cDNA cloning and sequence analysis of the Xenopus laevis egg envelope glycoprotein gp43

The glycoproteins of the Xenopus laevis egg envelope function in fertilization and development. As the unfertilizable coelomic egg transits the pars recta region of the oviduct, it is converted to a fertilizable egg by limited proteolysis of the envelope glycoprotein gp43 to gp41. This conversion is caused by an oviductally secreted serine active site protease, oviductin. We cloned a cDNA for gp43 from an oocyte cDNA library. The cDNA encoded a 454 amino acid protein homologous to the ZPC family of glycoproteins previously shown to be present in mammalian and fish egg envelopes. Conserved ZPC domains and motifs present in the Xenopus sequence included a signal peptide sequence, an N-linked glycosylation site, and 12 aligned Cys residues. In mammalian and Xenopus sequences, a furin-like (convertase) site and a C-terminal transmembrane domain were present reflecting the biosynthesis of ZPC in these species via the secretory glycoprotein pathway. However, fish envelope glycoproteins lack these sequences since they are synthesized via a different route (in the liver, transported to the ovary, and assembled into the egg envelope surrounding the oocyte). Consensus amino acid residues were identified by sequence comparisons of seven ZPC family members; 19% of the amino acid residues were invariant and 48% of the residues were identical in at least four of the seven sequences. The consensus sequence was used to make structure-fertilization function predictions for this phylogenetically conserved family of glycoproteins.     

A hatching enzyme substrate in the Xenopus laevis egg envelope is a high molecular weight ZPA homolog

The Xenopus laevis egg envelope is composed of six or more glycoproteins, three of which have been cloned and identified as the mammalian homologs ZPA (ZP2), ZPB (ZP1) and ZPC (ZP3). The remaining glycoproteins are a triplet of high molecular weight components that are selectively hydrolyzed by the hatching enzyme. We have isolated one of these proteins and cloned its cDNA. The mRNA for the protein was found to be expressed only in early stage oocytes, as are other envelope components. From the deduced amino acid sequence, it was indicated to be a secreted glycoprotein with a characteristic ZP domain in the C-terminal half of the molecule. The N-terminal half was unrelated to any known glycoprotein. Comparative sequence analysis of the ZP domain indicated that it was derived from an ancestor of ZPA and ZPB, with the greatest identity to ZPA. This envelope component has been designated ZPAX.     

Primary structure and developmental expression of Dp ZP2, a vitelline envelope glycoprotein homolog of mouse ZP2, in Discoglossus pictus, one of the oldest living Anuran species

A glycoprotein of the Xenopus vitelline envelope, gp 69/64, which mediates sperm binding, is closely related to the components of ZPA family, such as the mouse zona pellucida ZP2. To test the generality of these findings, we studied Discoglossus pictus, a species evolutionary distant from Xenopus and identified as a protein of 63 kDa in the vitelline envelope. Preliminary studies suggest that this protein may bind sperm at fertilization. We found that the 63-kDa protein is glycosylated and contains both N- and O-linked chains. We have cloned the cDNA encoding the Discoglossus protein of 63 kDa (Dp ZP2) by screening a Discoglossus cDNA library using Xenopus gp 69/64 cDNA as a probe. Analysis of the deduced sequence of Discoglossus protein revealed 48% identity with Xenopus gp 69/64 and 37-40% identity with mouse ZP2. The sequence conservation included a ZP domain, a potential furin cleavage site and a putative transmembrane domain. The N-terminus region of Dp ZP2 was 40% identical to the corresponding region of Xenopus gp 69/64 which has been shown to be essential for sperm binding to the VE. Although, as of yet, there is no evidence for sperm binding at the Dp ZP2 N-terminus, it is interesting that in this region three potential O-glycosylation sites are conserved in both species, in contrast to N-glycosylation sites. It was found that the Dp ZP2 mRNA is expressed in stage 1 oocytes and in the follicle cells surrounding the oocyte. Similarly, in Xenopus oocytes, the gp 69/64m RNA, was found in the oocytes, as well as in the somatic cells. Mol. Reprod. Dev. 59:133-143, 2001.     

