Specificities of heparin-binding sites from the amino-terminus and type 1 repeats of thrombospondin-1.

Interactions of heparin with intact human thrombospondin-1 (TSP1) and with two heparin-binding fragments of TSP1 were characterized using chemically modified heparins, a vascular heparan sulfate proteoglycan, and a series of heparin oligosaccharides prepared by partial deaminative cleavage. The avidity of TSP1 binding increased with oligosaccharide size, with plateaus at 4 to 6 and at 8 to 10 monosaccharide units. The dependence on oligosaccharide size for binding to the recombinant amino-terminal heparin-binding domain of TSP1 was the same as that of the intact TSP1 molecule but differed from that of a synthetic heparin-binding peptide from the type 1 repeats, suggesting that the interaction between intact TSP1 and heparin is primarily mediated by the amino-terminal domain. Based on activities of chemically modified heparins, binding to TSP1 depended primarily on 2-N- and 6-O-sulfation of glucosamine and to a lesser degree on 2,3-O-sulfation and the carboxyl residues of the uronic acids. In contrast, all of these modifications were required for binding of heparin to the type 1 repeat peptides. Affinity purification of heparin octasaccharides on immobilized TSP1 type 1 repeat peptides revealed a preference for oligosaccharides containing the disaccharide sequence IdoA(2-OSO(3))alpha1-4-GlcNS(6-OSO(3)). Binding of these oligosaccharides to the peptide required the Trp residues. These data demonstrate that the heparin-binding specificities of intact TSP1 and peptides from the type 1 repeats overlap with that of basic fibroblast growth factor (FGF2) and are consistent with the ability of these TSP1-derived molecules to inhibit FGF2-stimulated angiogenesis.

Selective inhibition of platelet macroaggregate formation by a recombinant heparin-binding domain of human thrombospondin.

Thrombospondin (TSP) is a platelet alpha-granule adhesive protein that plays a critical role in the stabilization of thrombus by promoting the formation of platelet macroaggregates. We have recently shown that a monoclonal antibody (mAb) to the NH2-terminal heparin-binding domain of TSP, MAII, inhibits platelet aggregation induced by thrombin in a dose-dependent manner. In this study, we have expressed in Escherichia coli two recombinant proteins comprising residues 1 to 174 (TSP18) and 1 to 242 (TSP28) of TSP. After purification, both proteins reacted equally well with mAb MAII, whereas the reactivity of TSP18 for heparin was lower than that of TSP28 or native TSP. At micromolar concentrations, TSP18 and TSP28 inhibited the second wave of platelet aggregation and the concomitant release of [14C]5-hydroxytryptamine induced by ADP in citrated platelet-rich plasma as well as aggregation and secretion induced by a low concentration of thrombin in washed platelet suspensions. The proteins did not inhibit surface expression of endogenous TSP on activated platelets, as measured by the binding of radiolabeled mAb 5G11, indicating that they did not interfere with the primary binding of TSP to the plasma membrane. In contrast, in a solid-phase binding assay, the proteins inhibited in a dose-dependent manner (IC50, 0.1 and 0.06 mumol/L for TSP18 and TSP28, respectively) the binding of radiolabeled TSP to surface-adsorbed fibrinogen. Furthermore, specific and saturable binding of the proteins to immobilized fibrinogen was demonstrated by enzyme-linked immunosorbent assay. The results suggest that interaction between the heparin-binding domain of TSP and membrane-bound fibrinogen may be critical in the platelet aggregation/secretion process.

Modulation of endothelial cell proliferation, adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin.

Thrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence-Trp-Ser-Pro-Trp- and a recombinant heparin binding fragment from the amino-terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin-binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin-dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin-binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time-dependent and reversible. Thus, the antiproliferative activities of the thrombospondin peptide and recombinant heparin-binding domain result at least in part from competition with heparin-dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp-Ser-Xaa-Trp sequences in the type I repeats and the amino-terminal domain play roles in the antiproliferative activity of thrombospondin.

Cloning and expression in Escherichia coli of an esterase-coding gene from the oil-degrading bacterium Acinetobacter calcoaceticus RAG-1.

