Identification of a linear B-cell epitope in the catalytic domain of bothropasin, a metalloproteinase from Bothrops jararaca snake venom.

Bothropasin is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca venom, the snake responsible for most bites in Southeastern Brazil. SVMPs, such as bothropasin, are involved in the main bothropic envenoming symptoms, which include hemorrhage, inflammation, necrosis and blood coagulation deficiency. B-cell epitope mapping of SVMPs can lead to the identification of peptides capable of inducing neutralizing antibodies without causing toxic effects, therefore improving anti-venom production. Here, using the SPOT synthesis technique, we have identified an epitope located in the catalytic domain of bothropasin (202KARMYELANIVNEILRYLYMH222) which was synthesized and named BotEp1. The peptide was used to immunize Swiss mice and Anti-BotEp1 serum cross-reacted with bothropasin and crude venoms from B. jararaca and B. atrox venoms. Furthermore, Anti-BotEp1 antibodies were able to completely neutralize the hemorrhagic activity of a chromatographic fraction from B. jararaca venom, which contains hemorrhagic SVMPs. In addition, the coagulation activity of the hemorrhagic fraction showed to be diminished when tested in serum from rabbit immunized with BotEp1 (compared to serum from non-immunized animal). Our results show the identification of neutralizing epitopes in bothropasin and provide basis for the use of synthetic peptides to improve the production of immunotherapeutics.

Drug evaluation: alfimeprase, a plasminogen-independent thrombolytic.

Alfimeprase is an analog of a fibrolase that disrupts formed thrombi through the hydrolysis of fibrin, rather than by activation of plasminogen. Nuvelo Inc, under license from Amgen Inc and together with Bayer AG, is developing this thrombolytic for the potential intravenous treatment of peripheral arterial occlusions and for other cardiovascular indications. Pharmacokinetic studies showed that alfimeprase was rapidly absorbed and achieved therapeutic concentrations at relatively low doses. Preclinical studies showed that adjunctive therapy with antiplatelet agents was necessary to maintain luminal patency. In phase I and II clinical trials alfimeprase effectively thombolysed clots with no drug-related adverse events. However, phase III clinical trials of alfimeprase did not meet their primary endpoints and enrollment in ongoing trials has been suspended pending further analyses and discussion with outside experts and regulatory agencies. In spite of this, the authors conclude that alfimeprase seems to be a lytic agent with much potential. Refinement in its use and dosing needs to be addressed, and further investigation into its pharmacokinetic properties may be worthwhile. Alfimeprase is a drug that is a 'work in progress'.

Artificial metalloprotease with active site comprising aldehyde group and Cu(II)cyclen complex.

To design artificial proteases that cleave peptide backbones of a wide range of proteins at selected sites, artificial active sites comprising the Cu(II) complex of cyclen (Cu(II)Cyc) and aldehyde group were synthesized on a cross-linked polystyrene. The aldehyde group was employed as the binding site in view of its ability of reversible formation of imine bonds with epsilon-amino groups of Lys residues exposed on the surface of proteins and Cu(II)Cyc as the catalytic group for peptide hydrolysis. The two polymeric artificial metalloproteases synthesized in the present study cleaved all of the protein substrates examined (myoglobin, gamma-globulin, bovine serum albumin, human serum albumin, lysozyme, and ovalbumin), manifesting saturation kinetic behavior. At 50 degrees C and pH 9.0 or 9.5, K(m) was (1.3-22) x 10(-)(4) M, comparable to those of natural proteases, and k(cat) was (6.0-25) x 10(-)(4) s(-)(1), corresponding to half-lives of 4.6-19 min. Intermediacy of the imine complexes formed between the aldehyde group of the catalyst and the epsilon-amino groups of Lys residues of the substrates was confirmed by the trapping experiment with NaB(OAc)(3)H. MALDI-TOF MS of the proteolytic reaction mixtures revealed formation of various cleavage products. Structures of some of the cleavage products were determined by using carboxypeptidase A and trypsin. Among various cleavage sites thus identified, Gln(91)-Ser(92) and Ala(94)-Thr(95) were the major initial cleavage sites in the degradation of myoglobin by the two catalysts. The selective cleavage of Gln(91)-Ser(92) and Ala(94)-Thr(95) was attributed to general acid assistance in peptide cleavage by Tyr(146) located in proximity to the two peptide bonds. Broad substrate selectivity, high cleavage-site selectivity, and high proteolytic rate are achieved, therefore, by positioning the aldehyde group in proximity to Cu(II)Cyc attached to a cross-linked polystyrene.

A facile synthetic method to prepare fluorescently labeled ROMP polymers.

