Jurphaas Van Rietschoten

Enzymatic N-glycosylation of synthetic ASN-X-THR containing peptides

The role of polyprenol sugar derivatives in the biosynthesis of certain eukaryotic glycoproteins has been partly described [ 11. Recently lipid pyrophospho-oligosaccharides, postulated as necessary intermediates to initiate N-glycosylation, were shown, when supplied to microsomes, to be effective donors to proteinic acceptors presenting at least one vacant Asn-X-zk sequence: unfolded forms of proteins [Z], polypeptide fragments from CNBr cleavages [3] or a synthetic heptapeptide [4]. It was proposed [S] on the grounds of structural examination of numerous glycoproteins, that this basic tripeptide sequence was a necessary, although probably not sufficient, requirement for an asparagine residue to be N-glycosylated. The data we report here extend our work [4] showing that thyroid rough microsomes catalyze the transfer of oligosaccharide from [Mrfa-r4C] oligosaccliaride-lipids to a synthetic human thyroglobulin heptapeptide and further to its d~itropl~enylated derivative. Our aim has been to further investigate specificity requirements of the oligosaccharide transferase, using as tools the following series of synthetic peptides: (I) H-Ala-Leu-Glu-Asn-Ala-Thr-Arg-NH, _~_._ (II) DNP-Ala-Leu-GluAsn-Ala-Thr-Arg-NH, (III) H Asn-Ala-Thr-NH, (IV) DNPAsn-Ala-Thr-NH, (V) DNP-Ala-Let.-Glu Asn-Pro-Thr-Arg-NH, __~_._ (VI) H-Tyr-Gln-SerAsn-Ser-~r-~et-NH~ ~--

Cobatoxin 1 from Centruroides noxius scorpion venom: chemical synthesis, three-dimensional structure in solution, pharmacology and docking on K+ channels

CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common alpha/beta scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 microg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat K(v)1.2 ( K(d) value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 microM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat K(v)1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K(v) channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.

Use of parvalbumin as a protecting protein in the sequenator: An easy and efficient way for sequencing small amounts of peptides

Since the description of the first automated apparatus for degradation of proteins by the phenylisothiocyanate method [l] it became obvious that the procedures worked out by Edman and Begg and later by Hermodson et al. [2] were not directly applicable to small and hydrophobic peptides. It has been shown that this was due to losses of the residual peptide during the extraction steps with organic solvents [ 1,3-61. Several alternative approaches have been developed to overcome the problem. One of these is to render the peptide suitable for the usual procedures by coupling polar groups either to lysine [4 ] and S-@-aminoethyl) cysteine residues [7] or to the C-terminal amino acid [.5,8]. A second approach is to adapt the procedure to the peptide either by decreasing the concentration of quadrol [6,8,9] or by using a volatile buffer [8] which, in both cases, limits the number and the volumes of organic solvents used for extraction. A third possibility is to attach the peptide to an insoluble resin and perform the degradation in an heterogeneous phase [lo] with the help of a specially designed apparatus [ 111. We wish to report the results we obtained in sequencing unmodified peptides in very small amounts (down to 20 nmol) in an Edman and Begg type sequenator using dimethylbenzylamine as coupling buffer but in the presence of a naturally aNHz-blocked protein (hake major parvalbumin) that acts as a protecting film. The technique proved also to be very efficient for the degradation of peptides attached to polystyrene resins: it prevents the loss

Lethal neurotoxicity in mice of the basic domains of HIV and SIV Rev proteins Study of these regions by circular dichroism

We have recently reported a basic domain‐mediated neurotoxic activity of HIV‐1 Tat [1991, J. Virol. 65, 961–965]. Here we have tested the neurotoxicity in vivo of several Rev‐related synthetic peptides and found that only those mimicking the basic regions of Rev from HIV‐1, HIV‐2 and SIV were lethal to mice. In contrast, the homologous domain of HTLV‐1 Rex was found to be inactive for lethal activity. Analysis of the tropism of these peptides for phospholipids has demonstrated a direct interaction of the basic domain‐containing peptides, except Rex, with acidic — but not neutral — phospholipids. As determined by circular dichroism, a possible correlation between the conformation of the basic regions and the toxicity is discussed.

