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.

Selective inhibition of factor Xa in the prothrombinase complex by the carboxyl-terminal domain of antistasin.

Studies of antistasin, a potent factor Xa inhibitor with anticoagulant properties, were performed wherein the properties of the full-length antistasin polypeptide (ATS-119) were compared with the properties of forms of antistasin truncated at residue 116 (ATS-116) and residue 112 (ATS-112). ATS-119 was 40-fold more potent than ATS-112 in prolonging the activated partial thromboplastin time (APTT), whereas ATS-119 inhibited factor Xa 2.2-fold less avidly and about 5-fold more slowly than did ATS-112. The decreased reactivity of ATS-119 suggests that the carboxyl-terminal domain of ATS-119 stabilizes an ATS conformation with a reduced reactivity toward factor Xa. The observation that calcium ion increases the reactivity of ATS-119 but not that of ATS-112 suggests that calcium ion may disrupt interactions involving the carboxyl terminus of ATS-119. Interestingly, ATS-119 inhibited factor Xa in the prothrombinase complex 2-6-fold more potently and 2-3-fold faster than ATS-112. These differences in affinity and reactivity might well account for the greater effectiveness of ATS-119 in prolonging the APTT and suggest that the carboxyl-terminal domain of ATS-119 disrupts interactions involving phospholipid, factor Va, and prothrombin in the prothrombinase complex. The peptide RPKRKLIPRLS, corresponding to the carboxyl domain of ATS-119 prolonged the APTT and inhibited prothrombinase-catalyzed processing of prothrombin, but it failed to inhibit the catalytic activity of isolated factor Xa. Thus, this novel inhibitor appears to exert its inhibitory effects at a site removed from the active site of factor Xa.

Characterization of recombinant hepatitis B surface antigen using surface plasmon resonance.

Two commercial monoclonal antibodies were shown to interact with distinct epitopes within the major antigenic a determinant region of yeast recombinant hepatitis B surface antigen (rHBsAg) particles, contained in the hepatitis B vaccine, by using the surface plasmon resonance phenomenon on a BIAcore system. Apparent avidity for these antibodies were compared among different rHBsAg batches, showing the consistency of these epitopes. Furthermore, the effect of the alteration of these epitopes, achieved by chemical modifications, was readily reflected by changes in the apparent avidity for these antibodies. The procedures used in the present study provide a novel and efficient way to characterize rHBsAg particles present in different hepatitis B vaccine products.

Chromatographic separation of low-molecular-mass recombinant proteins and peptides on Superdex 30 prep grade.

The chromatographic properties of Superdex 30 prep grade medium have been investigated in non-denaturing and denaturing mobile phases using commercially available proteins and peptides as well as low-molecular-mass (M(r)) recombinant polypeptides. The medium is a macroreticular gel composed of crosslinked agarose beads to which dextran has been covalently bound. The mean particle size is approximately 34 microns. Experimental results show a linear relation between the distribution coefficient (KD) and the log10 M(r) in the fractionation range 24,000-3000. The relationships between resolution and flow-rate or load volume were investigated and shown to be comparable with those of Superdex 75 and 200 prep grade media. Minimal loss of resolution occurred in the flow-range from 30-60 cm/h. Load volumes of up to 5% total column volume could be applied while maintaining baseline resolution of polypeptide mixtures. Non-specific interactions between the matrix and certain samples were characterized. The predominant interactions with the resin appear to be hydrophobic in nature rather than ionic. Hydrogen bonding may also play a role in the retardation of certain small molecules. The applicability of the resin for separating dimeric and oligomeric forms of low-molecular-mass recombinant proteins was shown.

Characterization of recombinant antistasin secreted by Saccharomyces cerevisiae.