Identification of membrane-active regions of the HIV-1 envelope glycoprotein gp41 using a 15-mer gp41-peptide scan

The identification of membrane-active regions of the ectodomain of the HIV-1 envelope glycoprotein gp41 has been made by determining the effect on membrane integrity of a 15-mer gp41-derived peptide library. By monitoring the effect of this peptide library on membrane leakage, we have identified three regions on the gp41 ectodomain with membrane-interacting capabilities: Region 1, which would roughly correspond to the polar sequence which follows the fusion domain and extends to the N-terminal heptad repeat region; Region 2, which would correspond to the immunodominant loop; and Region 3, which would correspond to the pre-transmembrane region of gp41. The identification of these three regions supports their direct role in membrane fusion as well as facilitating the future development of HIV-1 entry inhibitors.     

The interaction of a glycosaminoglycan heparin,with HIV-1 major envelope glycoprotein

We demonstrate in vitro the occurence of a specific but low-affinity interaction between soluble tetrameric rgp160 or soluble monomeric or tetrameric rgp120 and heparin-agarose (HA). This interaction is saturable, pH and temperature-dependent, and can be inhibited by soluble heparin, but not by soluble dextran. In buffer supplemented with 10 mM CaCl₂, the C₅₀ of soluble heparin, i.e., the concentration of soluble heparin which leads to 50% inhibition of the binding of [¹²⁵I]rgp160 or [¹²⁵I]rgp120 to HA, is 1.1. · 10^?4 disaccharidic molar concentration for rgp160 and 3.2 · 10^?4 disaccharidic molar concentration for rgp120, which indicates low-affinity interactions. Upon chromatography on HA, [¹²⁵I]rgp160 is repeatedly eluted as a retarded fraction when compared to the elutions volume of [¹²⁵I]rgp160-soluble heparin complex. Under the same experimental conditions, [¹²⁵I]rgp120 is also eluted, but as a less retarded fraction than [¹²⁵I]rgp160. Taken together, these results suggest that, at least part of the described anti HIV-1 activity of heparin might be mediated by interaction with HIV-1 major envelope glycoprotein.     

Identification and characterization of a unique Xenopus laevis egg envelope component,ZPD

We report the identification of a previously undetected Xenopus laevis egg envelope component discovered through cloning experiments. A cDNA sequence was found that represented a mature protein of 32 kDa. Peptide antibodies were generated to probe for the protein in egg envelope samples and reactivity was found to a glycoprotein of approximately 80 kDa. When deglycosylated egg envelope samples were probed, a 32 kDa protein was labeled, confirming the size of the translated cDNA sequence. A BLAST analysis showed that it is most closely related (34% amino acid identity) to the ZP domains of mammalian tectorin, uromodulin and ZPA. From a dendrogram of known egg envelope glycoproteins, the new glycoprotein was shown to be unique among egg envelope components and was designated ZPD. A similar glycoprotein was identified by immunocrossreactivity in Xenopus tropicalis and Xenopus borealis egg envelopes.     

Concentration of Ran on chromatin induces decondensation,nuclear envelope formation and nuclear pore complex assembly

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.     

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MYCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC₅₀ < 0.7 μM). MTCPP bound to the HIV-1 enveloope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to HIV-1 envelop glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anit-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.     

Discovery of novel anti-HIV-1 agents based on a broadly neutralizing antibody against the envelope gp120 V3 loop: a computational study

A computer-aided search for novel anti-HIV-1 agents able to mimic the pharmacophore properties of broadly neutralizing antibody (bNAb) 3074 was carried out based on the analysis of X-ray complexes of this antibody Fab with the MN, UR29, and VI191 peptides from the V3 loop of the HIV envelope protein gp120. Using these empirical data, peptidomimetic candidates of bNAb 3074 were identified by a public, web-oriented virtual screening platform (pepMMsMIMIC) and models of these candidates bound to the above V3 peptides were generated by molecular docking. The docking calculations identified four molecules exhibiting a high affinity to all of the V3 peptides. These molecules were selected as the most probable peptidomimetics of bNAb 3074. Finally, the stability of the complexes of these molecules with the MN, UR29, and VI191 V3 peptides was estimated by molecular dynamics and free energy simulations. Specific binding to the V3 loop was accomplished primarily by π–π interactions between the aromatic rings of the peptidomimetics and the conserved Phe-20 and/or Tyr-21 of the V3 immunogenic crown. In a mechanism similar to that of bNAb 3074, these compounds were found to block the tip of the V3 loop forming its invariant structural motif that contains residues critical for cell tropism. Based on these findings, the compounds selected are considered as promising basic structures for the rational design of novel, potent, and broad-spectrum anti-HIV-1 therapeutics.     

