Relationship between SU subdomains that regulate the receptor-mediated transition from the native (fusion-inhibited) to the fusion-active conformation of the murine leukemia virus glycoprotein

Envelope glycoproteins (Env) of retroviruses are trimers of SU (surface) and TM (transmembrane) heterodimers and are expressed on virions in fusion-competent forms that are likely to be metastable. Activation of the viral receptor-binding domain (RBD) via its interaction with a cell surface receptor is thought to initiate a cascade of events that lead to refolding of the Env glycoprotein into its stable fusion-active conformation. While the fusion-active conformation of the TM subunit has been described in detail for several retroviruses, little is known about the fusion-competent structure of the retroviral glycoproteins or the molecular events that mediate the transition between the two conformations. By characterizing Env chimeras between the ecotropic and amphotropic murine leukemia virus (MLV) SUs as well as a set of point mutants, we show that alterations of the conformation of the SU glycoprotein strongly elevate Env fusogenicity by disrupting the stability of the Env complex. Compensatory mutations that restored both Env stability and fusion control were also identified, allowing definition of interactions within the Env complex that maintain the stability of the native Env complex. We show that, in the receptor-unbound form, structural interactions between the N terminus of the viral RBD (NTR domain), the proline-rich region (PRR), and the distal part of the C-terminal domain of the SU subunit maintain a conformation of the glycoprotein that is fusion inhibitory. Additionally, we identified mutations that disrupt this fusion-inhibitory conformation and allow fusion activation in the absence of viral receptors, provided that receptor-activated RBD fragments are added in trans during infection. Other mutations were identified that allow fusion activation in the absence of receptors for both the viral glycoprotein and the trans-acting RBD. Finally, we found mutations of the SU that bypass in cis the requirement for the NTR domain in fusion activation. All these different mutations call for a critical role of the PRR in mediating conformational changes of the Env glycoprotein during fusion activation. Our results suggest a model of MLV Env fusion activation in which unlocking of the fusion-inhibitory conformation is initiated by receptor binding of the viral RBD, which, upon disruption of the PRR, allows the NTR domain to promote further events in Env fusion activation. This involves a second type of interaction, in cis or in trans, between the receptor-activated RBD and a median segment of the freed C-terminal domain.     

Evidence of an odorant-binding protein in the human olfactory mucus: location,structural characterization,and odorant-binding properties

Odorant-binding proteins (OBPs) are small abundant extracellular proteins belonging to the lipocalin superfamily. They are thought to participate in perireceptor events of odor detection by carrying, deactivating, and/or selecting odorant molecules. Putative human OBP genes (hOBP) have recently been described [Lacazette et al. (2000) Hum. Mol. Genet. 9, 289-301], but the presence of the corresponding proteins remained to be established in the human olfactory mucus. This paper reports the first evidence of such expression in the mucus covering the olfactory cleft, where the sensory olfactory epithelium is located. On the contrary, hOBPs were not observed in the nasal mucus covering the septum and the lower turbinate. To demonstrate the odorant binding activity of these proteins, a corresponding recombinant protein variant, hOBP(IIa)(alpha), was secreted by the yeast Pichia pastoris and thoroughly characterized. It appears as a monomer with one disulfide bond located between C59 and C151, a conservative feature of all other vertebrate OBPs. By measuring the displacement of several fluorescent probes, we show that hOBP(IIa)(alpha) is able to bind numerous odorants of diverse chemical structures, with a higher affinity for aldehydes and large fatty acids. A computed 3D model of hOBP(IIa)(alpha) is proposed and reveals that two lysyl residues of the binding pocket may account for the increased affinity for aldehydes. The relatively limited specificity of hOBP(IIa)(alpha) suggests that other human OBPs are expected to take into account the large diversity of odorant molecules.     

The occurrence of reactive oxygen species in the semen of males from infertile couples

The aim of this pilot study was to establish a reactive oxygen species (ROS) evaluation method as a step in the routine diagnosis of men from infertile couples, which attend the Centre of Assisted Reproduction at the Teaching Hospital in Olomouc. Standard semen analyses were performed manually according to WHO guidelines. The number of peroxidase-positive leukocytes in the semen was determined using the Endtz test. The levels of ROS were estimated by chemiluminescence assay using luminol (5-amino-2,3 dihydro-1,4 phthalazinedione) as a probe. The semen samples were collected from 68 patients. Normospermia was found in 15 patients (22.1 %). The semen samples of 3 normospermic patients were classified as ROS-positive. Elevated ROS production was recorded in all subgroups of patients irrespective of any pathology found. We confirmed that spermatozoa might be the source of ROS as well as the seminal leukocytes. Apart from the leukocytes, sperm cells with residual cytoplasm and immature spermatozoa are considered to be a major source of ROS. Thus it is suggested that sperm morphology abnormalities should be evaluated more carefully.     

Changes of electrical impedance in vaginal vestibule in cyclic sows

The changes of the electrical impedance in the vaginal vestibule during the oestrous cycle and the influence of sow parity on the vestibular impedance in oestrus were examined. Primiparous and multiparous sows of the Large White breed were used. Oestrus was tested via exposure of sows to a sexually mature boar. The criterion for conformation of ovulation was the increase in plasma progesterone levels above 12.5nmoll(-1) on day 8 and 12 after oestrus onset. A two-terminal method was used to measure the impedance. The vestibular impedance rose slightly in the first day after weaning. The impedance increased markedly during oestrus (P<0.01) and decreased during early dioestrus (P<0.01). No significant changes were observed thereafter. The individual sows reached the peak of vestibular impedance between 1 and 3 days after oestrus onset. The parity of sows did not significantly influence the impedance values during oestrus. The study showed that the impedance changes in the vaginal vestibule during peri-oestrus are considerably different from those described earlier in the vagina and that sow parity does not affect the vestibular impedance in oestrus.     