Secretion from recombinant yeast was used as a potential source of large quantities of the leech protein antistasin (ATS), a potent and highly specific inhibitor of the serine protease coagulation factor Xa. Mature recombinant ATS (r-ATS) is obtained after intracellular cleavage by the yscF protease of the mating factor alpha-1 pre-proleader from the fusion protein at the Lys-Arg sequence junction. Production levels are relatively low (ca. 1 mg/liter). Purification of the secreted product from a complex growth medium involved cell removal by microfiltration and diafiltration, cation-exchange capture and concentration on S-Sepharose Fast Flow, C-4 reverse-phase high-performance liquid chromatography (RP-HPLC), and HPLC cation-exchange chromatography step, and RP-HPLC concentration and desalting. The process was scaled up from the 16- to the 250-liter level with a corresponding increase in amount of r-ATS. From the 250-liter fermentation two major forms, r-ATS-I and r-ATS-II, distributed approximately 60:40, and a minor form, r-ATS-minor (ca. 1% of the purified r-ATS), were characterized. Limited N-terminal sequence analysis by Edman degradation indicated that r-ATS-I has the predicted mature N-terminus starting with Gln, that r-ATS-II is N-terminally blocked with pyroglutamate, and that r-ATS-minor is an incompletely processed form. RP-HPLC, hydrophilic-interaction HPLC, cation-exchange HPLC analysis, and electrophoresis results are consistent with the differences observed by sequencing. Preliminary in vitro characterization by intrinsic Ki determination for factor Xa inhibition indicated that the yeast r-ATS forms are indistinguishable from each other as well as from r-ATS expressed by the insect baculovirus host-vector system. Nevertheless, r-ATS-I and r-ATS-II appear less potent than insect-derived r-ATS in the activated partial thromboplastin time clotting assay. Further characterization indicated that C-terminal cleavage at Pro-116 had occurred in r-ATS-I and r-ATS-II as well as oxidation of methionine residues to methionine sulfoxide. The possible role of the C-terminus in inhibition of the prothrombinase complex is discussed.

Large-scale purification and characterization of recombinant tick anticoagulant peptide.

Recombinant tick anticoagulant peptide (r-TAP), a potent and specific inhibitor of blood coagulation factor Xa, was purified to greater than 99% homogeneity at the multi-gram scale. Genetically engineered yeast secreted 200-250 mg/l of the heterologous protein into the medium. Cells were separated from broth by diafiltration and purification was done by two chromatographic steps, both conducive to operation on a large scale. Analysis of the purified protein by several methods indicated that it was greater than 99% homogeneous and no incompletely processed or truncated proteins were detected. Physico-chemical characterization data of r-TAP show that it exists as a monomer in solution and no evidence of post-translational modification was observed. The purified protein was fully active in inhibiting human coagulation factor Xa.

1H-NMR studies of high-potential iron-sulfur protein from the purple phototrophic bacterium, Rhodospirillum tenue.

The high-potential iron-sulfur protein (HiPIP) from Rhodospirillum tenue (strain 3761) shows only a weak (20-25%) sequence similarity to HiPIPs from Chromatium vinosum, Ectothiorhodospira halophila and Ectothiorhodospira vacuolata, including the strict conservation of only two of the twelve residues assumed to be in the 4Fe-4S cluster packing region [Tedro, S. M., Meyer, T. E. and Kamen, M. D. (1979) J. Biol. Chem. 254, 1495-1500]. In spite of these differences, the general range and distribution of hyperfine-shifted 1H-NMR peaks of oxidized and reduced R. tenue HiPIP resemble those of E. halophila HiPIP I [Krishnamoorthi, R., Markley, J. L., Cusanovich, M. A., Pryzycieki, C. T. and Meyer, T. E. (1986) Biochemistry 25, 60-67]. Temperature- and pH-dependence and longitudinal relaxation behavior were determined for hyperfine-shifted peaks of the oxidized protein. Tentative assignments of peaks to ligands and aromatic residues suggest the presence of common apoprotein-active-site interactions in these proteins. Differences occur in the pattern of paramagnetically shifted peaks attributed to hydrogens bonded to the 4Fe-4S cluster. Hyperfine-shifted peaks of R. tenue HiPIP are not perturbed by pH changes in the range 5-9. In contrast, those of the C. vinosum protein exhibit a pH-dependence of chemical shifts that has been attributed to the titration of His42 [Nettesheim, D. G., Meyer, T. E., Feinberg, B. A. and Otvos, J. D. (1983) J. Biol. Chem. 258, 8235-8239]. Since R. tenue HiPIP contains no histidine, the present observation confirms the above hypothesis.

