A heme- and metal-binding hexapeptide from the sequence of rabbit plasma histidine-rich glycoprotein

Rabbit histidine-rich glycoprotein (HRG) binds low-spin heme and metals tightly at several sites that contain histidine. As part of an on-going effort to define and locate the binding sites for these and the other ligands of HRG, the sequence: NH2-Gly-His-Phe-Pro-Phe-His-Trp-... was found in a 16 kDa heme-binding peptide isolated from HRG. The spacing of the histidyl residues in this peptide, which contains the C-terminal 79 residues of HRG, together with molecular modeling suggested that this sequence might constitute one heme binding site of HRG by accommodating heme in a bis-histidyl linkage. Three peptides based on this sequence (I, HFPFHW; II, WHFPFH; and III, HFGFHW) were synthesized, and their ability to bind heme and metals examined. All three peptides bind heme as demonstrated by the changes produced in the absorbance of heme when mixed with the peptides. Substituting glycine for proline in the central position or moving the location of the tryptophan did not affect heme binding. The apparent Kd's of the mesoheme/peptide I, II and III complexes are 75 +/- 25 microM, indicative of heme binding approximately 100 times less avid than the mesoheme/HRG complex (Kd ca. 1 microM), but nearly 1000 times tighter than that of the mesoheme/histidine complex (Kd ca. 60 mM). The absorbance spectra of the mesoheme/peptide complexes, the loss of binding caused by modification of histidine residues, and the pH dependence of heme binding, all indicate that heme forms a low spin, bis-histidyl type of complex with these peptides, like that formed with HRG itself. Copper, but not cadmium or nickel, was an effective inhibitor of heme binding by the peptides. The sequence of HRG congruent with the sequence of peptide I is proposed to be one heme- and metal-binding site of rabbit HRG.     

Paramagnetic probes of the domain structure of histidine-rich glycoprotein

The interaction of Cu2+ and Fe3+-mesoporphyrin with histidine-rich glycoprotein (HRG) from rabbit serum was examined spectroscopically. The first equivalent of Cu2+ binds to HRG producing a type II electron paramagnetic resonance (EPR) spectrum with g[[ = 2.25, gm = 2.05, A[[ = 0.019 cm-1 (180 G), and superhyperfine along gm. These spectral parameters suggest moderately covalent coordination of Cu2+ to the protein by nitrogens. With increasing Cu2+ the superhyperfine disappears; however, the g and A values change only marginally. The increase in EPR signal amplitude throughout the addition of 1-15 equiv of Cu2+ is linear and thereafter maximizes, suggesting 18-22 equiv are bound. In contrast, changes in the circular dichroism spectrum at 280 nm appear sigmoidal and can be interpreted as the binding of Cu2+ to two structurally distinct regions of the protein. Evidence for two structurally distinct binding domains is found by comparing EPR spectra of Cu2+ complexes of HRG with spectra from complexes of two of its major proteolysis products (peptides). After binding 1 equiv of Cu2+, both the 30-kDa histidine-rich peptide and the native protein exhibit identical spectra including the pronounced superhyperfine. In contrast, the spectrum of the histidine-normal 45-kDa peptide with 1 equiv of Cu2+ bound lacks superhyperfine and parallels closely that of the native protein with 20 equiv bound. Finally, Fe3+-mesoporphyrin binds to HRG exhibiting both high-spin (g = 6.05) and low-spin (gz = 2.94, gy = 2.25, gx = 1.50) EPR resonances, and the latter imply bis(histidine) coordination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Further characterization of the interaction of histidine-rich glycoprotein with heparin: evidence for the binding of two molecules of histidine-rich glycoprotein by high molecular weight heparin and for the involvement of histidine residues in heparin binding

