Binding of the G domains of laminin alpha1 and alpha2 chains and perlecan to heparin, sulfatides, alpha-dystroglycan and several extracellular matrix proteins.
The C-terminal G domain of the mouse laminin alpha2 chain consists of five lamin-type G domain (LG) modules (alpha2LG1 to alpha2LG5) and was obtained as several recombinant fragments, corresponding to either individual modules or the tandem arrays alpha2LG1-3 and alpha2LG4-5. These fragments were compared with similar modules from the laminin alpha1 chain and from the C-terminal region of perlecan (PGV) in several binding studies. Major heparin-binding sites were located on the two tandem fragments and the individual alpha2LG1, alpha2LG3 and alpha2LG5 modules. The binding epitope on alpha2LG5 could be localized to a cluster of lysines by site-directed mutagenesis. In the alpha1 chain, however, strong heparin binding was found on alpha1LG4 and not on alpha1LG5. Binding to sulfatides correlated to heparin binding in most but not all cases. Fragments alpha2LG1-3 and alpha2LG4-5 also bound to fibulin-1, fibulin-2 and nidogen-2 with Kd = 13-150 nM. Both tandem fragments, but not the individual modules, bound strongly to alpha-dystroglycan and this interaction was abolished by EDTA but not by high concentrations of heparin and NaCl. The binding of perlecan fragment PGV to alpha-dystroglycan was even stronger and was also not sensitive to heparin. This demonstrated similar binding repertoires for the LG modules of three basement membrane proteins involved in cell-matrix interactions and supramolecular assembly. (+info)
The L1 major capsid protein of human papillomavirus type 11 recombinant virus-like particles interacts with heparin and cell-surface glycosaminoglycans on human keratinocytes.
The L1 major capsid protein of human papillomavirus (HPV) type 11, a 55-kDa polypeptide, forms particulate structures resembling native virus with an average particle diameter of 50-60 nm when expressed in the yeast Saccharomyces cerevisiae. We show in this report that these virus-like particles (VLPs) interact with heparin and with cell-surface glycosaminoglycans (GAGs) resembling heparin on keratinocytes and Chinese hamster ovary cells. The binding of VLPs to heparin is shown to exhibit an affinity comparable to that of other identified heparin-binding proteins. Immobilized heparin chromatography and surface plasmon resonance were used to show that this interaction can be specifically inhibited by free heparin and dextran sulfate and that the effectiveness of the inhibitor is related to its molecular weight and charge density. Sequence comparison of nine human L1 types revealed a conserved region of the carboxyl terminus containing clustered basic amino acids that bear resemblance to proposed heparin-binding motifs in unrelated proteins. Specific enzymatic cleavage of this region eliminated binding to both immobilized heparin and human keratinocyte (HaCaT) cells. Removal of heparan sulfate GAGs on keratinocytes by treatment with heparinase or heparitinase resulted in an 80-90% reduction of VLP binding, whereas treatment of cells with laminin, a substrate for alpha6 integrin receptors, provided minimal inhibition. Cells treated with chlorate or substituted beta-D-xylosides, resulting in undersulfation or secretion of GAG chains, also showed a reduced affinity for VLPs. Similarly, binding of VLPs to a Chinese hamster ovary cell mutant deficient in GAG synthesis was shown to be only 10% that observed for wild type cells. This report establishes for the first time that the carboxyl-terminal portion of HPV L1 interacts with heparin, and that this region appears to be crucial for interaction with the cell surface. (+info)
Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II.
Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors. (+info)
Orientation of heparin-binding sites in native vitronectin. Analyses of ligand binding to the primary glycosaminoglycan-binding site indicate that putative secondary sites are not functional.
A primary heparin-binding site in vitronectin has been localized to a cluster of cationic residues near the C terminus of the protein. More recently, secondary binding sites have been proposed. In order to investigate whether the binding site originally identified on vitronectin functions as an exclusive and independent heparin-binding domain, solution binding methods have been used in combination with NMR and recombinant approaches to evaluate ligand binding to the primary site. Evaluation of the ionic strength dependence of heparin binding to vitronectin according to classical linkage theory indicates that a single ionic bond is prominent. It had been previously shown that chemical modification of vitronectin using an arginine-reactive probe results in a significant reduction in heparin binding (Gibson, A., Baburaj, K., Day, D. E., Verhamme, I. , Shore, J. D., and Peterson, C. B. (1997) J. Biol. Chem. 272, 5112-5121). The label has now been localized to arginine residues within the cyanogen bromide fragment-(341-380) that contains the primary heparin-binding site on vitronectin. One- and two-dimensional NMR on model peptides based on this primary heparin-binding site indicate that an arginine residue participates in the ionic interaction and that other nonionic interactions may be involved in forming a complex with heparin. A recombinant polypeptide corresponding to the C-terminal 129 amino acids of vitronectin exhibits heparin-binding affinity that is comparable to that of full-length vitronectin and is equally effective at neutralizing heparin anticoagulant activity. Results from this broad experimental approach argue that the behavior of the primary site is sufficient to account for the heparin binding activity of vitronectin and support an exposed orientation for the site in the structure of the native protein. (+info)
Depolarization-evoked Ca2+ release in a non-excitable cell, the rat megakaryocyte.