人星形细胞HIV—1膜蛋白gp120新受体的免疫学鉴定

自1994年首次报导HIV－1外膜蛋白gp120与人胎儿星形细胞膜蛋白质位点（推测分子量为260kD,命名为PAG）结合以来[1],这项工作持续集中于研究该蛋白的功能与作用,本研究藉助杂交瘤技术建立了一系列不鼠抗人星形细胞PAG）的单克隆抗体,这些抗体成功的抑制了gp120与PAG的结合,抑制可达50％以上,并几乎完全阻断了gp120介导的由摄入所星表细胞风钙离子的升高,通过ELISA可证实抗体与星形细胞的特异反应,蛋白印迹和免疫沉淀试验结果表明PAG作为实体的存在,试验表明PAG对于gp120与星形细胞的结合,对于gp120个导的星形细胞摄入所致钙离子的升高均有决定性作用,许多方向报导和研究表明gp120可与多种细胞结合而导致HIV－1感染,由此推论PAG是HIV－1gp120在人星形细胞上的新受体,同时也可能对HIV－1脑病的治疗开辟一条崭新的途径,PAG是否为HIV－1感染人星形细胞的受体,仍有等进一步实验证明。     

Characterization and HIV-1 fusion inhibitory properties of monoclonal Fabs obtained from a human non-immune phage library selected against diverse epitopes of the ectodomain of HIV-1 gp41

Using a human non-immune phage library comprising more than 10(9) functional human antibody specificities in Fab format, we have been able to select a set of eight monoclonal Fabs targeted against diverse epitopes of the ectodomain of gp41 from HIV-1. The antigens used for panning the antibodies comprised two soluble, disulfide-linked, trimeric polypeptides derived from gp41, N(CCG)-gp41 and N35(CCG)-N13. The former comprises an exposed trimeric coiled-coil of the N-helices of gp41 fused in helical phase to the minimal thermostable ectodomain of gp41, while the latter comprises only the trimeric coiled-coil of N-helices. The selected Fabs were probed by Western blot analysis against four antigens: N(CCG)-gp41, N35CCG-N13, N34CCG (a smaller version of N35CCG-N13), and the minimal thermostable ectodomain core of gp41 in its six-helix bundle conformation (6-HB). Three classes of Fabs were found: class A (two Fabs) interact predominantly with the 6-HB; class B (four Fabs) interact with both the 6-HB and the internal trimeric coiled-coil of N-helices; and class C (two Fabs) interact specifically with the internal trimeric coiled-coil of N-helices. The IC50 values for the Fabs, expressed as bivalent mini-antibodies, ranged from 6 microg/ml to 60 microg/ml in a quantitative vaccinia virus-based reporter gene assay for HIV-1 envelope-mediated cell fusion using the envelope from the HIV-1 T tropic strain LAV. The two most potent fusion inhibitors belonged to class B. This panel of Fabs provides a set of useful probes for studying HIV-1 envelope-mediated cell fusion and may serve as a basis for developing Fab-based anti-HIV-1 therapeutics.     

Exploitation of the HIV-1 coat glycoprotein, gp120, in neurodegenerative studies in vivo

Neuronal loss has often been described at post-mortem in the brain neocortex of patients suffering from AIDS. Neuroinvasive strains of HIV infect macrophages, microglial cells and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products that, in conjunction with potentially neurotoxic molecules generated by the host, might initiate a complex network of events which lead neurones to death. In particular, the HIV-1 coat glycoprotein, gp120, has been proposed as a likely aetiologic agent of the described neuronal loss because it causes death of neurones in culture. More recently, it has been shown that brain neocortical cell death is caused in rat by intracerebroventricular injection of a recombinant gp120 coat protein, and that this occurs via apoptosis. The latter observation broadens our knowledge in the pathophysiology of the reported neuronal cell loss and opens a new lane of experimental research for the development of novel therapeutic strategies to limit damage to the brain of patients suffering from HIV-associated dementia.     

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibit decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.