Andrey A. Komissarov

Atomic structure at 2.5 Å resolution of uridine phosphorylase from E. coli as refined in the monoclinic crystal lattice

Uridine phosphorylase from E. coli (Upase) has been crystallized using vapor diffusion technique in a new monoclinic crystal form. The structure was determined by the molecular replacement method at 2.5 Å resolution. The coordinates of the trigonal crystal form were used as a starting model and the refinement by the program XPLOR led to the R‐factor of 18.6%. The amino acid fold of the protein was found to be the same as that in the trigonal crystals. The positions of flexible regions were refined. The conclusion about the involvement in the active site is in good agreement with the results of the biochemical experiments.

Effects of extracellular DNA on plasminogen activation and fibrinolysis.

Background: Elevated levels of extracellular DNA and aberrant fibrinolysis occur in a range of severe diseases. Results: DNA competes with fibrin for fibrinolytic enzymes. DNA stimulates fibrin-independent plasminogen activation and increases enzyme susceptibility to serpins. Conclusion: DNA is a macromolecular template that both potentiates and inhibits fibrinolysis. Significance: Understanding the interaction of DNA with the fibrinolytic system could improve the outcomes of fibrinolytic therapy. The increased levels of extracellular DNA found in a number of disorders involving dysregulation of the fibrinolytic system may affect interactions between fibrinolytic enzymes and inhibitors. Double-stranded (ds) DNA and oligonucleotides bind tissue-(tPA) and urokinase (uPA)-type plasminogen activators, plasmin, and plasminogen with submicromolar affinity. The binding of enzymes to DNA was detected by EMSA, steady-state, and stopped-flow fluorimetry. The interaction of dsDNA/oligonucleotides with tPA and uPA includes a fast bimolecular step, followed by two monomolecular steps, likely indicating slow conformational changes in the enzyme. DNA (0.1–5.0 μg/ml), but not RNA, potentiates the activation of Glu- and Lys-plasminogen by tPA and uPA by 480- and 70-fold and 10.7- and 17-fold, respectively, via a template mechanism similar to that known for fibrin. However, unlike fibrin, dsDNA/oligonucleotides moderately affect the reaction between plasmin and α2-antiplasmin and accelerate the inactivation of tPA and two chain uPA by plasminogen activator inhibitor-1 (PAI-1), which is potentiated by vitronectin. dsDNA (0.1–1.0 μg/ml) does not affect the rate of fibrinolysis by plasmin but increases by 4–5-fold the rate of fibrinolysis by Glu-plasminogen/plasminogen activator. The presence of α2-antiplasmin abolishes the potentiation of fibrinolysis by dsDNA. At higher concentrations (1.0–20 μg/ml), dsDNA competes for plasmin with fibrin and decreases the rate of fibrinolysis. dsDNA/oligonucleotides incorporated into a fibrin film also inhibit fibrinolysis. Thus, extracellular DNA at physiological concentrations may potentiate fibrinolysis by stimulating fibrin-independent plasminogen activation. Conversely, DNA could inhibit fibrinolysis by increasing the susceptibility of fibrinolytic enzymes to serpins.

Equilibrium Binding Studies of Recombinant Anti-single-stranded DNA Fab ROLE OF HEAVY CHAIN COMPLEMENTARITY-DETERMINING REGIONS

We previously isolated nucleic acid-binding antibody fragments (Fab) from bacteriophage display libraries representing the immunoglobulin repertoire of autoimmune mice to expedite the analysis of antibody-DNA recognition. In the present study, the binding properties of one such anti-DNA Fab, high affinity single-stranded (ss) DNA-binding Fab (DNA-1), were defined using equilibrium gel filtration and fluorescence titration. Results demonstrated that DNA-1 had a marked preference for oligo(dT) (100 nM dissociation constant) and required oligo(dT) >5 nucleotides in length. A detailed analysis of the involvement of the individual heavy chain (H) complementarity-determining regions (CDR) ensued using previously constructed HCDR transplantation mutants between DNA-1 and low affinity ssDNA-binding Fab (D5), a Fab that binds poorly to DNA (Calcutt, M. J. Komissarov, A. A., Marchbank, M. T., and Deutscher, S. L.(1996) Gene (Amst.) 168, 9-14). Circular dichroism studies indicated that the wild type and mutant Fab studied were of similar overall secondary structure and may contain similar combining site shapes. The conversion of D5 to a high affinity oligo(dT)-binding Fab occurred only in the presence of DNA-1 HCDR3. Results with site-specific mutants in HCDR1 further suggested a role of residue 33 in interaction with nucleic acid. The results of these studies are compared with previously published data on DNA-antibody recognition.

