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ABSTRACT: SPC(3) is a multiple antigen peptide derived from the V(3) loop of human immunodeficiency virus (HIV) envelope (Env). It exerts a potent anti-HIV activity whereas it alters neither Env expression nor binding to CD(4). Here, SPC(3) binding characteristics, its subsequent intracellular fate and the fact that it inhibited SDF(1)alpha binding to the lymphocyte surface provided strong arguments to conclude that it exerts its anti-HIV activity through interference with the CXCR(4) coreceptor. In contrast, it interferes with none of the other major surface proteins and mechanisms involving V(3) and implicated in infection, as shown here. This work identifies the target mechanism of SPC(3).
FEMS Microbiology Letters 03/2000; 183(2):235-40. · 2.04 Impact Factor
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ABSTRACT: Aah VI was isolated from the venom of the North African scorpion, Androctonus australis hector. It is the first glycosylated neurotoxin from scorpion venom to be described. It was not toxic to mice, when injected intracerebroventricularly at a dose of 1.2 microg per animal. However, it had typical activity in Blatella germanica cockroaches resulting in gradual paralysis and very low toxicity (LD50 = 8.5 microg/g of animal). It consists of 66 amino acid residues and is heterogeneously N-glycosylated at a single site, on asparagine 9, of the Asn-Gly-Thr sequence. The potential N-glycosylation site was deduced from automatic Edman degradation and amino acid analysis, and glycan heterogeneity was evidenced by ESMS. Determination of the N-glycan structures (dHex, Hex and HexNAc) was assessed by nanoESMS/MS with picomolar amounts of sample. Current knowledge of N-glycan structure and composition suggests that the glycan structures are derived from a common core.
FEBS Letters 02/1999; 443(2):175-80. · 3.54 Impact Factor
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ABSTRACT: A multibranched peptide construct (SPC3) derived from the conserved sequence of the third variable domain (V3) of the human immunodeficiency virus (HIV) envelope (Env) inhibits HIV infectivity. It is being tested in phase II clinical trials (FDA protocol 257A). Because some Env-derived peptides inhibit HIV infectivity through alteration of Env biosynthetic pathway, we studied whether SPC3 displays its activity through interference with Env biosynthesis or with its functions at the membrane. Syncytium formation was impaired when human CD4+ cells expressed recombinant HIV Env in the presence of SPC3. This inhibition was not due to an effect of SPC3 on the amount of Env expressed at the cell membrane. As assessed using antibodies, the conformation of the receptor binding site and of V3 presented on membrane Env was not affected by the presence of SPC3 during biosynthesis. Finally, despite the ability of SPC3 to bind to CD4+ cell membrane, SPC3 did not interfere with Env binding to CD4. These data suggest that SPC3 interferes with the infection process at a post-CD4 binding step, and not with the folding of Env.
European Journal of Allergy and Clinical Immunology 11/1998; 52(4):283-8. · 1.30 Impact Factor
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ABSTRACT: alpha-Glucosidase inhibitors-e.g., 1-deoxynojirimycin (DNM)-interfere with HIV infectivity in CD4+ cell cultures but have proven unsuccessful in clinical trials. In vitro, several HIV Env properties, including the cleavage of the Env precursor gp 160, the immunoreactivity of the third variable domain (V3) of Env, the binding to the CD4 receptor, and the induction of the membrane fusion between the virus and the host cell, have been reported to be altered by such inhibitors. We have studied these properties for Env expressed via a recombinant vaccinia virus in two Chinese hamster ovary cell lines, an alpha-glucosidase I-deficient cell line and its parental cell line, treated with DNM under conditions that have been reported to alter Env properties. The glycosylation of Env, but not the quantity produced, varied in accordance with the experimental conditions. However, irrespective of these conditions, Env cleavage, V3 immunoreactivity, CD4 binding, membrane expression, and ability to induce syncytium formation were similar. Thus, neither the alpha-glucosidase I deficiency nor DNM treatment had a significant effect on the properties of Env produced here. Cellular mechanisms that may allow the normal expression of Env are discussed and may offer an explanation for the many discrepant results obtained to date on the effects of DNM on HIV Env.
