Oligomerization of the Macrophage Mannose Receptor Enhances gp120-mediated Binding of HIV-1

C-type lectin receptors expressed on the surface of dendritic cells and macrophages are able to bind glycoproteins of microbial pathogens via mannose, fucose, and N-acetylglucosamine. Langerin on Langerhans cells, dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin on dendritic cells, and mannose receptor (MR) on dendritic cells and macrophages bind the human immunodeficiency virus (HIV) envelope protein gp120 principally via high mannose oligosaccharides. These C-type lectin receptors can also oligomerize to facilitate enhanced ligand binding. This study examined the effect of oligomerization of MR on its ability to bind to mannan, monomeric gp120, native trimeric gp140, and HIV type 1 BaL. Mass spectrometry analysis of cross-linked MR showed homodimerization on the surface of primary monocyte-derived dendritic cells and macrophages. Both monomeric and dimeric MR were precipitated by mannan, but only the dimeric form was co-immunoprecipitated by gp120. These results were confirmed independently by flow cytometry analysis of soluble monomeric and trimeric HIV envelope and a cellular HIV virion capture assay. As expected, mannan bound to the carbohydrate recognition domains of MR dimers mostly in a calcium-dependent fashion. Unexpectedly, gp120-mediated binding of HIV to dimers on MR-transfected Rat-6 cells and macrophages was not calcium-dependent, was only partially blocked by mannan, and was also partially inhibited by N-acetylgalactosamine 4-sulfate. Thus gp120-mediated HIV binding occurs via the calcium-dependent, non-calcium-dependent carbohydrate recognition domains and the cysteine-rich domain at the C terminus of MR dimers, presenting a much broader target for potential inhibitors of gp120-MR binding.The mannose receptor (MR)² is a C-type lectin receptor that is expressed on the surface of a variety of cells, including immature monocyte-derived dendritic cells (MDDC), dermal dendritic cells, macrophages, and hepatic endothelial cells. It is a multifunctional protein, involved in antigen recognition and internalization during the early stages of the innate immune response (1) as well as physiological clearance of the endogenous pituitary hormones lutropin and thyrotropin (2, 3). Recognition of foreign antigens occurs via mannose, fucose, and GlcNAc residues (4, 5), which are generally not found as terminal residues on mammalian glycoproteins but are highly abundant on surface proteins of pathogens such as the HIV-1 envelope gp120 (6, 7). Once bound, pathogens can be internalized by endocytosis or phagocytosis, where they are targeted to lysosomes for proteolytic degradation and presentation on major histocompatibility complex class II (8). In immature DCs, soluble recombinant HIV envelope proteins are processed by this pathway, initially binding to both dendritic cell-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) and MR and ultimately co-localizing with MR but not DC-SIGN in lysosomes (9). Furthermore, in immature DCs and to a greater extent mature DCs, a proportion of intact HIV-1 enters a unique vesicular compartment that co-localizes with tetraspanin proteins such as CD81 (10, 11). Recently, this compartment has been shown to be continuous with the plasma membrane (11) and does not represent a continuation of the endolysosomal network. Interestingly, this compartment can translocate virus from DCs to CD4 T cells, upon the formation of a virological synapse (10–12). Although viral uptake can occur in DCs independent of HIV env (2), the efficiency of HIV binding and uptake is greatly enhanced by the presence of C-type lectin-env interactions. At least initial binding to DC-SIGN (and most likely also MR) is required for T cell trans-infection (13).Structurally, the extracellular domain of MR consists of an N-terminal cysteine-rich domain (Cys-RD), followed by a fibronectin type II domain and eight carbohydrate recognition domains (CRD) on a single polypeptide backbone (1). Of the eight CRDs, CRD 4–8 have been shown to be required for high affinity binding of ligands containing terminal mannose/fucose/GlcNAc residues, with CRD 4 having demonstrable monosaccharide binding in isolation (14). Binding and release of ligand within the low pH environment of the endolysosomal compartment are also Ca²⁺-dependent. Acid-induced removal of Ca²⁺ binding in CRD 4 and 5 was shown to cause a conformational rearrangement of the domain, resulting in a loss of carbohydrate binding activity (15). In contrast, binding of sulfated carbohydrates to the Cys-RD appears to be Ca²⁺-independent as no Ca²⁺-binding sites were observed in its crystal structure (2, 16).Oligomerization of CLRs such as DC-SIGN (17), Langerin (18), and mannose-binding protein (19) has been reported to be essential for binding of oligosaccharide-bearing ligands. Early studies on MR suggested that it exists solely as a monomeric molecule and that clustering of multiple CRDs within the single polypeptide backbone was necessary for high affinity binding of oligosaccharide moieties (20). However, more recent studies have shown that dimerization is possible in the presence of Ca²⁺ (21) and that an equilibrium may exist between monomeric and dimeric forms on the cell surface (22). It is currently unclear what effect dimerization has on ligand binding to the CRDs; however, there is evidence that dimerization of MR is required for high affinity binding of ligands bearing terminal N-acetylgalactosamine 4-sulfate (GalNAc-4-SO₄) such as lutropin and thyrotropin (22) to the Cys-RD.To date, studies on the oligomerization and ligand binding activity of MR have used solubilized protein from cell lysates (20) or purified recombinant fragments (21). Because the membrane microenvironment can influence protein associations, soluble forms of MR may not necessarily be a true model of the quaternary structure and function of the native protein. Here, we used a well established method of cross-linking (23) on MDDCs, monocyte-derived macrophages (MDMs), and MR-transfected Rat-6 cells to preserve lateral protein-protein interactions between MR on the cell surface prior to solubilization. Mass spectrometry analysis of affinity-purified complexes showed they were homo-oligomers, and further resolution of the complex on a low percentage polyacrylamide gel by SDS-PAGE strongly indicates that they are dimers. Dimerization of MR was also found to be essential for binding mannan, monomeric gp120, native trimeric gp140, and HIV-1 viral particles. Persistence of monomeric gp120 and trimeric gp140 binding to dimeric MR in the presence of EGTA and various CRD and other inhibitors, however, suggested that gp120-mediated HIV-1 binding is not Ca²⁺-dependent and that at least binding probably occurs to both Ca²⁺-dependent and -independent CRDs and also the Cys-RD.     