Rabbit histidine-rich glycoprotein (HRG, 94 kDa) binds heparin with high affinity (apparent Kd 60-110 nM). Eosin Y (1 equiv) bound to HRG was used as a reporter group to monitor associations of HRG with heparins of molecular mass 10, 17.5, and 30 kDa. The stoichiometries of the heparin-HRG complexes were determined by fluorescence and absorbance measurements as well as by analytical ultracentrifugation. Two types of complex form: complexes of 1 heparin:1 HRG and of 1 heparin:2 HRG. The 1:2 complex formation requires a minimum heparin chain length since 17.5-kDa but not 10-kDa heparin binds two HRG molecules. The formation of the 1:2 complexes of the larger heparin fractions is enhanced by divalent copper or zinc (1-10 equiv) bound to HRG. However, metal is not required for complex formation since all sizes of heparin examined interact tightly with HRG in the presence of ethylenediaminetetraacetic acid. Between 0.1 and 0.3 M ionic strength, both 1:1 and 1:2 complexes of heparin with HRG are progressively destabilized. No heparin-HRG complex is found at ionic strengths of 0.5 M. Between pH 8.5 and pH 6.5 both 1:2 and 1:1 complexes are found with 17.5-kDa heparin, but at pH 5.5 only 1:1 complexes are formed. The heparin-HRG interaction is progressively decreased by modification of the histidine residues of HRG, whereas modification of 22 of the 33 lysine residues of HRG has little effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

The histidine-rich glycoprotein of serum has a domain rich in histidine, proline, and glycine that binds heme and metals

Histidine-rich glycoprotein (HRG) from rabbit serum was digested with plasmin, reduced, and carboxymethylated, and the fragments produced were resolved by reverse-phase high-performance liquid chromatography. Several peptide fractions were obtained that contain unusually high contents of histidine, proline, and glycine. One His-Pro-Gly-rich peptide (apparent Mr 30 000) was obtained in sufficient yield and purity for further study. This peptide is 29 mol % histidine, 37% proline, and 16% glycine, indicating that most of these three amino acids are located in one region of HRG. The peptide contains 9% by weight carbohydrate and is devoid of tyrosine or tryptophan. The far-ultraviolet circular dichroism spectrum of the peptide has a minimum at 203 nm, indicating that the peptide contains polyproline II helical sections. The peptide represents a binding domain of HRG since it retains much of the ability of intact HRG to bind heme and metals including Zn2+, Ni2+, and Cu2+. As with the parent HRG molecule, interaction of the peptide with heme and metals is dependent on pH and intact histidine residues.     

Characterization of receptors for vasoactive intestinal peptide solubilized from the lung

The zwitterionic detergent CHAPS was used to solubilize functional receptors for vasoactive intestinal peptide (VIP) from guinea pig lung. The solubilized receptors were resolved by high performance gel filtration in 3 mM CHAPS into two active fractions with apparent Stokes radii of 5.9 +/- 0.1 and 2.3 +/- 0.1 nm. The binding of 125I-VIP to the two receptor fractions was time-dependent, reversible, and saturable. Trypsin destroyed the binding activity of the receptor fractions, indicating their proteinic nature. Unlabeled VIP competitively displaced the binding of 125I-VIP to the 5.9-nm fraction (IC50 = 240 pM) and the 2.3-nm fraction (IC50 = 1.2 microM). Scatchard analysis indicated a single class of binding sites in each receptor fraction, with Kd values 300 pM and 0.97 microM for the 5.9- and 2.3-nm Stokes radii fractions, respectively. When the high affinity, 5.9-nm Stokes radius fraction was rechromatographed in 9 nM CHAPS, 46% of the binding activity eluted in the low affinity, 2.3-nm Stokes radius fraction, indicating that the latter is a product of dissociation of the high affinity receptor complex. GTP inhibited the binding of 125I-VIP to the high affinity complex but not the low affinity species. Scatchard plots of VIP binding by the high affinity receptors treated with GTP suggested the presence of two distinct binding sites (Kd 4.4 and 153 nM), compared to a single binding site (Kd = 0.3 nM) obtained in untreated receptors. The nonhydrolyzable GTP analog, guanyl-5'-yl-imidodiphosphate, inhibited VIP binding by the high affinity receptor fraction with potency nearly equivalent to that of GTP. These observations suggest that GTP-binding regulatory proteins are functionally coupled to the VIP-binding subunit in the high affinity receptor complex. The peptide specificity characteristics of the two receptor fractions were different. Peptide histidine isoleucine and growth hormone releasing factor, peptides homologous to VIP, were 87.5- and 22.9-fold less potent than VIP in displacing 125I-VIP binding by the high affinity receptor complex, respectively. On the other hand, growth hormone-releasing factor was more potent (22.7-fold) and peptide histidine isoleucine was less potent (31.3-fold) than VIP in displacing the binding by the low affinity species.     

