An apolipoprotein E4 fragment affects matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1 and cytokine levels in brain cell lines.

Apolipoprotein (apo) E4 isoform, a major risk factor for Alzheimer disease (AD), is more susceptible to proteolysis than apoE2 and apoE3 isoforms. ApoE4 fragments have been found in AD patients' brain. In the present study, we examined the effect of full-length apoE4 and apoE4 fragments apoE4[Δ(186-299)] and apoE4[Δ(166-299)] on inflammation in human neuroblastoma SK-N-SH and human astrocytoma SW-1783 cells. Western blot and zymography analysis showed that treatment of SK-N-SH cells with apoE4[Δ(186-299)], but not full-length apoE4 or the shorter apoE4[Δ(166-299)] fragment, leads to increased extracellular levels of matrix metalloproteinase 9 (MMP9) and tissue inhibitor of metalloproteinase 1 (TIMP1). Real-time PCR showed that interleukin (IL)-1ß gene expression is also increased in SK-N-SH cells treated with apoE4[Δ(186-299)]. Treatment of SK-N-SH cells with IL-1ß leads to increased MMP9 and TIMP1 extracellular levels, suggesting that the induction of IL-1ß may be the mechanism by which apoE4[Δ(186-299)] regulates MMP9 and TIMP1 levels in these cells. In contrast to SK-N-SH cells, treatment of SW-1783 cells with apoE4[Δ(186-299)], and to a lesser extent with apoE4, leads to increased TIMP1 extracellular levels without affecting MMP9 levels. Additionally, apoE4[Δ(186-299)] leads to decreased IL-10 gene expression in SK-N-SH cells, whereas both apoE4 and apoE4[Δ(186-299)] lead to decreased TNFα gene expression without affecting IL-1ß and IL-10 gene expression in SW-1783 cells. Overall, our findings indicate that a specific apoE4 fragment (apoE4[Δ(186-299)]), with molecular mass similar that of apoE4 fragments detected in AD patients' brain, can influence the level of inflammatory molecules in brain cell lines. It is possible that these phenomena contribute to AD pathogenesis.

Solution structures of the fibronectin-like Leishmania gp63 SRYD-containing sequence in the free and antibody-bound states--transferred NOE and molecular dynamics studies.

The anti-SRYD monoclonal antibody (mAbSRYD) raised against the IASRYDQL synthetic octapeptide, the 250-257 sequence of the Leishmania major surface glycoprotein gp63 recognizes both SRYD-containing peptides and the whole cognate major surface protein on intact parasites. Two SRYD-containing peptides, which antigenically and functionally mimic the RGDS sequence of fibronectin and efficiently inhibit parasite attachment to the macrophage receptors, were studied by two-dimensional transferred nuclear Overhauser effect experiments in the presence of mAbSRYD. The antibody-bound IASRYDQL octapeptide solution conformation was determined on the basis of 55 interproton-distance restraints, derived from NMR measurements. Eighteen structures which were first generated using an approach combining distance geometry and molecular dynamics, converge by energy minimization toward a folded structure with an average rmsd from the experimental data of less than 0.05 nm for the overall backbone and 0.025 nm for the SRYD motif. A distorted gamma-turn was found, stabilized by the backbone-backbone D255-NH to R253-CO hydrogen bond, while the R253 and D255 side chains are pointing in opposite directions. This latter antibody-bound structure is compared with that of the free octapeptide in dimethylsulfoxide solution, and with the crystal structure of the RYD fragment in OPG2 Fab, an antireceptor antibody that mimics the RGD cell adhesion site. On this basis, a mechanism for IASRYDQL-receptor interaction is discussed.

Use of sequential oligopeptide carriers (SOCn) in the design of potent Leishmania gp63 immunogenic peptides.

The antigenic sequence Ac-IASRYDQL (gp63-SRYD) of the major surface glycoprotein of Leishmania, gp63, was covalently attached to the Lys-N epsilon H2 groups of a new sequential oligopeptide carrier (SOCn), namely, (Lys-Aib-Gly)n (n = 5.6), in order to obtain potent immunogens and site-specific antibodies. It was shown, using 1H-NMR spectroscopy, that the gp63-SRYD octapeptides bound to the SOCn retain their original structural profile outlined by an ionic interaction between R and D side chains and a type 1 beta-turn involving the QNH-->RCO hydrogen bonding. Also, the gp63-SRYD octapeptides linked to the carrier do not experience conformational restrictions, probably because of the favorable conformation of the SOCn. Immunizations of outbred rabbits with the peptide carriers designed resulted in high-titered antibody response to the gp63-SRYD octapeptide and the gp63 cognate protein. Thus, this chemically defined model may be used for incorporating "protective" Leishmania epitopes and ultimately for the design of a multivalent synthetic vaccine against leishmaniosis.