Site-specific mutagenesis of a recombinant anti-single-stranded DNA Fab. Role of heavy chain complementarity-determining region 3 residues in antigen interaction

The heavy chain complementarity-determining region 3 (HCDR3) of the anti-oligo(dT) recombinant antibody fragment, DNA-1, contributes significantly to antigen binding (Komissarov, A. A., Calcutt, M. J., Marchbank, M. T., Peletskaya, E. N., and Deutscher, S. L. (1996) J. Biol. Chem. 271, 12241–12246). In the present study, the role of separate HCDR3 residues of DNA-1 in interaction with oligo(dT) was elucidated. Based on a molecular model of the combining site, residues at the base (Arg98 and Asp108) and in the middle (Tyr101-Arg-Pro-Tyr-Tyr105) of HCDR3 were predicted to support the loop conformation and directly contact the ligand, respectively. Twenty-five site-specific mutants were produced as hexahistidine-tagged proteins, purified, and examined for binding to (dT)15 using two independent methods. All mutations in the middle of HCDR3 led to either abolished or diminished affinity. Tyr101 likely participates in hydrogen bonding, while Tyr104 and Tyr105 may be involved in aromatic-aromatic interactions with the ligand. The residues Arg102 and Pro103 were not as critical as the tyrosines. It is speculated that HCDR3 interacts with the thymines, rather than the phosphates, of the ligand. A 3-fold increase in affinity was observed by mutation of Asp108 to alanine. The highly conserved Arg98 and Asp108 do not appear to form a salt bridge.

Plasminogen Activator Inhibitor-1 Deficiency Augments Visceral Mesothelial Organization, Intrapleural Coagulation, and Lung Restriction in Mice with Carbon Black/Bleomycin–Induced Pleural Injury

Local derangements of fibrin turnover and plasminogen activator inhibitor (PAI)-1 have been implicated in the pathogenesis of pleural injury. However, their role in the control of pleural organization has been unclear. We found that a C57Bl/6j mouse model of carbon black/bleomycin (CBB) injury demonstrates pleural organization resulting in pleural rind formation (14 d). In transgenic mice overexpressing human PAI-1, intrapleural fibrin deposition was increased, but visceral pleural thickness, lung volumes, and compliance were comparable to wild type. CBB injury in PAI-1(-/-) mice significantly increased visceral pleural thickness (P < 0.001), elastance (P < 0.05), and total lung resistance (P < 0.05), while decreasing lung compliance (P < 0.01) and lung volumes (P < 0.05). Collagen, α-smooth muscle actin, and tissue factor were increased in the thickened visceral pleura of PAI-1(-/-) mice. Colocalization of α-smooth muscle actin and calretinin within pleural mesothelial cells was increased in CBB-injured PAI-1(-/-) mice. Thrombin, factor Xa, plasmin, and urokinase induced mesothelial-mesenchymal transition, tissue factor expression, and activity in primary human pleural mesothelial cells. In PAI-1(-/-) mice, D-dimer and thrombin-antithrombin complex concentrations were increased in pleural lavage fluids. The results demonstrate that PAI-1 regulates CBB-induced pleural injury severity via unrestricted fibrinolysis and cross-talk with coagulation proteases. Whereas overexpression of PAI-1 augments intrapleural fibrin deposition, PAI-1 deficiency promotes profibrogenic alterations of the mesothelium that exacerbate pleural organization and lung restriction.

Proteolytic regulation of epithelial sodium channels by urokinase plasminogen activator: cutting edge and cleavage sites

Background: Depressed fibrinolysis and edema concurrently exist in edematous injury. Results: Divergent regulation of ENaC by urokinase and tPA was observed. Both the catalytic domain of urokinase and cleavage sites in ENaC were identified. Conclusion: Urokinase activates ENaC through catalytic activity-dependent proteolytic modification of γ subunit. Significance: Activation of ENaC by urokinase but not tPA provides a novel mechanism for the alleviation of lung edema and pleural effusion. Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human αβγ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, γ but not α subunit of ENaC was proteolytically cleaved at (177GR↓KR180) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ ENaC, incremental increase in opening rate, and activation of closed (electrically “silent”) channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.