Resonance Raman investigation of the effects of copper binding to iron-mesoporphyrin.histidine-rich glycoprotein complexes.

Histidine-rich glycoprotein (HRG) binds both hemes and metal ions simultaneously with evidence for interaction between the two. This study uses resonance Raman and optical absorption spectroscopies to examine the heme environment of the 1:1 iron-mesoporphyrin.HRG complex in its oxidized, reduced and CO-bound forms in the absence and presence of copper. Significant perturbation of Fe(3+)-mesoporphyrin.HRG is induced by Cu2+ binding to the protein. Specifically, high frequency heme resonance Raman bands indicative of low-spin, six-coordinate iron before Cu2+ binding exhibit monotonic intensity shifts to bands representing high-spin, five-coordinate iron. The latter coordination is in contrast to that found in hemoglobin and myoglobin, and explains the Cu(2+)-induced decrease and broadening of the Fe(3+)-mesoporphyrin.HRG Soret band concomitant with the increase in the high-spin marker band at 620 nm. After dithionite reduction, the Fe(2+)-mesoporphyrin.HRG complex displays high frequency resonance Raman bands characteristic of low-spin heme and no iron-histidine stretch, which together suggest six-coordinate iron. Furthermore, the local heme environment of the complex is not altered by the binding of Cu1+. CO-bound Fe(2+)-mesoporphyrin.HRG exhibits bands in the high and low frequency regions similar to those of other CO-bound heme proteins except that the iron-CO stretch at 505 cm-1 is unusually broad with delta nu approximately 30 cm-1. The dynamics of CO photolysis and rebinding to Fe(2+)-mesoporphyrin.HRG are also distinctive. The net quantum yield for photolysis at 10 ns is low relative to most heme proteins, which may be attributed to very rapid geminate recombination. A similar low net quantum yield and broad iron-CO stretch have so far only been observed in a dimeric cytochrome c' from Chromatium vinosum. Furthermore, the photolytic transient of Fe(2+)-mesoporphyrin.HRG lacks bands corresponding to high-spin, five-coordinate iron as is found in hemoglobin and myoglobin under similar experimental conditions, suggesting iron hexacoordination before CO recombination. These data are consistent with a closely packed distal heme pocket that hinders ligand diffusion into the surrounding solvent.     

Plasmon waveguide resonance spectroscopic evidence for differential binding of oxidized and reduced Rhodobacter capsulatus cytochrome c2 to the cytochrome bc1 complex mediated by the conformation of the Rieske iron-sulfur protein

The dissociation constants for the binding of Rhodobacter capsulatus cytochrome c2 and its K93P mutant to the cytochrome bc1 complex embedded in a phospholipid bilayer were measured by plasmon waveguide resonance spectroscopy in the presence and absence of the inhibitor stigmatellin. The reduced form of cytochrome c2 strongly binds to reduced cytochrome bc1 (Kd = 0.02 microM) but binds much more weakly to the oxidized form (Kd = 3.1 microM). In contrast, oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a biphasic fashion with Kd values of 0.11 and 0.58 microM. Such a biphasic interaction is consistent with binding to two separate sites or conformations of oxidized cytochrome c2 and/or cytochrome bc1. However, in the presence of stigmatellin, we find that oxidized cytochrome c2 binds to oxidized cytochrome bc1 in a monophasic fashion with high affinity (Kd = 0.06 microM) and reduced cytochrome c2 binds less strongly (Kd = 0.11 microM) but approximately 30-fold more tightly than in the absence of stigmatellin. Structural studies with cytochrome bc1, with and without the inhibitor stigmatellin, have led to the proposal that the Rieske protein is mobile, moving between the cytochrome b and cytochrome c1 components during turnover. In one conformation, the Rieske protein binds near the heme of cytochrome c1, while the cytochrome c2 binding site is also near the cytochrome c1 heme but on the opposite side from the Rieske site, where cytochrome c2 cannot directly interact with Rieske. However, the inhibitor, stigmatellin, freezes the Rieske protein iron-sulfur cluster in a conformation proximal to cytochrome b and distal to cytochrome c1. We conclude from this that the dual conformation of the Rieske protein is primarily responsible for biphasic binding of oxidized cytochrome c2 to cytochrome c1. This optimizes turnover by maximizing binding of the substrate, oxidized cytochrome c2, when the iron-sulfur cluster is proximal to cytochrome b and minimizing binding of the product, reduced cytochrome c2, when it is proximal to cytochrome c1.     