Inhibitors of 2-ketoglutarate-dependent dioxygenases block aspartyl beta-hydroxylation of recombinant human factor IX in several mammalian expression systems.

While a role has been ascribed to the gamma-carboxyglutamate (Gla) residues in vitamin K-dependent coagulation proteins and the enzyme catalyzing this posttranslational modification has been identified and partially characterized, both the functional significance of a second posttranslationally synthesized amino acid found in these proteins, beta-hydroxyaspartate (Hya), and the aspartyl beta-hydroxylating enzyme remain to be determined. We now report that inhibitors of 2-ketoglutarate-dependent dioxygenases, such as dipyridyl, o-phenanthroline, and pyridine 2,4-dicarboxylate, block hydroxylation of Asp64 in recombinant factor IX molecules produced in three different mammalian expression systems. This hydroxylation was not inhibited by the specific copper chelators 2,9-dimethylphenanthroline or D-penicillamine. The Gla levels in these proteins were unaffected by these compounds and demonstrate that carboxylation proceeds independently of hydroxylation. Using these Hya-deficient recombinant factor IX molecules we demonstrate that this residue does not play a significant role in factor IX binding to endothelial cells under equilibrium conditions. From additional binding studies we have concluded that the Gla domain of factor IX is a major cell binding domain of factor IX. Furthermore, in contrast to studies demonstrating a marked loss of one-stage clotting activity in recombinant factors IX following site-directed mutations of Asp64 to neutral or basic residues (Rees, D. J. G., Jones, I. M., Handford, P. A., Walter, S. J., Esnouf, M. P., Smith, K. J., and Brownlee, G. J. (1988) EMBO J. 7, 2053-2061), we have not found a decrease of one-stage clotting activity with Hya-deficient factor IX. Hya-deficient proteins produced in this manner may prove to be more appropriate to elucidate the function of Hya than those produced by site-directed mutagenesis.

Occurrence of beta-hydroxylated asparagine residues in non-vitamin K-dependent proteins containing epidermal growth factor-like domains.

Vitamin K-dependent bovine protein S has been shown to contain a posttranslationally hydroxylated asparagine within a conserved sequence in three of its epidermal growth factor (EGF)-like domains. In a review of amino acid sequences deduced from cDNA data, we have observed that a conserved sequence containing a potential asparagine hydroxylation site exists within EGF-like domains of a variety of functionally diverse proteins. We have studied a number of these and report the presence of erythro-beta-hydroxyasparagine (e-beta Hyn) in three non-vitamin K-dependent proteins: the plasma complement proteins C1r and C1s (where overbar indicates activated form) and the urinary protein uromodulin. For each protein, e-beta Hyn was identified in enzyme digests following the initial observation of erythro-beta-hydroxyaspartic acid (e-beta Hya) in acid hydrolysates of the proteins. e beta Hya and e-beta Hyn residues are detected by a postcolumn derivatization cation-exchange HPLC method herein described. HPLC isolation of the presumptive e-beta Hyn residue from enzyme digests of intact C1r allowed confirmation of its structure by GC/MS. Based upon available cDNA sequence data and observation of e-beta Hya in acid hydrolysates, we suggest other proteins in which e-beta Hyn may occur.

Hydrogen-1 nuclear magnetic resonance investigation of Clostridium pasteurianum rubredoxin: previously unobserved signals.