Antigenicity and conformational analysis of the Zn(2+)-binding sites of two Zn(2+)-metalloproteases: Leishmania gp63 and mammalian endopeptidase-24.11.

The antigenic properties of the Zn(2+)-binding region of two Zn(2+)-metalloproteases, Leishmania surface protease gp63 and mammalian endopeptidase-24.11 (E-24.11), possessing in their active site the characteristic amino acid sequence HEXXH, were investigated by using oligoclonal antibodies raised against two synthetic peptides, V1VTHEMAHALG11 (pepgp63) and V1IGHEITHGFD11 (pepE-24.11), containing the respective Zn(2+)-binding sites of the cognate protein. The affinity-purified antibodies, tested on synthetic peptides modelled from the active sites of ten different Zn(2+)-metalloproteases, showed high selectivity for their respective peptides. However, cross-reactivity was revealed when the antibodies were tested against the gp63 and E-24.11 molecules. A panel of synthetic peptide analogues and peptides of various size was synthesized and used for the fine antigenic characterization of pepgp63 and pepE-24.11. The shortest peptides capable of significant antibody binding were the pentapeptides V1VTHE5 and E5ITHG9 for pepgp63 and pepE-24.11 respectively. His4 and Glu5 were found to be indispensable for anti-pepgp63 binding to pepgp63, whereas in the case of pepE-24.11, Glu5 and His8 were found to be critical. The conformational characteristics of the two peptides correlate well with the observed differences in their antigenicity. 1H-NMR studies showed that pepgp63 adopts a folded structure whereas pepE-24.11 takes up a rather flexible conformation. Moreover, the antigenically critical His4 of pepgp63 contributes to the structural stabilization of the peptide. Similarly, the antigenically critical His8 of pepE-24.11 is involved in partial structural stabilization of its C-terminal region. The generated antibodies may be useful tools for identifying and classifying proteins possessing similar Zn(2+)-binding motifs and/or environments.

Expression of the major surface glycoprotein of Leishmania, gp63, in wild-type and sinefungin-resistant promastigotes.

In this study, we have surveyed gp63 expression in sinefungin-(SF)-resistant and wild-type Leishmania promastigotes. Documentation of gp63 expression in Leishmania promastigotes was carried out by Western blotting, purification of the protein and assessment of gp63 protease activity. We demonstrated a 3-4-fold and 1.5-2-fold increase of gp63 protein in SF-resistant Leishmania donovani and Leishmania tropica promastigotes compared to wild-type, respectively. Northern blot analysis showed that the increase in the amount of gp63 protein in SF-resistant compared to wild-type parasites was concomitant with an increase in gp63 mRNA. No extrachromosomal DNA was identified by alkaline lysis of isolated DNA samples and Southern blot analysis. Treatment of SF-resistant and wild-type L. donovani promastigotes with cycloheximide resulted in an increase of the steady state levels of gp63 mRNA in the SF-resistant parasites to approximately fivefold that of the wild type. After treating parasites with actinomycin D, estimated gp63 mRNA t1/2 in the wild type was 40 min and increased to 83 min in SF-resistant promastigotes. Therefore, the overexpression of gp63 may be mediated, at least in part, by post-transcriptional stabilization of a gp63 transcript by a protein factor. Down regulation of the latter factor may account for the observed increase in gp63 expression in SF-resistant promastigotes. Attempts to correlate gp63 expression with promastigote virulence suggested that the observed increase in gp63 expression did not result in a significant change in the virulence of SF-resistant compared to wild-type L. donovani promastigotes.

The Ser-Arg-Tyr-Asp region of the major surface glycoprotein of Leishmania mimics the Arg-Gly-Asp-Ser cell attachment region of fibronectin.