Further localization of the gelatin-binding determinants within fibronectin. Active fragments devoid of type II homologous repeat modules

Digestion of a 42-kDa gelatin-binding fragment (GBF) of fibronectin with pepsin followed by affinity chromatography on gelatin-Sepharose produces three fractions, a drop-through non-binding fraction, a retarded fraction that is dominated by a 13-kDa fragment whose NH2 terminus is identical to that of 42-kDa GBF, and a binding fraction that contains a homogeneous fragment of apparent mass 21 kDa with an NH2 terminus corresponding to Arg484. This 21-kDa GBF binds repeatedly to gelatin-Sepharose, eluting near 2.6 M in a urea gradient. It also binds in the fluid phase to a fluorescent-labeled collagen peptide with Kd = 10 microM and inhibits the binding of 42-kDa GBF to the same peptide with KI = 7.3 microM. Thus, major gelatin-binding determinants of fibronectin are located within a 21-kDa region that contains two type I homologous "finger" modules and is devoid of the type II "kringle-like" modules that were previously thought to be essential for this activity.     

Histidine placement in de novo-designed heme proteins.

The effects of histidine residue placement in a de novo-designed four-alpha-helix bundle are investigated by placement of histidine residues at coiled coil heptad a positions in two distinct heptads and at each position within a single heptad repeat of our prototype heme protein maquette, [H10H24]2 [[Ac-CGGGELWKL x HEELLKK x FEELLKL x HEERLKK x L-CONH2]2]2 composed of a generic (alpha-SS-alpha)2 peptide architecture. The heme to peptide stoichiometry of variants of [H10H24]2 with either or both histidines on each helix replaced with noncoordinating alanine residues ([H10A24]2, [A10H24]2, and [A10A24]2) demonstrates the obligate requirement of histidine for biologically significant heme affinity. Variants of [A10A24]2, [[Ac-CGGGELWKL x AEELLKK x FEELLKL x AEERLKK x L-CONH2]2]2, containing a single histidine per helix in positions 9 to 15 were evaluated to verify the design based on molecular modeling. The bis-histidine site formed between heptad positions a at 10 and 10' bound ferric hemes with the highest affinity, Kd1 and Kd2 values of 1.5 and 800 nM, respectively. Placement of histidine at position 11 (heptad position b) resulted in a protein that bound a single heme with moderate affinity, Kd1 of 9.5 microM, whereas the other peptides had no measurable apparent affinity for ferric heme with Kd1 values >200 microM. The bis-histidine ligation of heme to [H10A24]2 and [H11A24]2 was confirmed by electron paramagnetic resonance spectroscopy. The protein design rules derived from this study, together with the narrow tolerances revealed, are applicable for improving future heme protein designs, for analyzing the results of randomized heme protein combinatorial libraries, as well as for implementation in automated protein design.     

Histidine-rich glycoprotein binds to cell-surface heparan sulfate via its N-terminal domain following Zn2+ chelation