1. The effect of membrane potential on [Ca2+]i in rat megakaryocytes was studied using simultaneous whole-cell patch clamp and fura-2 fluorescence recordings. 2. Depolarization from -75 to 0 mV had no effect on [Ca2+]i in unstimulated cells, but evoked one or more spikes of Ca2+ increase (peak increase: 714 +/- 95 nM) during activation of metabotropic purinoceptors by 1 microM ADP. 3. The depolarization-evoked Ca2+ increase was present in Ca2+-free medium and also following removal of Na+. Thus depolarization mobilizes Ca2+ from an intracellular store without a requirement for altered Na+-Ca2+ exchange activity. 4. Intracellular dialysis with heparin blocked the depolarization-evoked Ca2+ increase, indicating a role for functional IP3 receptors. 5. Under current clamp, ADP caused the membrane potential to fluctuate between -43 +/- 1 and -76 +/- 1 mV. Under voltage clamp, depolarization from -75 to -45 mV evoked a transient [Ca2+]i increase (398 +/- 91 nM) during exposure to ADP. 6. We conclude that during stimulation of metabotropic purinoceptors, membrane depolarization over the physiological range can stimulate Ca2+ release from intracellular stores in the rat megakaryocyte, a non-excitable cell type. This may represent an important mechanism by which electrogenic influences can control patterns of [Ca2+]i increase. (+info)
Fructose 1,6-bisphosphate aldolase is a heparin-binding protein.
Proteins with affinity to heparin under physiological conditions were isolated from bovine cerebral cortex. First, the extract of cerebral cortex was applied to a chondroitin polysulfate column under physiological conditions. Then, the pass-through fraction was applied to a heparin column. Among the bands on SDS polyacrylamide gel electrophoresis of the fraction bound to the heparin column, the major one was identified as fructose 1,6-bisphosphate aldolase (FPA), a cytosolic enzyme involved in the glycolytic pathway. The results indicated that FPA is a heparin-binding protein which exhibits no affinity to chondroitin polysulfate. The results of affinity chromatographies revealed that FPA binds to intact heparin and modified heparins desulfated at C2 OH of the iduronic acid residue or at C6 OH or C2 NH2 of the glucosamine residue. When 6-O-desulfated heparin was employed as the affinity ligand, a single peak having FPA activity was isolated from the extract of bovine cerebral cortex. By further Mono Q chromatography and Superdex gel-filtration, five isoenzymes were purified with more than 50% recovery. These isoenzymes were identified as FPA A4, A3C1, A2C2, A1C3, and C4 by native electrophoresis with and without 4 M urea and subsequent amino acid sequence analysis. The use of 6-O-desulfated heparin affinity chromatography thus facilitated the purification of FPA. (+info)
Characterisation of the conformational and quaternary structure-dependent heparin-binding region of bovine seminal plasma protein PDC-109.
PDC-109, the major heparin-binding protein of bull seminal plasma, binds to sperm choline lipids at ejaculation and modulates capacitation mediated by heparin. Affinity chromatography on heparin-Sepharose showed that polydisperse, but not monomeric, PDC-109 displayed heparin-binding capability. We sought to characterise the surface topology of the quaternary structure-dependent heparin-binding region of PDC-109 by comparing the arginine- and lysine-selective chemical modification patterns of the free and the heparin-bound protein. A combination of reversed-phase peptide mapping of endoproteinase Lys-C-digested PDC-109 derivatives and mass spectrometry was employed to identify modified and heparin-protected residues. PDC-109 contains two tandemly arranged fibronectin type II domains (a, Cys24-Cys61; b, Cys69-Cys109). The results show that six basic residues (Lys34, Arg57, Lys59, Arg64, Lys68, and Arg104) were shielded from reaction with acetic anhydride and 1,2-cyclohexanedione in heparin-bound PDC-109 oligomers. In the 1H-NMR solution structures of single fibronectin type II domains, residues topologically equivalent to PDC-109 Arg57 (Arg104) and Lys59 lay around beta-strand D on the same face of the domain. In full-length PDC-109, Arg64 and Lys68 are both located in the intervening polypeptide between domains a and b. Our data suggest possible quaternary structure arrangements of PDC-109 molecules to form a heparin-binding oligomer. (+info)
Communication between spermatozoa and egg before contact by chemotaxis appears to be prevalent throughout the animal kingdom. In non-mammalian species, sperm chemotaxis to factors secreted from the egg is well documented. In mammals, sperm chemotaxis to follicular factors in vitro has been established in humans and mice. The attractants of female origin in non-mammalian species are heat-stable peptides or proteins of various sizes, or other small molecules, depending on the species. Species specificity of the attractants in non-mammalian species may vary from high species specificity, through specificity to families with no specificity within a family, to absence of specificity. The mammalian sperm attractants have not been identified but they appear to be heat-stable peptides. The claim that progesterone is the attractant for human spermatozoa has failed to be substantiated, neither have claims for other mammalian sperm attractants been verified. The molecular mechanism of sperm chemotaxis is not known. Models involving modulation of the intracellular Ca2+ concentration have been proposed for both mammalian and non-mammalian sperm chemotaxis. The physiological role of sperm chemotaxis in non-mammalian species appears to differ from that in mammals. In non-mammalian species, sperm chemotaxis strives to bring as many spermatozoa as possible to the egg. However, in mammals, the role appears to be recruitment of a selective population of capacitated ('ripe') spermatozoa to fertilize the egg. (+info)