A putative esterase gene (est) from Acinetobacter calcoaceticus RAG-1 has been cloned into Escherichia coli. Esterase-positive clones exhibited high levels of esterase activity even in intact cells. In addition, expression of the est gene conferred on E. coli the ability to grow on simple triglycerides such as triacetin (TAC). The original esterase-positive plasmid pRA17 carried a 2.2-kb insert from a partial MboI digest of RAG-1 DNA, which gave a single band with RAG-1 DNA following Southern hybridization. By subcloning and sequencing the est gene was found to contain a sequence of 870 bp which could be translated to yield a protein of Mr 32,700. In support of the sequencing results was the finding that when pRA17 was expressed in minicells, a unique peptide of Mr 32,500 was identified. This peptide was not found in minicells transformed with esterase-negative plasmids, such as pRA176, which contained a Tn5 insertion in the est gene. The fact that the production of active esterase depended on the orientation of the est gene within the vector suggested that transcription proceeded from the tet promoter in pBR322.

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus.

A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5' termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.

Regulatory mutants of Streptomyces clavuligerus affected in free diaminopimelic acid content and antibiotic biosynthesis.

The composition of intracellular free amino acid pools was determined in Streptomyces clavuligerus mutants possessing an altered aspartokinase which is insensitive to concerted feedback inhibition by threonine and lysine. These mutants contained total free amino acid pool contents that were considerably higher than those found in the wild-type strain. Diaminopimelic acid accounted for 10 to 20% of the total free amino acid pools, depending on the individual mutant and its culture growth phase, whereas diaminopimelic acid contained in the wild-type strain accounted for only 0.5% of the total free amino acid pool.

beta-lactam antibiotic production by Streptomyces clavuligerus mutants impaired in regulation of aspartokinase.

This work describes isolation and characterization of Streptomyces clavuligerus mutants resistant to the lysine analogue S-(2-aminoethyl)-L-cysteine (AEC). The mutation to AEC resistance was shown to affect the feedback regulation of aspartokinase; 70% of the mutants isolated had aspartokinase activity insensitive to concerted feedback inhibition by lysine plus threonine. Among these mutants, 70% (about 50% of the total AEC-resistant strains isolated) showed significant overproduction of beta-lactam antibiotics.

Regulation of cephamycin C synthesis, aspartokinase, dihydrodipicolinic acid synthetase, and homoserine dehydrogenase by aspartic acid family amino acids in Streptomyces clavuligerus.

The effect of the cephalosporin precursors and amino acids of the aspartic acid family on antibiotic production by Streptomyces clavuligerus was investigated DL-meso-Diaminopimelate and L-lysine each stimulated specific antibiotic production by 75%. A fourfold increase in specific production was obtained by simultaneous addition of the two compounds. The stimulation could be further increased by adding valine to the two effectors. In the streptomycetes the alpha-aminoadipyl side chain of the cephalosporin antibiotics is derived from lysine. Streptomycetes, like other bacteria, are expected to produce lysine from aspartic acid; therefore, the feedback control mechanisms operating in the aspartic acid family pathway of S. clavuligerus, which may affect the flow of carbon to alpha-aminoadipic acid, were investigated. Threonine inhibited antibiotic production by 41% when added to minimal medium at a concentration of 10 mM. Simultaneous addition of 10 mM lysine completely reversed this inhibition. The aspartokinase of S. clavuligerus was found to be subject to concerted feedback inhibition by threonine and lysine. Threonine may act to limit the supply of lysine available for cephamycin C biosynthesis via this concerted mechanism. Single or simultaneous addition of any other amino acid of the aspartate family in the in vitro assay did not inhibit aspartokinase activity. Activity was stimulated by lysine. Aspartokinase biosynthesis was partially repressed by methionine or isoleucine at concentrations higher than 10 mM. Methionine, but not isoleucine, inhibited cephamycin C synthesis by 27% when added to minimal medium at a concentration of 10 mM. Dihydrodipicolinate synthetase, the first specific enzyme of the lysine branch, was not inhibited by lysine but was partially inhibited by high concentrations of 2,6-diaminopimelate and alpha-aminoadipate; it was slightly repressed by diaminopimelic acid. Homoserine dehydrogenase activity was inhibited by threonine and partially repressed by isoleucine. It appears that S. clavuligerus aspartokinase is a key step in the control of carbon flow toward alpha-aminoadipic acid.