Histidine-rich glycoprotein (HRG) is an alpha2-glycoprotein found in mammalian plasma at high concentrations (approximately 150 microg/ml) and is distinguished by its high content of histidine and proline. Structurally, HRG is a modular protein consisting of an N-terminal cystatin-like domain (N1N2), a central histidine-rich region (HRR) flanked by proline-rich sequences, and a C-terminal domain. HRG binds to cell surfaces and numerous ligands such as plasminogen, fibrinogen, thrombospondin, C1q, heparin, and IgG, suggesting that it may act as an adaptor protein either by targeting ligands to cell surfaces or by cross-linking soluble ligands. Despite the suggested functional importance of HRG, the cell-binding characteristics of the molecule are poorly defined. In this study, HRG was shown to bind to most cell lines in a Zn(2+)-dependent manner, but failed to interact with the Chinese hamster ovary cell line pgsA-745, which lacks cell-surface glycosaminoglycans (GAGs). Subsequent treatment of GAG-positive Chinese hamster ovary cells with mammalian heparanase or bacterial heparinase III, but not chondroitinase ABC, abolished HRG binding. Furthermore, blocking studies with various GAG species indicated that only heparin was a potent inhibitor of HRG binding. These data suggest that heparan sulfate is the predominate cell-surface ligand for HRG and that mammalian heparanase is a potential regulator of HRG binding. Using recombinant forms of full-length HRG and the N-terminal N1N2 domain, it was shown that the N1N2 domain bound specifically to immobilized heparin and cell-surface heparan sulfate. In contrast, synthetic peptides corresponding to the Zn(2+)-binding HRR of HRG did not interact with cells. Furthermore, the binding of full-length HRG, but not the N1N2 domain, was greatly potentiated by physiological concentrations of Zn2+. Based on these data, we propose that the N1N2 domain binds to cell-surface heparan sulfate and that the interaction of Zn2+ with the HRR can indirectly enhance cell-surface binding.     

Differential binding of histidine-rich glycoprotein (HRG) to human IgG subclasses and IgG molecules containing kappa and lambda light chains.

In previous studies we showed that the plasma protein histidine-rich glycoprotein (HRG) binds strongly to pooled human IgG. In the present work myeloma proteins consisting of different human IgG subclasses were examined for their ability to interact with human HRG. Using an IAsys optical biosensor we found initially that IgG subclasses differ substantially in their affinity of interaction with HRG. However, the most striking finding was the observation that the kinetics of the HRG interaction was dramatically affected by whether the IgG subclasses contained the kappa or lambda light (L)-chains. Thus, the on-rate for the binding of HRG to the kappa L-chain containing IgG1 and IgG2 (IgG1kappa and IgG2kappa) was approximately 4- and approximately 10-fold faster than that for the binding of HRG to lambda L-chain containing IgG1 and IgG2 (IgG1lambda and IgG2lambda), respectively, with the dissociation constants (K(d)) in the range 3-5 nM and 112-189 nM for the kappa and lambda isoforms, respectively. In contrast, the on-rate for the binding of HRG to IgG3kappa and IgG4kappa was found to be 9- and 20-fold slower than that for the binding of HRG to IgG3lambda and IgG4lambda, respectively, with the K(d) in the range 147-268 nM and 96-109 nM for the kappa and lambda isoforms, respectively. The binding of HRG to immunoglobulins containing the kappa L-chain (particularly IgG1kappa) was generally potentiated in the presence of a physiological concentration (20 microM) of Zn(2+) (K(d) decreased to 0.60 +/- 0.01 for IgG1kappa), but Zn(2+) had no effect or slightly inhibited the binding of HRG to immobilized IgG subclasses possessing the lambda L-chain. Interestingly, HRG also bound differentially to Bence Jones (BJ) proteins containing kappa and lambda L-chains, with HRG having a 14-fold lower K(d) for BJkappa than for BJlambda when 20 microM Zn(2+) was present. HRG also bound to IgM (IgMkappa), but the affinity of this interaction (K(d) approximately 1.99 +/- 0.05 microM) was markedly lower than the interaction with IgG, and the affinity was actually decreased 4-fold in the presence of Zn(2+). The results demonstrate that both the heavy (H)- and L-chain type have a profound effect on the binding of HRG to different IgG subclasses and provide the first evidence of a functional difference between the kappa and lambda L-chains of immunoglobulins.     