Previously unobserved signals were located in the 470-MHz 1H NMR spectra of oxidized and reduced rubredoxin (Rd) from Clostridium pasteurianum. When the protein was oxidized, some of the resonances broadened beyond detection. Longitudinal relaxation (T1) measurements identified a number of these peaks as arising from residues close to the paramagnetic iron; these resonances exhibited short T1 values attributable to the dominant electron-nuclear dipolar relaxation mechanism. The chemical shifts of these peaks were not strongly dependent on the oxidation state of the protein, although relative ratios of line widths of several peaks in the spectra of oxidized and reduced Rd suggested localized conformational changes of the protein as a result of oxidation. Furthermore, spectra of the oxidized protein collected in the range 8-60 degrees C revealed no appreciable changes in the chemical shifts of these peaks with temperature. These results seem to point out a negligible dipolar contribution, due to either magnetic anisotropy or zero field splitting, to the observed shifts in the spectrum of oxidized Rd. Resonances were assigned to tyrosine-11 or phenylalanine-49 (but not to either specifically) on the basis of their T1 values and the X-ray diffraction data of the protein molecule [Watenpaugh, K. D., Sieker, L. C., Herriott, J. R., & Jensen, L. H. (1973) Acta Crystallogr., Sect. B: Struct. Crystallogr. Cryst. Chem. B29, 943-956; and a further refinement deposited with the Protein Data Bank]. An upfield-shifted peak at about -1.1 ppm in the spectra of both oxidized and reduced Rd was assigned to a methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen-1 nuclear magnetic resonance investigation of high-potential iron-sulfur proteins from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata: a comparative study of hyperfine-shifted resonances.

Proton NMR spectra of the oxidized and reduced forms of high-potential iron-sulfur proteins (HiPIPs) were recorded at 200 MHz. The proteins studied were the HiPIPs I and II from Ectothiorhodospira halophila and Ectothiorhodospira vacuolata. Hyperfine-shifted peaks in spectra of the oxidized proteins were assigned to some of the protons of the cysteinyl ligands and aromatic residues at the active site on the basis of their chemical shifts, longitudinal relaxation times, and temperature-dependent behavior. The cysteinyl C beta-H protons were found to resonate downfield (about 100 ppm) and the C alpha-H protons upfield (about-25 ppm). This hyperfine shift pattern is consistent with the observed isotropic shift being contact in origin; it probably results from a pi-spin-transfer mechanism. The large magnitudes of the chemical shifts of peaks assigned to aromatic residues suggest that these residues interact with the iron-sulfur cluster via pi-pi overlap. Some of the hyperfine-shifted peaks observed in water were found to disappear in 2H2O solution. Such resonances probably arise from exchange-labile hydrogens of amino acid residues directly hydrogen bonded to the iron-sulfur cluster. In the case of HiPIPs I and II from E. vacuolata, whose spectra are similar except for the number of such peaks, the relative number of hydrogen bonds inferred to be present in the oxidized and reduced proteins qualitatively explains the difference between their midpoint redox potentials. On the other hand, for E. halophila HiPIPs I and II, consideration of the inferred number of hydrogen bonds alone fails to predict the sign of the difference between their midpoint redox potentials.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of reduction of high redox potential ferredoxins by the semiquinones of Clostridium pasteurianum flavodoxin and exogenous flavin mononucleotide. Electrostatic and redox potential effects.