Virology 05/1997; 231(1):89-95. · 3.35 Impact Factor
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ABSTRACT: Alkaline (pI 8.6-7.5) and neutral (pI 7.0-6.0) isoforms of human TSH have been isolated from a highly purified intrapituitary preparation by isoelectric focusing and compared for their respective actions on thyroid cell proliferation. Both TSH isoforms displayed the same ability to bind to porcine thyroid membranes as the original hormone preparation, indicating a similar recognition at the receptor sites. Alkaline forms showed a higher potency in inducing either cyclic AMP (cAMP) production or [3H]thymidine incorporation in FRTL-5 cells (half-maximal effective doses (ED50 values) = 0.25 and 0.29 nM respectively) compared with their neutral counterparts (ED50 values = 0.66 and 0.70 nM respectively). Increasing the concentration of alkaline forms in the presence of a half-maximal concentration of neutral TSH resulted in a profound inhibition of cell growth without a significant change in cAMP. Conversely, increasing the amount of neutral forms in the presence of a half-maximal dose of alkaline TSH resulted in an additive response for cAMP production but not in cell proliferation. To assess whether glycosylation might be responsible for the variation in hormone action, both alkaline and neutral TSH isoforms were tested for recognition of their carbohydrate chains by concanavalin A (Con A) and ricin. No major difference was found in binding to Con A, indicating that the contribution of carbohydrates to changes in hormone pI was not related to core branching. Very few galactose residues were accessible in either hormone fraction since little binding to ricin was observed. Isoelectric focusing of TSH forms before and after neuraminidase treatment revealed that neutral forms had a higher sialic acid content than alkaline TSH. In conclusion, the current findings show that TSH isoforms differentially affect cAMP production and cell growth. TSH fractions with a high sialic acid content and a low mitogenic activity behave as antagonists to the more active forms for cell proliferation. It is suggested that physiological control of TSH action at the thyroid gland may reside in the respective amounts of various TSH forms which, once bound to their receptor, can induce variable activation of post-receptor events while controlling cell proliferation.
Journal of Molecular Endocrinology 11/1994; 13(2):187-98. · 3.48 Impact Factor
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ABSTRACT: Enzymatic deglycosylation of human thyroid-stimulating hormone (hTSH) was shown to result in a mixture of partially and fully deglycosylated forms of the hormone by gel electrophoresis, silver staining and immunoblotting. Radioiodination of the enzymatic digest, followed by gel filtration and concanavalin A-Sepharose chromatography allowed to separate two different forms of partially deglycosylated [125I]hTSH and a fully deglycosylated hormone. The final recovery was of approx. 60% for [125I]hTSH deglycosylated in its beta-subunit, of 30% for [125I]hTSH missing the oligosaccharide in beta and one in alpha but only of 10% for [125I]hTSH deglycosylated in both the alpha- and beta-subunits. Gel electrophoresis under non-denaturing conditions showed that each form migrated distinctly from free subunits and reverse-phase high performance liquid chromatography after reduction and carboxymethylation identified the presence of the two subunits. Mapping of [125I]hTSH derivatives with polyclonal, monoclonal and anti-peptide antibodies allowed to identify two novel glycosylation-independent epitopes preserved in deglycosylated hTSH while the main immunogenic determinant was lost. When assayed in a bioassay with FRTL-5 cells, the hormone deprived of its beta-linked carbohydrate chain was found to be as effective as the native hormone on cAMP production and cell growth. In contrast, the fully deglycosylated derivative proved to stimulate cAMP release but appeared to be definitely less potent on thyroid cell growth. Our findings thus demonstrate that glycosylation of the alpha-subunit but not that of the beta-subunit is essential to express the domains involved in hTSH immunoreactivity as well as those controlling the post-receptor biological activity of the hormone.
Molecular and Cellular Endocrinology 07/1991; 78(1-2):137-50. · 4.19 Impact Factor
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ABSTRACT: The glycoprotein hormones are a family of four proteins: LH, FSH, TSH and CG. These molecules are glycosylated dimers, sharing a common alpha-subunit and differing by their beta-subunit which confers to the hormone its immunological and biological specificity. The biological function of these hormones is mediated through the recognition of specific receptors at the target organ. Although still controversial, it appears that both subunits of the hormone are required to bind to the receptor and induce cAMP release. Furthermore, these hormones exhibit natural variability in their bioactivity and the molecular basis of this process are poorly understood at the moment. Recent data relative to the mapping of glycoprotein hormones, were obtained by site-directed mutagenesis as well as by the use of synthetic peptides. These two approaches allowed to elucidate several linear peptide sequences involved in the biologically active conformation and immunoreactivity of these molecules. Furthermore, these hormones exist in different molecular forms with a variable biological activity and immunological ratio, and this polymorphism is probably due to the glycan moities. The presence of these glycans are necessary for full expression of their biological activity as well as immunoreactivity, and both the biosynthesis and the secretion of these various glycoforms are probably under physiological regulation. We therefore propose that glycosylation may alter the expression of several domains at the surface of the hormone to modulate its plasmatic clearance as well as the action of each individual glycoform at the receptor and this will ultimately control its biological function.
Annales d Endocrinologie 02/1991; 52(4):254-68. · 0.74 Impact Factor
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International Journal of Radiation Applications and Instrumentation Part B Nuclear Medicine and Biology 02/1990; 17(7):651-6.