Characterization of Cu2+ and Fe3+ -mesoporphyrin complexes with histidine-rich glycoprotein: evidence for Cu2+ -Fe3+ -mesoporphyrin interaction

One equivalent of Fe3+ -mesoporphyrin (heme) is coordinated by two axial histidine ligands to a preferred site on histidine-rich glycoprotein (HRG). This study shows that titration of this stochiometric heme.HRG complex with 0-20 equivalents of Cu2+ produces a series of pronounced spectral changes indicative of multiple, sequential alterations of the heme environment. A monotonic low- to high-spin heme transition characterized by a decrease in resonance amplitude at g = 2.99, an increase at g = 6.0, and an increase in absorptivity at 620 nm is induced with the addition of the first 10 Cu2+ equivalents. Furthermore, optical absorption and circular dichroism spectra exhibit isosbestic and isodichroic points throughout the addition of the first 8 and 12 equivalents, respectively. The isosbestic points imply a transition between two optically well defined axial heme coordinations, and the isodichroic points suggest that these axial coordinations also represent two distinct protein conformations. A second isosbestic is formed during the addition of 14-20 equivalents of Cu2+, again suggesting well-defined coordinations; however, changes in the EPR spectra over this range are more complex. Whereas the amount of low-spin (g = 2.99) heme.HRG complex continues to decrease with the addition of 10-20 Cu2+ equivalents, the amount of the high-spin (g = 6.0) complex reaches a maximum near 14 equivalents and decreases markedly thereafter. Of potentially greater significance is the appearance of signals at g = 9.3 (maximum), 7.7 (maximum), 4.8 (crossover), and 1.61 (minimum) after addition of 10 or more Cu2+ equivalents. Some of these signals are similar to those exhibited by cardiac cytochrome c oxidase upon reduction and reoxidation. Thus, even without the addition of exogenous reductants and oxygen, the interaction of Cu2+ with the stoichiometric heme.HRG complex may produce structural features similar to those found in a mechanistically important but poorly understood form of cardiac cytochrome c oxidase.     

Histidine-rich glycoprotein specifically binds to necrotic cells via its amino-terminal domain and facilitates necrotic cell phagocytosis

Cells that become necrotic or apoptotic through tissue damage or during normal cellular turnover are usually rapidly cleared from the circulation and tissues by phagocytic cells. A number of soluble proteins have been identified that facilitate the phagocytosis of apoptotic cells, but few proteins have been defined that selectively opsonize necrotic cells. Previous studies have shown that histidine-rich glycoprotein (HRG), an abundant (approximately 100 microg/ml) 75-kDa plasma glycoprotein, binds to cell surface heparan sulfate on viable cells and cross-links other ligands, such as plasminogen, to the cell surface. In this study we have demonstrated that HRG also binds very strongly, in a heparan sulfate-independent manner, to cytoplasmic ligand(s) exposed in necrotic cells. This interaction is mediated by the amino-terminal domain of HRG and results in enhanced phagocytosis of the necrotic cells by a monocytic cell line. In contrast, it was found that HRG binds poorly to and does not opsonize early stage apoptotic cells. Thus, HRG has the unique property of selectively recognizing necrotic cells and may play an important physiological role in vivo by facilitating the uptake and clearance of necrotic, but not apoptotic, cells by phagocytes.     

Identification of a nucleotide-binding site on glycoprotein IIb. Relationship to ADP-induced platelet activation

Formalin-fixed platelets have been used to study the binding of adenine nucleotides in order to avoid the complications of nucleotide metabolism and to achieve steady-state binding. Sp-adenosine-5'-(1-thiotriphosphate) (Sp-ATP-alpha-S) binds to platelets at two sites (Kd1 3 nM; 31,000 sites/platelet; Kd2 200 nM; 300,000 sites/platelet) as compared with values for ADP under these conditions (Kd1 30 nM; 25,000 sites/platelet and Kd2 3 microM; 400,000 sites/platelet) (bound/total approximately 0.1). Competition binding experiments showed that both of the ATP-alpha-S sites were accessible to ADP and vice versa. [35S]ATP-alpha-S was photoaffinity cross-linked to unfixed platelets by direct irradiation with ultraviolet light. A single radiolabeled component (120 kDa) was identified and shown to be identical with the alpha subunit of GPIIb based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting with anti-GPIIb monoclonal antibodies, by isoelectric focusing (pI 4.5-5.5), by immunoaffinity adsorption using monoclonal anti-GPIIb/IIIa antibodies coupled to Sepharose, and by crossed immunoelectrophoresis. Amino-terminal sequencing of a tryptic fragment labeled with [35S]ATP-alpha-S identified an 18-kDa domain beginning at Tyr-198 in the primary sequence of GPIIb alpha. These studies demonstrate the presence of an adenine nucleotide-binding site on GPIIb alpha.     

