molecule to heparin, suggesting a direct role in heparin recognition within the intact protein. The collective data suggest a complex interaction between prion protein and heparin/heparan sulfate and has implications for the cellular and pathological functions of prion proteins.     

Characterization of the adenine binding sites of two Dolichos biflorus lectins.

The seed lectin and a stem and leaf lectin (DB58) from Dolichos biflorus have high-affinity hydrophobic sites that bind to adenine. The present study employs a centrifugal filtration assay to characterize these sites. The seed lectin contains two identical sites with Ka's of 7.31 x 10(5) L/mol whereas DB58 has a single site with a Ka of 1.07 x 10(6) L/mol. The relative affinities of these sites for a host of adenine analogs and derivatives were determined by competitive displacement assays. The most effective competitors for adenine were the cytokinins, a class of plant hormone, for which the lectins had apparent Ka's of 1.96 x 10(5)-4.90 x 10(4) L/mol. Direct binding of the cytokinin 6-(benzylamino)purine (BAP) to both lectins showed positive cooperativity for only the seed lectin, indicating the interaction of this ligand with more than one class of hydrophobic binding site. Fluorescence enhancement assays demonstrate cooperativity between hydrophobic sites of the seed lectin and also suggest that BAP binds to more than one class of site.     

Characterization of the structure of antithrombin-binding heparan sulfate generated by heparan sulfate 3-O-sulfotransferase 5

The 3-O-sulfation of glucosamine is a key modification step during the biosynthesis of anticoagulant heparan sulfate (HS). Both heparan sulfate 3-O-sulfotransferase -1 (3-OST-1) and 3-O-sulfotransferase-5 (3-OST-5) transfer sulfate to the 3-OH group of glucosamine to generate antithrombin-binding heparan sulfate (HS(act)). Here, we reported the isolation and characterization of the antithrombin-binding HS oligosaccharides generated by 3-OST-5 (3-OST-5 oligo(act)). (3)H-labeled HS of Chinese hamster ovary cells was exhaustively modified by 3-OST-1 to remove the 3-OST-1 modification sites followed by antithrombin-affinity fractionation. The non-antithrombin-binding fraction of 3-OST-1 pretreated HS was further modified by 3-OST-5 to generate additional antithrombin-binding HS, which was designated as 3-OST-5 HS(act). Structural analysis of 3-OST-5 HS(act) revealed that the antithrombin-binding site of 3-OST-5 HS(act) is located within a domain clustered with N-sulfated glucosamine units. We also isolated 3-OST-5 antithrombin-binding oligosaccharides (3-OST-5 oligo(act)) from high pH nitrous acid degraded 3-OST-5 HS(act). A disaccharide analysis revealed that 3-OST-5 oligo(act) were composed of multiple 3-O-sulfated glucosamine units. Our results provide additional insights on the relationship between the anticoagulant activity and structure of HS.     

Characterization of two types of binding sites of substrate-like inhibitors in the heavy meromyosin molecule

The effects of several phosphorylating and alkylating analogs of the substrate on the ATPase activity of myosin and heavy meromyosin were compared. The data obtained confirmed the previously made assumption on the existence of two types of substrate-like inhibitor binding sites in the enzyme molecule. In one of the sites, presumably in the active one, there occurs a reversible competitive inhibition characterized by a high affinity for the inhibitors, which are mixed anhydrides of various mononucleotides and mesitylcarboxylic acid or its derivatives. An enhancement of hydrophobicity of these compounds causes an increase in their affinity for this site. At much higher concentrations of the inhibitors an irreversible inhibition takes place, the rate of inhibition being decreased with an increase in the phosphorylating capacity of the compound. This site possesses a far lower affinity for the inhibitors and reveals a certain specificity with respect to the analog mononucleotide moiety structure, i.e. a replacement of the 6-NH2-group by the 6-OH-group or an increase in the number of the phosphate residues result in a decrease of the efficiency of inhibition. No correlation between the analog capacity to cause irreversible inhibition and to act as an effective competitive inhibitor of reversible type has been shown to exist, thus allowing to use inhibitors of preferable action in one of the two types of the binding sites. No irreversible inhibition site was revealed when the ATPase activity of myosin subfragment I with and without the DTNB chains was investigated. Actin protects myosin against the inhibiting action of the analogs tested.     