Enzyme‐catalyzed uridine phosphorolysis: SN2 mechanism with phosphate activation by desolvation

The rate of uridine phosphorolysis catalyzed by uridine phosphorylase from Escherichia coli decreases with increasing ionic strength. In contrast, the rate was increased about twofold after preincubation of uridine phosphorylase with 60% acetonitrile. These data correlate with known effects of polar and bipolar aprotic solvents on SN2 nucleophilic substitution reactions. The enzyme modified with fluorescein‐5′‐isothiocyanate (fluorescein residue occupies an uridine‐binding subsite [Komissarov et al., (1994) Biochim. Biophys. Acta 1205, 54–58]) was selectively modified with irreversible inhibitor SA‐423, which reacts near the phosphate‐binding subsite. The double‐modified uridine phosphorylase is assumed to imitate the enzyme—substrate complex. Modification with SA‐423 was accompanied with dramatic changes in the absorption spectrum of active site‐linked fluorescein, which were identical to those for fluorescein in a hydrophobic medium, namely 80% acetonitrile. The data obtained suggest that an increase in active site hydrophobicity leads to phosphate desolvation and facilitates the enzymatic SN2 uridine phosphorolysis reaction.

Analysis of a nucleic-acid-binding antibody fragment: Construction and characterization of heavy-chain complementarity-determining region switch variants.

The display of antibody (AB) fragments (Fab) on the surface of filamentous bacteriophage (phage) and selection of phage that interact with a particular antigen (Ag) has enabled the isolation of Fab that bind nucleic acids. Nucleic acid (NA) binding Ab occur in vivo in connective tissue disease patients and certain inbred strains of mice and are thought to be pathogenic. Although there is ample data concerning the amino acid (aa) sequence of murine monoclonal Ab (mAb) reactive with DNA, significantly less is known about how autoAb interact with NA. The complementarity-determining regions (CDR) contained in the Fab contribute to most Ag binding, especially through heavy (H)-chain CDR 3. We have examined the role of individual H-chain CDR of a previously isolated recombinant single-stranded DNA-binding Fab (DNA-1) in nucleic acid interaction using a combination of H-chain CDR switching and solution-binding experiments. The three H-chain CDR of DNA-1 Fab were independently switched with the H-chain CDR of a Fab (D5) with very similar sequence and framework (FR) that binds DNA poorly in order to create all possible H-chain CDR combinations. The chimeric Fab genes were bacterially expressed, and their products were purified and analyzed. Results indicated that the H-chain CDR 3 of DNA-1 Fab, in the context of the remainder of the H-chain of D5 Fab, restored binding to oligo(dT)15 to 60% of DNA-1 levels, whereas H-chain CDR 1 and 3 of DNA-1 with CDR 2 of D5 Fab restored binding to 100% A combination of H-chain CDR 2 and 3 of DNA-1 Fab with H-chain CDR 1 of D5, unexpectedly resulted in the ability of the chimeric Fab to bind RNA preferentially over DNA. These studies demonstrate the importance of both H-chain CDR 1 and 3 in DNA recognition and further suggest that the specificity of the type of NA recognized by a particular Fab can be drastically altered by exchanging CDR.

Crystallization and molecular-replacement studies of a recombinant antigen-binding fragment complexed with single-stranded DNA

Anti-DNA antibodies have been implicated in autoimmune diseases and also serve as models for understanding protein-DNA recognition. Crystals of a recombinant antigen-binding fragment (Fab) complexed with dT(5) have been obtained and initial phases have been determined using molecular replacement. The crystals diffract to 2.1 A resolution and occupy space group P6(5)22, with unit-cell parameters a = 171.8, c = 144.6 A; there are two Fabs per asymmetric unit. X-PLORdirect rotation-function calculations followed by Patterson correlation filtering were successful when using a Fab search model; however, they failed when using the individual variable and conserved domains of the Fab as search models. AMoRe successfully identified the correct solution in cases where X-PLOR failed.

selective modification of putative uridine-binding site of uridine phosphorylase from E. coli with fluorescein 5′-isothiocyanate

A putative uridine-binding site of uridine phosphorylase (EC 2.4.2.3) from E. coli was modified with fluorescein 5-isothiocyanate (FITC). Treatment with FITC irreversibly inactivates the enzyme (Ki = 1.0 mM, k2 = 0.15 min-1). Under the conditions of 90% inactivation the incorporation of the reagent reaches about 1 mol per mol of the enzyme subunit. Addition of uridine prevents the enzyme inactivation by FITC. In contrast to this, addition of a second substrate phosphate increases the rate of inactivation by 2.3-fold (k2 = 0.34 min-1), but has no effect on the affinity of the reagent to the enzyme. The modified protein retains the ability to bind phosphate but not uridine. According to differential absorption spectroscopy data, the binding of phosphate to the active site of the enzyme is accompanied by conformational changes which may accelerate the inactivation rate. The data presented suggest that in the UPase FITC occupies the putative uridine-binding site, while the phosphate-binding site still retains the ability to interact with the second substrate.