Histidine-rich glycoprotein modulation of the anticoagulant activity of heparin. Evidence for a mechanism involving competition with both antithrombin and thrombin for heparin binding

Heparin binding to rabbit histidine-rich glycoprotein (HRG) was studied in a purified system, allowing for determination of a heparin dissociation constant of approximately 5.5 X 10(-8) M for the interaction with HRG at pH 7.0. The strong interaction between heparin and HRG was demonstrated to be competitive with the binding of both antithrombin and thrombin to the heparin chain. HRG was further tested as a modulator of the anticoagulant activity of heparin by comparing rates of the heparin-catalyzed reaction between antithrombin and thrombin in the presence and absence of added HRG. The heparin-antithrombin-thrombin reaction was modeled using the formalism of a two-substrate enzyme-catalyzed reaction with heparin as the enzyme and HRG analyzed as an enzyme inhibitor. HRG was shown to compete with both antithrombin and thrombin for binding to heparin by this kinetic analysis. Thus, both the kinetic and heparin-binding data indicate that the mechanism by which HRG modulates heparin anticoagulant activity involves competition for heparin with both the inhibitor and the protease. Inhibition by HRG of the heparin-catalyzed reaction was found to be highly dependent on pH, with a sharp increase in inhibition from about 15% to greater than 90% observed as pH was lowered from 7.4 to 7.0. Since little change in the rate of the heparin-catalyzed inhibition of thrombin by antithrombin occurs in this pH region, the dramatic change in HRG inhibition seen upon pH titration may reflect increasing ionic interaction between heparin and HRG due to the protonation of histidine residues which occurs in this pH region.     

Spectroscopic characterization of metal binding by Klebsiella aerogenes UreE urease accessory protein

 The urease accessory protein encoded by ureE from Klebsiella aerogenes is proposed to function in Ni(II) delivery to the urease apoprotein. Wild-type UreE contains a histidine-rich region at its carboxyl terminus and binds 5–6 Ni per dimer, whereas the functionally active but truncated H144*UreE lacks the histidine-rich motif and binds only two Ni per dimer [Brayman TG, Hausinger RP (1996) J Bacteriol 178 : 5410-5416]. For both proteins, Cu(II), Co(II), and Zn(II) ions compete for the Ni-binding sites. In order to characterize the coordination environments of bound metals, especially features that are unique to Ni, the Ni-, Cu-, and Co-bound forms of H144*UreE were studied by a combination of EPR, ESEEM, hyperfine-shifted ¹H-NMR, XAS, and RR spectroscopic methods. For each metal ion, the two binding sites per homodimer were spectroscopically distinguishable. For example, the two Ni-binding sites each have pseudo-octahedral geometry in an N/O coordination environment, but differ in their number of histidine donors. The two Cu-binding sites have tetragonal geometry with two histidine donors each; however, the second Cu ion is bound by at least one cysteine donor in addition to the N/O-type donors found for the first Cu ion. Two Co ions are bound to H144*UreE in pseudo-octahedral geometry with N/O coordination, but the sites differ in the number of histidine donors that can be observed by NMR. The differences in coordination for each type of metal ion are relevant to the proposed function of UreE to selectively facilitate Ni insertion into urease in vivo. Received: 8 October 1997 / Accepted: 30 December 1997     

Heme sulfuric anhydrides as soybean leghemoglobin structure probes

Mesoheme monosulfuric anhydride reacts at three distinct sites in soybean apoleghemoglobin a, at lysine-6, lysine-19 and lysine-57, the last one being the major site of reaction. The heme peptides obtained from thermolytic and pronase hydrolysates of the anhydride-leghemoglobin a were purified and correlated with the known amino acid sequence of the protein. Mesoheme bissulfuric anhydride also reacts with soybean apoleghemoglobin a giving a complex mixture of hemepeptides after hydrolysis with pronase. The visible spectrum of anhydride leghemoglobin is that of low spin heme. This suggests that anhydride leghemoglobin has a conformation with a covalent attachment via propionic acid side chain to lysine-57 and the sixth coordination position of the heme iron occupied by the distal histidine at position 61. Native leghemoglobin is assumed to exist in a similar type of configuration at low temperature, but with the heme propionate side chain being involved in a salt bridge with lysine-57.     