Agrin is a chimeric proteoglycan with the attachment sites for heparan sulfate/chondroitin sulfate located in two multiple serine-glycine clusters

Agrin is a large extracellular matrix protein that plays a key role in the formation and maintenance of the vertebrate neuromuscular junction. The amino acid sequence of agrin encodes a protein with a molecular size of 220 kDa, whereas SDS-PAGE shows a diffuse band around 400 kDa. Further studies showed that agrin is highly glycosylated and belongs to the family of heparan sulfate proteoglycans. By expressing different protein fragments, we localized the glycosaminoglycan (GAG) attachment sites to two locations within the agrin molecule. One site that is located between the seventh and eight follistatin-like domain includes 3 closely spaced serine-glycine (SG) consensus sequences and carries exclusively heparan sulfate side chains. The second site is located further downstream in the centrally located serine-threonine-rich domain and contains a cluster of 4 closely packed SG consensus sequences. This site predominantly carries chondroitin sulfate side chains. Investigating the contribution of individual serines in GAG priming by site-directed mutagenesis showed that each serine of the two SG clusters has the potential to carry GAGs. In accordance with the mixed GAG glycosylation of agrin peptide fragments, it was found that recombinant and in vivo-derived full-length agrin are not exclusively heparan sulfate proteoglycans but also carry chondroitin sulfate side chains.     

Decorin-binding sites in the adhesin DbpA from Borrelia burgdorferi: a synthetic peptide approach

Lyme disease is caused by the spirochete Borrelia burgdorferi following transmission from infected Ixodes ticks to human hosts. Following colonization of the skin, spirochetes can disseminate throughout the body, resulting in complications that can include ocular, cardiac, neural, and skeletal disease. We have previously shown that B. burgdorferi expresses two closely related decorin-binding adhesins (DbpA and DbpB) of the MSCRAMM (microbial surface component recognizing adhesive matrix molecule) type that can mediate bacterial attachment to extracellular matrices in the host. Furthermore, three Lys residues in DbpA appear to be critical for the binding of DbpA to decorin. We have now characterized the interaction of DbpA and decorin further by using a synthetic peptide approach. We synthesized a panel of peptides that spanned the DbpA sequence and examined their ability to inhibit the binding of intact DbpA to decorin. From these studies, we identified a decorin-binding peptide that lost this activity if the sequence was either scrambled or if a critical Lys residue was chemically modified. A minimal decorin-binding peptide was identified by examining a set of truncated peptides. One peptide is proposed to contain the primary decorin-binding site in DbpA. By comparing the amino acid sequences of 29 different DbpA homologs from different B. burgdorferi sensu lato isolates, we discovered that the identified decorin-binding sequence was quite variable. Therefore, we synthesized a new panel of peptides containing the putative decorin-binding sequence of the different DbpA homologs. All of these peptides were active in our decorin-binding assay, and consensus decorin binding motifs are discussed.     

Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.     

Characterization of nucleotide-binding sites on the chloroplast coupling factor 1 using two photolabile analogs

Nucleotide-binding sites on the chloroplast coupling factor 1 (CF1) have been probed using two photoreactive ADP analogs: 2-azido-ADP (2-N3-ADP) and 2',3'-O-(4-benzoyl)benzoyl-ADP (Bz-ADP). Photolabeling of the isolated CF1 with 2-N3-ADP results in incorporation of the analog exclusively into the beta-subunit of the enzyme. The location of the nucleotide-binding site(s) within the beta-subunit of the CF1 was investigated using peptide mapping. Within the discrimination limits of this technique, it is concluded that the azido- and benzoyl-modified analogs both bind to the same conformation of the nucleotide-binding site(s) of soluble CF1. Bz-ADP, however, labels the binding site(s) on membrane-bound CF1 in a slightly different manner.     

Characterization of the two eIF4A-binding sites on human eIF4G-1

Eukaryotic translation initiation factor 4G-1 (eIF4G) plays a critical role in the recruitment of mRNA to the 43 S preinitiation complex. eIF4G has two binding sites for the RNA helicase eIF4A, one in the central domain and one in the COOH-terminal domain. Recombinant eIF4G fragments that contained each of these sites separately bound eIF4A with a 1:1 stoichiometry, but fragments containing both sites bound eIF4A with a 1:2 stoichiometry. eIF3 did not interfere with eIF4A binding to the central site. Interestingly, at the same concentration of free eIF4A, more eIF4A was bound to an eIF4G fragment containing both eIF4A sites than the sum of binding to fragments containing the single sites, indicating cooperative binding. Binding of eIF4A to an immobilized fragment of eIF4G containing the COOH-terminal site was competed by a soluble eIF4G fragment containing the central site, indicating that a single eIF4A molecule cannot bind simultaneously to both sites. The association rate constant, dissociation rate constant, and dissociation equilibrium constant for each site were determined by surface plasmon resonance and found to be, respectively, 1.2 x 10(5) m(-1) s(-1), 2.1 x 10(-3) s(-1), and 17 nm for the central site and 5.1 x 10(3) m(-1) s(-1), 1.7 x 10(-3) s(-1), and 330 nm for the COOH-terminal site.     