We have measured the ionic strength dependence of the rate constants for the electron-transfer reactions of flavin mononucleotide (FMN) and flavodoxin semiquinones with 10 high redox potential ferredoxins (HiPIP's). The rate constants were extrapolated to infinite ionic strength by using a theoretical model of electrostatic interactions developed in our laboratory. In all cases, the sign of the electrostatic interaction was the same as the protein net charge, but the magnitudes were much smaller. The results are consistent with a model in which the electrical charges are approximately uniformly distributed over the HiPIP surface and in which there are both short- and long-range electrostatic interactions. An electrostatic field calculation for Chromatium vinosum HiPIP is consistent with this. The presumed site of electron transfer includes that region of the protein surface to which the iron-sulfur cluster is nearest and appears to be relatively hydrophobic. The principal short-range electrostatic interaction would involve the negative charge on the iron-sulfur cluster. For some net negatively charged proteins, this effect is magnified, and for net positively charged HiPIP's, it is counterbalanced. The rate constants extrapolated to infinite ionic strength can be correlated with redox potential differences between the reactants, as has previously been shown for cytochrome-flavin semiquinone reactions. Both electrostatic and redox potential effects are magnified for the flavodoxin semiquinone as compared to the FMN semiquinone-HiPIP reactions. This was also observed previously for the flavin semiquinone-cytochrome reactions.(ABSTRACT TRUNCATED AT 250 WORDS)

Circular dichroism and redox properties of high redox potential ferredoxins.

The circular dichroism (CD) spectra of 13 examples of high-potential iron-sulfur proteins (HiPIPs), a class of [4Fe-4S] ferredoxins, have been determined. In contrast to the proposal of Carter [Carter, C. W., Jr. (1977) J. Biol. Chem. 252, 7802-7811], no strict correlation between visible CD features and utilization of the [4Fe-4S]2+/[4Fe-4S]3+ oxidation levels was found. Although most HiPIPs have these features, the model requires their presence in all species. There is also no simple relationship between CD spectral features and the presence of conserved tyrosine-19. In addition, no apparent correlation between CD properties and oxidation-reduction potential could be detected. However, amino acid side chains in close contact to the iron-sulfur cluster appear to be important in modulating spectral and oxidation-reduction properties. In particular, the negative shoulder at 290 nm and negative maximum at 230 nm correlate with the presence of Trp-80 (Chromatium vinosum numbering). Two HiPIPs that do not have Trp at this position have positive bands at 290 and 230 nm. These bands in the Ectothiorhodospira halophila HiPIPs are apparently associated with Trp-49, which is located on the opposite side of the effective mirror plane of the cluster from Trp-80. The effect of pH on circular dichroism and redox potential in Thiocapsa roseopersicina HiPIP, which has a histidine at position 49, is consistent with the interaction of the side chain with the cluster. Despite specific differences in their CD spectra, the various HiPIPs studied show general similarity consistent with structural homology within this class of iron-sulfur proteins.

Effects of pH and exocyclic substitution on flavosemiquinone reactivity with redox proteins and inorganic oxidants.

The effect of pH on the reaction of free flavosemiquinone analogs generated by laser-flash photolysis with oxidized Chromatium vinosum high-potential iron-sulfur protein, other iron-containing redox proteins, and nonbiological one-electron oxidants has been investigated. The results demonstrate that the second-order rate constant for the oxidation of lumiflavin flavosemiquinone increases dramatically with increasing pH for the redox proteins and some of the other oxidants. The pH-rate constant profiles for the redox proteins closely follow the ionization of the proton at the N-5 position of the neutral lumiflavin flavosemiquinone, suggesting a higher intrinsic reactivity for the anionic lumiflavin flavosemiquinone. This increased reactivity apparently results from changes in the redox potential and in the electron spin density distribution between the two protonic forms of the semiquinone. Similar pH dependencies are observed for a number of other flavin structural analogs, yielding estimates of the N-5 pK values for these analogs. The data are consistent with the involvement of both the N-5-dimethylbenzene ring portion and the C-4a position of the flavin macrocycle in flavosemiquinone oxidation by one-electron oxidants.

Kinetics of reduction of Clostridium pasteurianum rubredoxin by laser photoreduced spinach ferredoxin:NADP+ reductase and free flavins. Electron transfer within a protein-protein complex.