Evidence for the Interaction between (−)-Epigallocatechin Gallate and Human Plasma Proteins Fibronectin,Fibrinogen, and Histidine-rich Glycoprotein

Human plasma proteins were subjected to affinity chromatography with (–)-epigallocatechin gallate (EGCg)-agarose, and the bound proteins were examined by sodium dodecylsulfate–polyacrylamide gel electrophoresis. A molecular weight evaluation of the protein bands suggested the presence of three proteins, fibronectin, fibrinogen, and a 75-kDa protein. When human serum was used, the 75-kDa protein dominated the bound fraction. The determination of the partial amino acid sequence of a peptide derived by endopeptidase digestion of this fraction suggested the 75-kDa protein to be histidine-rich glycoprotein (HRG). The presence of these proteins in the bound fraction was confirmed by the immunoblotting method. Affinity chromatography of the individual proteins indicated that fibrinogen and HRG had direct affinity for EGCg. Dot binding assays demonstrated the interaction of EGCg with these proteins. The method also showed that only gallate-containing catechins were bound by these proteins. These data suggest that when EGCg is absorbed in the body through the digestive system, it may interact with these proteins in blood plasma.     

Binding of bile acids by glutathione S-transferases from rat liver

Binding of bile acids and their sulfates and glucuronides by purified GSH S-transferases from rat liver was studied by 1-anilino-8-naphthalenesulfonate fluorescence inhibition, flow dialysis, and equilibrium dialysis. In addition, corticosterone and sulfobromophthalein (BSP) binding were studied by equilibrium and flow dialysis. Transferases YaYa and YaYc had comparable affinity for lithocholic (Kd approximately 0.2 microM), glycochenodeoxycholic (Kd approximately to 60 microM), and cholic acid (Kd approximately equal 60 microM), and BSP (Kd approximately 0.09 microM). YaYc had one and YaYa had two high affinity binding sites for these ligands. Transferases containing the Yb subunit had two binding sites for these bile acids, although binding affinity for lithocholic acid (Kd approximately 4 microM) was lower than that of transferases with Ya subunit, and binding affinities for the other bile acids were comparable to the Ya family. Sulfated bile acids were bound with higher affinity and glucuronidated bile acids with lower affinity by YaYa and YaYc than the respective parent bile acids. In the presence of GSH, binding of lithocholate by YaYc was unchanged and binding by YbYb' was inhibited. Conversely, GSH inhibited the binding of cholic acid by YaYc but had less effect on binding by YbYb'. Cholic acid did not inhibit the binding of lithocholic acid by YaYa.     

富组氨酸糖蛋白(HRG)的结构与功能

富组氨酸糖蛋白(HRG)为一种多结构域血浆糖蛋白,可与多种配体结合而行使多种功能.HRG配体包括锌离子、肝素和硫酸肝素、纤溶酶原、纤溶酶、纤维蛋白原、凝血酶敏感素、原肌球蛋白、IgG、FcγR及补体.在锌离子存在或在低pH的环境中(如组织损伤或肿瘤生长),HRG的富含组氨酸结构域与配体的结合能力加强.HRG的多结构域特点及其与多种配体结合的性质表明,其可以作为细胞外衔接蛋白衔接细胞表面的不同配体.除了细胞表面分子,HRG还可以结合IgG,从而阻止可溶性免疫复合物的产生.HRG与大多数细胞发生结合的功能是在锌离子存在或低pH环境下,通过与细胞表面硫酸肝素蛋白聚糖相互作用实现的.HRG还具有加强凋亡细胞、坏死的吞噬细胞和免疫复合物的清除、抗血管新生、细胞的粘附和迁移、纤维蛋白溶解作用、血凝固、补体激活等生理活动调节等功能.本文针对HRG的分子结构与功能及其在临床上的研究进展进行概述.