Coral reefs in a high-latitude,siliciclastic barrier island setting: reef framework and sediment production at Inhaca Island,southern Mozambique

Inhaca Island (southern Mozambique) is located in a high-latitude setting along the seaward margins of the estuarine Maputo Bay and is subject to fluctuations in temperature and salinity, and high sedimentation and turbidity levels. Coral reefs are developed sporadically along the margins of intertidal channels, but framework development is severely restricted. Coral growth is bathymetrically limited (never exceeding 6-m depth), and framework accumulation is only present in the upper 1–2 m. Massive Porites sp. produce a basic reef structure, with other coral genera (mainly Acropora sp., Favia sp., Platygyra sp., Pocillopora sp., and Montipora sp.) colonizing available substrata. Sediment samples also indicate restricted carbonate sediment production, with siliciclastics (mainly quartz) a major sediment contributor (often >90%) and carbonate grain assemblages differing from those normally associated with lower-latitude reefs. Although corals, molluscs and coralline algae (including rhodoliths) represent dominant grain constituents, Halimeda is absent and there is a low diversity (four species identified) of benthic foraminifera (mainly Amphistegina sp.). Grain associations are therefore somewhat transitional in character, comprising elements of both tropical (chlorozoan) and temperate (foramol) grain assemblages. These patterns of reef and associated carbonate production emphasize the marginal character of these reef environments, which form one end member in a broad spectrum of marginal reef systems that are now being identified in a range of both high- and low-latitude settings.     

Fusion with HDEL protects cell wall invertase from early degradation when N-glycosylation is inhibited

Previous data obtained in different suspension-cultured plant cells have clearly illustrated that N-glycans are absolutely required for transport of glycoproteins to the extracellular compartment, regardless of their oligosaccharide structure [see Lerouge et al. (1998) Plant Mol. Biol. 38: 31 for review]. In the present study the role of N-glycosylation in the transport of glycoproteins to the cell surface was studied in BY2 tobacco cells using both endogenous and recombinant cell wall invertases as markers. When synthesized without their N-glycans, both invertases were very rapidly degraded. This degradation did not occur in an acidic compartment and was brefeldin A-insensitive. Therefore, it most probably represents a pre-Golgi event. However, the low efficiency of specific inhibitors did not favor a strong contribution of proteasomes in this proteolysis. In contrast, addition of a C-terminal His-Asp-Glu-Leu (HDEL) extension prevented arrival of these non-glycosylated glycoproteins in the compartment where they are degraded. These results argue for the presence of an endoplasmic reticulum (ER) domain specialized in protein degradation. Consistent with our results and the well-known stabilization of recombinant proteins retained in the ER, the addition of an ER retention signal to a protein would prevent its targeting to an ER domain devoted to degradation.     

Rational design of a CD4 mimic that inhibits HIV-1 entry and exposes cryptic neutralization epitopes

The conserved surfaces of the human immunodeficiency virus (HIV)-1 envelope involved in receptor binding represent potential targets for the development of entry inhibitors and neutralizing antibodies. Using structural information on a CD4-gp120-17b antibody complex, we have designed a 27-amino acid CD4 mimic, CD4M33, that presents optimal interactions with gp120 and binds to viral particles and diverse HIV-1 envelopes with CD4-like affinity. This mini-CD4 inhibits infection of both immortalized and primary cells by HIV-1, including primary patient isolates that are generally resistant to inhibition by soluble CD4. Furthermore, CD4M33 possesses functional properties of CD4, including the ability to unmask conserved neutralization epitopes of gp120 that are cryptic on the unbound glycoprotein. CD4M33 is a prototype of inhibitors of HIV-1 entry and, in complex with envelope proteins, a potential component of vaccine formulations, or a molecular target in phage display technology to develop broad-spectrum neutralizing antibodies.     

Translokin is an intracellular mediator of FGF-2 trafficking

Basic fibroblast growth factor (bFGF or FGF-2) exerts its pleiotropic activities both as an exogenous and an intracellular factor. FGF-1 and FGF-2 are prototypes for this dual signalling, but the mechanisms of their intracellular actions remain unknown. Here we show that Translokin, a cytoplasmic protein of relative molecular mass 55,000 (M(r) 55K), interacts specifically with the 18K form of FGF-2. Translokin is ubiquitously expressed and colocalizes with the microtubular network. As Translokin does not interact with FGF-1, we used a strategy based on FGF-1-FGF-2 chimaeras to map the interacting regions in FGF-2 and to generate Nb1a2, a non-interacting variant of FGF-2. Although most of the FGF-2 properties are preserved in Nb1a2, this variant is defective in intracellular translocation and in stimulating proliferation. The fusion of a nuclear localization signal to Nb1a2 restores its mitogenic activity and its nuclear association. Inhibiting Translokin expression by RNA interference reduces the translocation of FGF-2 without affecting the intracellular trafficking of FGF-1. Our data show that the nuclear association of internalized FGF-2 is essential for its mitogenic activity and that Translokin is important in this translocation pathway.     

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.