Characterization of heparan sulfate from the unossified antler of Cervus elaphus

The antler is the most rapidly growing tissue in the animal kingdom. According to previous reports, antler glycosaminoglycans (GAGs) consist of all kinds GAGs except for heparan sulfate (HS). Chondroitin sulfate is the major antler GAG component comprising 88% of the total uronic acid content. In the current study, we have isolated HS from antler for the first time and characterized it based on both NMR spectroscopy and disaccharide composition analysis. Antler GAGs were isolated by protease treatment and followed by cetylpyridinium chloride precipitation. The sensitivity of antler GAGs to heparin lyase III showed that this sample contained heparan sulfate. After incubation of antler GAGs with chondroitin lyase ABC, the HS-containing fraction was recovered by ethanol precipitation. The composition of HS disaccharides in this fraction was determined by its complete depolymerization with a mixture of heparin lyase I, II, and III and analysis of the resulting disaccharides by the reversed-phase (RP) ion pairing-HPLC, monitored by the fluorescence detection using 2-cyanoacetamide as a post-column labeling reagent. Eight unsaturated disaccharides (DeltaUA-GlcNAc, DeltaUA-GlcNS, DeltaUA-GlcNAc6S, DeltaUA2S-GlcNAc, DeltaUA-GlcNS6S, DeltaUA2S-GlcNS, DeltaUA2S-GlcNAc6S, DeltaUA2S-GlcNS6S) were produced from antler HS by digestion with the mixture of heparin lyases. The total content of 2-O-sulfo disaccharide units in antler HS was higher than that of heparan sulfate from most other animal sources.     

Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A.

Bacterial superantigens (SAgs) are potent activators of T lymphocytes and play a pathophysiological role in Gram-positive septic shock and food poisoning. To characterize potential MHC class II binding sites of the bacterial SAg staphylococcal enterotoxin (SE) A, we performed alanine substitution mutagenesis throughout the C-terminus and at selected sites in the N-terminal domain. Four amino acids in the C-terminus were shown to be involved in MHC class II binding. Three of these amino acids, H225, D227 and H187, had a major influence on MHC class II binding and appeared to be involved in coordination of a Zn2+ ion. Alanine substitution of H225 and D227 resulted in a 1000-fold reduction in MHC class II affinity. Mutation at F47, which is equivalent to the F44 previously shown to be central in the MHC class II binding site of the SAg, SEB, resulted in a 10-fold reduction in MHC class II affinity. The combination of these mutations in the N- and C-terminal sites resulted in a profound loss of activity. The perturbation of MHC class II binding in the various mutants was accompanied by a corresponding loss of ability to induce MHC class II-dependent T cell proliferation and cytotoxicity. All of the SEA mutants were expressed as Fab-SEA fusion proteins and found to retain an intact T cell receptor (TCR) epitope, as determined in a mAb targeted MHC class II-independent T cell cytotoxicity assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a galactose specific adhesin of enteroaggregative Escherichia coli

A fimbrial adhesin was identified from an enteroaggregative Escherichia coli strain. The adhesin was purified to 740-fold by sequential chromatography on an affinity matrix and gel filtration column in the FPLC system. The homogeneity of the purified protein was established by analytical isoelectrofocussing (pI 7.25). The native adhesin appeared as a high-molecular-weight aggregative protein as revealed by gel filtration chromatography on Superose 12HR10/30 column. However, in sodium dodecyl sulfate-polyacrylamide gel electrophoresis the molecular weight of the adhesin was found to be 18 kDa and this was further confirmed by gel filtration chromatography on Superose 6HR 10/30 column presence of 6 M guanidine hydrochloride. The N-terminal 15-amino-acid sequence of the adhesin did not show homology with any of the previously reported fimbrial adhesins. The purified adhesin showed adhesion to human erythrocytes in the presence of Ca(2+) (5 mM). The optimum temperature and pH for the hemadhesion activity was found to be 25 degrees C and 6.5, respectively. The inhibition study clearly suggested that the binding site of the adhesin could recognize galactose as the specific sugar. The fluorescence of 4-methylumbelliferyl-alpha-D-galactopyranoside was quenched on binding to the adhesin and maximum reversal of fluorescence quenching was observed by competitive substitution titration with raffinose. The adhesin was found to contain one binding site per monomer for its specific sugar residue. The association constant and the free energy of binding were obtained as 3.98 x 10(5) M(-1) and -31.97 kJ/mol, respectively. The adherence of the bacteria to HEp-2 monolayer was inhibited in presence of galactose and this was further supported by a significant reduction in the bacterial adherence to the HEp-2 cells, pretreated with beta-D-galactosidase.     

Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels

L-type Ca channels from porcine cardiac sarcolemma were incorporated into planar lipid bilayers. We characterized interactions of permeant and blocking ions with the channel's pore by (a) studying the current-voltage relationships for Ca2+ and Na+ when equal concentrations of the ions were present in both internal and external solutions, (b) testing the dose-dependent block of Ba2+ currents through the channels by internally applied cadmium, and (c) examining the dose and voltage dependence of the block of Na+ currents through the channels by internally and externally applied Ca2+. We found that the I-V relationship for Na+ appears symmetrical through the origin when equal concentrations of Na+ are present on both sides of the channel (gamma = 90 pS in 200 mM NaCl). The conductance for outward Ca2+ currents with 100 mM Ca2+ on both sides of the channel is approximately 8 pS, a value identical to that observed for inward currents when 100 mM Ca2+ was present outside only. This provides evidence that ions pass through the channel equally well regardless of the direction of net flux. In addition, we find that internal Cd2+ is as effective as external Cd2+ in blocking Ba2+ currents through the channels, again suggesting identical interactions of ions with each end of the pore. Finally, we find that micromolar Ca2+, either in the internal or in the external solution, blocks Na+ currents through the channels. The affinity for internally applied Ca2+ appears the same as that for externally applied Ca2+. The voltage dependence of the Ca(2+)-block suggests that the sites to which Ca2+ binds are located approximately 15% and approximately 85% of the electric field into the pore. Taken together, these data provide direct experimental evidence for the existence of at least two ion binding sites with high affinity for Ca2+, and support the idea that the sites are symmetrically located within the electric field across L-type Ca channels.     

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.     

Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa

A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa. By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P. aeruginosa strains. Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur. Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P. aeruginosa strains. Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F. Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells. Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F. Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected. This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F.     

Characterization at nucleotide resolution of the homogeneously staining region sites of insertion in two cancer cell lines

The mechanisms of formation of intrachromosomal amplifications in tumours are still poorly understood. By using quantitative polymerase chain reaction, DNA sequencing, chromosome walking, in situ hybridization on metaphase chromosomes and whole-genome analysis, we studied two cancer cell lines containing an MYC oncogene amplification with acquired copies ectopically inserted in rearranged chromosomes 17. These intrachromosomal amplifications result from the integration of extrachromosomal DNA molecules. Replication stress could explain the formation of the double-strand breaks involved in their insertion and in the rearrangements of the targeted chromosomes. The sequences of the junctions indicate that homologous recombination was not involved in their formation and support a non-homologous end-joining process. The replication stress-inducible common fragile sites present in the amplicons may have driven the intrachromosomal amplifications. Mechanisms associating break-fusion-bridge cycles and/or chromosome fragmentation may have led to the formation of the uncovered complex structures. To our knowledge, this is the first characterization of an intrachromosomal amplification site at nucleotide resolution.     

Characterization of the binding sites of two proteins involved in the bacteriophage P2 site-specific recombination system.

Integration of the bacteriophage P2 genome into the Escherichia coli host chromosome occurs by site-specific recombination between the phage attP and E. coli attB sites. The phage-encoded 38-kDa protein, integrase, is known to be necessary for both phage integration as well as excision. In order to begin the molecular characterization of this recombination event, we have cloned the int gene and overproduced and partially purified the Int protein and an N-terminal truncated form of Int. Both the wild-type Int protein and the integration host factor (IHF) of E. coli were required to mediate integrative recombination in vitro between a supercoiled attP plasmid and a linear attB substrate. Footprint experiments revealed one Int-protected region on both of the attP arms, each containing direct repeats of the consensus sequence TGTGGACA. The common core sequences at attP and attB were also protected by Int from nuclease digestion, and these contained a different consensus sequence, AA T/A T/A C/A T/G CCC, arranged as inverted repeats at each core. A single IHF-protected site was located on the P (left) arm, placed between the core- and P arm-binding site for Int. Cooperative binding by Int and IHF to the attP region was demonstrated with band-shift assays and footprinting studies. Our data support the existence of two DNA-binding domains on Int, having unrelated sequence specificities. We propose that P2 Int, IHF, attP, and attB assemble in a higher-order complex, or intasome, prior to site-specific integrative recombination analogous to that formed during lambda integration.