The major surface glycoprotein of Leishmania, gp63, a fibronectin-like molecule, plays a key role in parasite-macrophage interaction. Binding of gp63 to macrophage receptors is inhibited by Arg-Gly-Asp-Ser (RGDS)-containing synthetic peptides of fibronectin and by antibodies to these peptides. However, gp63 lacks an RGDS tetrapeptide. We sought to identify the region of gp63 that antigenically and functionally mimics the RGDS-containing region of fibronectin. We thus synthesized on polyethylene rods overlapping tetracosapeptides covering the whole sequence of Leishmania major gp63. gp63 affinity-purified antibodies raised against fibronectin and against the RGDS-containing fibronectin decapeptide RGDSPASSKP bound specifically to gp63 residues 241-264. Subsequently, by the use of smaller peptides, the gp63 tetrapeptide 252-255 (SRYD) was identified as the minimum antibody binding segment. Single residue substitution peptide analogues showed that indeed Tyr and Gly can be alternatively substituted in the SRYD- and RGDS-containing peptides of gp63 and fibronectin, respectively, without major effects on their antibody binding capacity. Subsequently, we investigated the effect of an SRYD peptide on promastigote-macrophage interaction in vitro; treatment of macrophages with an SRYD-containing gp63 octapeptide efficiently inhibited parasite attachment to macrophage receptors. Thus, the conserved among species sequence SRYD of gp63, with significant hydrophilicity, flexibility, and beta-turn propensity features, mimics antigenically and functionally the RGDS sequence of fibronectin. We suggest that this segment constitutes the putative gp63 adhesion site.

Substrate-dependent pH optima of gp63 purified from seven strains of Leishmania.

The major surface glycoprotein of Leishmania, gp63, is a membrane-bound metalloprotease. Contradictory data supporting a neutral or acidic nature of this enzyme have been presented. Seven strains of Old and New World Leishmania, including Leishmania donovani complex (Leishmania infantum and L. donovani), Leishmania major, Leishmania tropica and Leishmania mexicana amazonensis were used for the purification and comparative study of gp63. The protein was extracted from promastigotes by phase separation in Triton X-114 and purified by anion exchange chromatography. In agreement with previous reports, all purified gp63 were found to be structurally and immunologically related. Both membrane-bound gp63, on the surface of promastigotes, and the purified proteases had optimal activity at neutral to alkaline pH on azocasein, whereas their activity was optimal at acidic to neutral pH against 125I-insulin B-chain. The IC50 concentrations of 1,10-phenathroline against the two substrates, at the optimal pH, were comparable, suggesting that both activities measured were associated with gp63 rather than another contaminating enzyme. This was further supported by the comparable enrichment values, estimated from the specific activity of the enzyme during purification, using both assays. These results explain the earlier apparent discrepancies and suggest that the optimum pH of gp63 is substrate-dependent and not related to species differences or to the different purification procedures applied.

Identification of monomeric and oligomeric forms of a major Leishmania infantum antigen by using monoclonal antibodies.

Ten monoclonal antibodies (MAbs) produced against isolated Leishmania infantum membranes were used as probes of L. infantum membrane antigens. Western blots of L. infantum membranes, sodium dodecyl sulfate solubilized and heated at 100 degrees C before analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed that all 10 MAbs recognized a band at 58 kilodaltons (kDa). However, when solubilized membranes were not heated, 2 of the 10 MAbs recognized, in addition to the 58-kDa band, bands of higher molecular weight. Limited digestion of heated or nonheated membranes showed that both groups of MAbs (i.e., not capable or capable of binding to the high-molecular-weight bands) recognized the same proteolytic digests. Hydrophilic forms of the above proteins, possessing proteolytic activity, were detected and isolated by gel filtration. Protein staining of the isolated monomer analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under reducing and heating conditions, revealed incomplete reduction of the 58-kDa protein. The reduced form of the 58-kDa protein migrated at 63 to 65 kDa and was not recognized by the MAbs. These results suggest the existence of a monomeric and an oligomeric form of the 58-kDa antigen. The observed inhibition of Leishmania promastigote-macrophage binding caused by MAbs representative of the two groups (capable of oligomeric and/or monomeric antigen recognition) suggest that the 58-kDa monomer and oligomer play an important role in promastigote-macrophage interaction. We suggest that the 58-kDa L. infantum antigen is the major surface Leishmania antigen (p63) identified by others.