Cytotoxic effect on lymphocytes of Tat from human immunodeficiency virus (HIV-1)

The human immunodeficiency virus type 1 (HIV‐1) genome codes for trans‐activator Tat, an 86‐residue protein whose expression is critical for viral replication. Full‐length Tat and Tat peptides from HIV‐1 were chemically synthesized using optimized solid phase technique. Synthetic Tat 2 in86, was found not only to inhibit antigen‐induced human peripheral blood lymphocyte (PBL) proliferation in vitro, as described by Viscidi et al. [1989, Science 246, 1606‐1608], but also mitogen‐induced PBL proliferation, with 50% inhibition obtained at 0.9 and 8 μ;M, respectively. To assess the mechanism by which Tat exert its inhibitory effect, we analysed its interaction and effect on CD4+‐cells. Direct fluorescence and indirect immunofluorescence assays analysed by flow cytometry showed that fluorescein isothiocyanate‐labeled and ‐unlabeled Tat interact (>0.2 μ;M) with CD4‐expressing lymphoid cells (CEM cell line). Experiments of chromium‐51 release and Trypan blue exclusion on these tumor cells in vitro have demonstrated the capacity of Tat to modify cellular membrane permeability and cell viability, in a dose‐dependent manner. The use of Tat peptides revealed that those containing the Tat basic region from 49 to 57 were able to bind to the cell membrane and to exhibit a cytotoxic activity on lymphocytes. Together, the data suggest that the potential cytotoxicity of Tat on lymphocytes could be directly implicated in virus‐induced immune dysfunction observed in HIV‐1 infected patients.

Liposomal encapsulation enhances antiviral efficacy of SPC3 against human immunodeficiency virus type-1 infection in human lymphocytes

Because encapsulation of antiviral drugs in liposomes resulted generally in improved activity against retroviral replication in vivo, the antiviral effects of free-SPC3 and liposome-associated SPC3 were compared in cultured human lymphocytes infected with HIV-1. SPC3 was entrapped in various liposomal formulations, either different in size (mean diameter of 100 and 250 nm), SPC3 concentration or cholesterol content. Liposome-associated SPC3 were tested for both inhibition of cell-cell fusion and infection with HIV-1 clones. SPC3 inhibited HIV-1-induced fusion at a micromolar concentration range. When associated with liposomes, SPC3 was found to be about 10-fold more potent than free SPC3 in inhibiting syncytium formation. Continuous treatment with free SPC3 also inhibited virus production in a dose-dependent manner, with inhibition of HIV infection of C8166 T-cells or human peripheral blood lymphocytes (PBLs) at micromolar concentrations. Liposomal entrapment was found to increase the antiviral efficacy of SPC3 by more than 10- and 5-fold in C8166 and PBLs, respectively. These data suggest that the liposome approach may be used to improve SPC3 antiviral efficacy.

Dipole moments of scorpion toxins direct the interaction towards small- or large-conductance Ca2+-activated K+ channels

Ca2+-activated K+ channels consist of a large family of membrane proteins, among which two groups have been characterized by electrophysiological criteria, the small conductance (SK) and the large conductance (BK) Ca2+-activated K+ channels. Scorpion toxins that block K+ channels exhibit a common three-dimensional structure constituted of a short α-helix connected by disulfide bonds to a β-sheet. The leiurotoxin I (LTX1) related toxins interact specifically with the SK channel via basic residues of their α-helix, while the charybdotoxin (ChTX) family recognizes the BK channel with basic residues of their β-sheet. In an attempt to better understand the structure-activity relationships of these toxins and the characteristics of the electrostatic interactions with the receptor site, we investigated the electrostatic potential supported by natural toxins and a synthetic analogue to find out if it may help in understanding the molecular mechanisms involved in this peptide-protein interaction.