The kinetics of reduction of oxidized Clostridium pasteurianum rubredoxin (Rdox) by free flavin semiquinones generated by the laser flash photolysis technique and by spinach ferredoxin:NADP+ reductase (FNR) semiquinone (also produced by flavin semiquinone reduction) have been investigated under anaerobic conditions. 5-Deazariboflavin semiquinone (5-dRf) rapidly reduces oxidized rubredoxin (Rdox) (k = 3.0 X 10(8) M-1 S-1) and oxidized ferredoxin:NADP+ reductase (FNRox) to the semiquinone level (k = 5.5 X 10(8) M-1 S-1). Lumiflavin semiquinone reduces Rdox more slowly (k = 1.3 X 10(7) M-1 S-1) and is not measurably reactive with FNRox. Absorption difference spectroscopy and difference CD indicate that Rdox and FNRox form a 1:1 complex at low ionic strength (10 mM), which is completely dissociated at higher ionic strength (310 mM). Apparent second order rate constants for reduction of Rdox in its free and complexed state by lumiflavin semiquinone are the same. Reduction of Rdox (both free and complexed) by free FNR semiquinone and intracomplex electron transfer were investigated using 5-dRf as the reductant. At I = 10 mM, a first order rate constant of 2.0 X 10(3) S-1 was obtained, which corresponds to the processes involved in intracomplex electron transfer from FNR semiquinone to Rdox. A second order reaction between free FNR semiquinone and complexed Rdox was also observed to occur (k = 5 X 10(7) M-1 S-1). At I = 310 mM, these reactions are not observed and the reaction of FNR semiquinone with free Rdox is second order (k = 4 X 10(6) M-1 S-1).

Electron-transfer reactions of photoreduced flavin analogues with c-type cytochromes: quantitation of steric and electrostatic factors.

We have found correlations between rate constants and the difference in redox potential of the reactants for electron-transfer reactions between oxidized cytochromes and either photoproduced riboflavin or flavin mononucleotide (FMN) semiquinones (the latter rate constants extrapolated to infinite ionic strength). The riboflavin-cytochrome rate constants are about 70% of those for reduction by lumiflavin, probably because of steric interference by the ribityl side chain. Reduction of cytochromes by FMN semiquinone was ionic strength dependent in all cases, due to electrostatic interactions. Extrapolation of rate constants to infinite ionic strength shows that the phosphate exerts a significant steric effect as well (rate constants average about 27% of those for lumiflavin, although part of this decrease is due to a difference in the semiquinone pK value). Differences in the magnitude of the FMN steric effect correlate well with surface topology differences for those cytochromes whose three-dimensional structures are known. Mitochondrial cytochromes c and the cytochromes c2 all showed attractive (plus-minus) interaction with FMN in spite of the fact that some of these proteins have large net negative charges. Four small c-type cytochromes (including Pseudomonas cytochrome c-551) show a weak repulsive interaction with FMN semiquinone. We conclude that flavosemiquinones interact at a site on the cytochromes that is near the exposed heme edge. There is a large positive electrostatic field at this site in mitochondrial cytochrome c and the cytochromes c2, but this region is primarily hydrophobic in Pseudomonas cytochrome c-551 and in the other small bacterial cytochromes.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between rate constant for reduction and redox potential as a basis for systematic investigation of reaction mechanisms of electron transfer proteins.

Rate constants for the reduction of approximately 40 electron transfer proteins by photoreduced flavins have been determined by laser flash photolysis techniques. The data for a series of 12 homologous cytochromes and 10 homologous high redox potential ferredoxins (HiPIPs) are in excellent agreement with semi-empirical equations relating rate constant and thermodynamic redox potential that have proven applicable to nonbiological electron transfer systems. These correlations allow the establishment of relative reactivities within structurally homologous classes of biological oxidation-reduction proteins, including cytochromes and HiPIPs, and a variety of nonhomologous heme-, iron-sulfur-, copper-, and flavin-containing proteins. A qualitative correspondence is shown to exist between such relative reactivity and the extent of solvent exposure of the redox centers in a particular structural class. The implications of these results are considered, and it is concluded that free energy relationships provide a sound basis for systematic analysis of reaction mechanisms of electron transfer proteins.