[structure: see text] To probe the activities of sperm ADAM protein (fertilinbeta), we devised a general synthetic strategy to generate fluorescently labeled fertilinbeta oligopeptide polymers. Immunofluorescence studies with these polymers demonstrated that fertilinbeta polymers bind specifically to a protein receptor on the mouse egg plasma membrane.

Copper II complex of a tridentate ligand: an artificial metalloprotease for bovine serum albumin.

A copper(II) complex of 2, 6-bis(benzimidazo-2-yl) pyridine was synthesized and its binding properties with bovine serum albumin (BSA) has been evaluated. The binding plot obtained from the absorption titration data gives a binding constant of 2.4 (+/-0.3) x10(3) M(-1). It was found that the charge transfer band of the metal complex was perturbed in the presence of BSA. The gel electrophoresis pattern of BSA incubated with copper(II) complex shows the metalloproteolytic activity of the metal complex. In the presence of oxygen, protein undergoes site-specific cleavage by binding to the histidine residues of domain III, with the resultant formation of four fragments of molecular weight 49, 45, 22 and 17 kDa. This indicates the presence of two specific binding sites in the protein molecule. In the absence of molecular oxygen, the metal complex was found unable to cleave the protein. The circular dichroism (CD) spectrum of the isolated fragments shows nearly 38% and 32% of alpha helical content in 49 and 45 kDa fragments, respectively, which shows that the cleavage leads to no changes in the secondary structure of the protein fragments.

Origin of rate-acceleration in ester hydrolysis with metalloprotease mimics.

Mimics of carboxypeptidase A, a prototypical metalloprotease, were synthesized by linking macrocyclicpolyamines to the primary side of beta-cyclodextrin followed by complexing with Zn(II). These enzyme mimics exhibit saturation kinetics in hydrolysis of p-nitrophenyl acetate (PNPA) and enhance the rate of hydrolysis reaction by almost 300-fold. The effective molarities (EM) of the mimics range from 0.2 to 1.9 M. Origin of the rate acceleration was examined: the reactivity of Zn(II) complexes of [12]aneN3 [12]aneN4, and [14]aneN4 for hydrolyzing PNPA increases with increase in basicity of the zinc bound hydroxides [Zn(II)-OH], yielding a linear Brönsted plot. Free hydroxide fits well on this plot. A similar plot was obtained with the enzyme mimics. The Brönsted relationships indicate that the Zn(II)-OH in the catalytic systems hydrolyzes the ester by direct nucleophilic attack on the ester carbonyl of cyclodextrin-bound but not Zn(II)-coordinated PNPA.

Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

Functional molecules based on polyazometals. (1). Artificial metalloproteinases prepared by conjugation of polyazometals with poly(allylamine).

Polyazometals (PAMs) conjugated with poly(allylamine) manifested high catalytic activity in the hydrolytic cleavage of bovine serum albumin. Based on cleavage sites, the mechanism for the PAM-catalyzed peptide hydrolysis is suggested.

Vaccination of the Leishmania major susceptible BALB/c mouse. I. The precise selection of peptide determinant influences CD4+ T cell subset expression.

BALB/c mice are susceptible to cutaneous leishmaniasis upon infection with Leishmania major while C57BL/6 are not. There is a major promastigote surface protease (PSP or gp63) which is available in both native and recombinant forms, and for which the primary amino acid sequence is known. Immunization with PSP has been shown to offer some protection against challenge with the live organism. Therefore, we attempted to develop a peptide vaccine with PSP peptides. In the first experiments, recall proliferative responses to PSP were measured using a set of 15mer peptides spanning the entire PSP molecule which allowed designation of major determinant regions in BALB/c, C57BL/6, and CBA mice. Several of these determinants were promiscuous and shared almost the identical core amino acid residues in the different strains. Immunization with major determinant peptides was recalled vigorously with L. major soluble antigen as well as with PSP. The response to peptide was almost entirely Th1 as measured by a localized ELISA assay for single-cell production of IFN-gamma. A similar assay for IL-5, which overcomes problems of sensitivity and inhibition by lymphokines produced by Th1 cells, indicates very little production of Th2 cells even by BALB/c. It was found that if a major responsive peak was examined by recall with overlapping peptides, the highest, central peptide gave a mainly Th1 response while the boundary, less efficient peptides gave more of a Th2 response. Possible reasons for this were discussed. These results point to the importance of selecting the exactly appropriate peptide in considering a vaccinogen that might protect susceptible individuals. Even the choice of a somewhat immunogenic peptide within the determinant envelope might actually exacerbate infection by steering the response in a Th2 direction.