V3 loop-derived multibranched peptides as inhibitors of HIV infection in CD4+ and CD4− cells

The V3 loop is one of the variable domains of the HIV-1 surface envelope glycoprotein gp120 that mainly generates isolate-specific neutralizing antibodies. These anti-V3 antibodies presumably interfere with the function of the V3 loop that is thought to be involved in virus-cell fusion. In an approach to neutralize HIV infection, monomeric linear or cyclic V3 loop-related peptides have experimentally been used to impair the fusion process. The reported results are contradictory, including peptide-induced enhancement of viral infectivity. In order to develop a new strategy, synthetic polymeric constructions (SPCs) including the V3 loop consensus sequence of HIV-1 North American/European isolates (i.e. GPGRAF) have been synthesized and tested for antiviral activity. The rationale for using SPCs instead of monomeric peptides was a presumed enhancement of ligand avidity due to multivalence. Among the SPCs tested, SPC3 (eight GPGRAF motifs radially branched on an uncharged polylysine core matrix) was found to inhibit the infection of human CD4+ lymphocytes and macrophages, as well as CD4−/galactosylceramide (GalCer)-expressing epithelial cells by distantly related laboratory strains and clinical isolates of HIV-1 and HIV-2. SPC3 affected HIV-1 infection by two distinct mechanisms, depending on the cell-type: (i) postbinding inhibition of HIV-1 entry into CD4+ lymphocytes; and (ii) prevention of GalCer-mediated HIV-1 attachment to the surface of CD4−/GalCer+ cells. SPC3 may therefore represent the first of a novel class of anti-HIV therapeutic agents able to neutralize a wide range of HIV isolates in both CD4+ and CD4− susceptible cells. The antiviral properties of this peptide are currently being evaluated in HIV-1-infected patients (phase II clinical trials).

SPC3, a nontoxic peptide inhibitor of HIV infection.

Dear Editor: The third variable region of the type 1 human immunodeficiency virus (HIV-1) surface glycoprotein gpl20 (V3 loop) is essential for virus infectivity and tropism (5). This domain seems to be involved in the postbinding events necessary for viral entry into the cells (4). Several attempts have been made to inhibit HIV infection by V3 loop-related peptides. The results have been various and contradictory. For instance, De Rossi et al. (1) found that some V3 peptides enhanced viral infectivity, while Nehete et al. (6), using other V3 peptides, found an inhibition of HIV-1 infection in lymphocytes. Recently, we reported that an octameric form of the HIV-1 V3 loop consensus motif GPGRAF ([GPGRAF]s-(K)4-(K)2-K-I~A , i.e. SPC3),

Specificity of anti-P25 antibodies produced against whole HIV-1 particles or soluble forms of the protein

Specificity of anti-p25 antibodies produced against either whole Human Immunodeficiency Virus type 1 (HIV-1) particles in humans and chimpanzees, or against soluble forms of the protein in chimpanzees and rabbits was analyzed by ELISA using a panel of 37 long (> or = 30 residues) or shorter (9-21 residues) overlapping peptides covering the entire p25 sequence. Antibodies elicited by intact virions presented similar reactivity patterns in HIV-1-infected humans and in HIV-1-infected or immunized chimpanzees and recognized only a limited region mostly the C-terminus of the molecule. Moreover, 8 of the human sera (36%), which nonetheless reacted with high titers and avidity with native p25, did not bind to any long or short peptide. These results suggest that the majority of antibodies elicited by viral particles are presumably directed to conformational epitopes. In contrast, antibodies raised against soluble forms of p25 could react against all long peptides but one (residues 211-245) and against some short peptides, indicating that most of p25 sequence may be immunogenic under these conditions. These results suggest that the reactivity spectrum of anti p25 antibodies is rather different if they are produced against intact HIV-1 particles or the soluble protein. They also indicate that it may be possible to manipulate the specificity of the humoral immune response by using either intact virions or purified proteins.