Protection of photosynthetic O2 evolution against heat inactivation: the role of chloride,pH and coupling status

Heat inactivation of photosynthetic O₂ evolution was studied in isolated thylakoids from spinach (Spinacia oleracea) and mangrove (Avicennia marina) leaves. Different temperatures, salt, pH and uncoupler effects were investigated. From these results and others in the literature it was concluced that chloride loss from the membrane and, more specifically, the oxygen-evolving complex of photosystem II, may be the cause of inhibition of oxygen evolution during heat inactivation.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - Tricine N-2-hydroxy-1, 1-bis (hydroxymethyl) ethyl glycine - EDTA ethylenediaminetetraacetic acid - FeCN K-ferricyanide     

Lysozyme-promoted association of protein I molecules in the outer membrane of Escherichia coli.

Incubation of whole envelopes prepared from sonically oscillated Escherichia coli K-12 cultures with lysozyme in vitro resulted in the appearance of a protein species with an apparent molecular weight double that of outer membrane protein I. Similar dimers were also detected in purified outer membranes and whole envelopes from lysozyme-induced spheroplasts of E. coli K-12. This was confirmed by two-dimensional electrophoresis in which the dimers were resolved in the second dimension to run as single polypeptides of protein I. Formation of dimers was correlated with peptidoglycan degradation, but the ability of protein I molecules to associate may vary between strains of E. coli, since dimers were found only in outer membranes from E. coli W7. We suggest that extensive degradation of peptidoglycan leads to nonspecific formation of protein I aggregates, but that these aggregates do not occur in vivo.     

Global absolute quantification of a proteome: Challenges in the deployment of a QconCAT strategy

In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.     

Screening for MCL-PHA-Producing Fluorescent Pseudomonads and Comparison of MCL-PHA Production Under Iso-osmotic Conditions Induced by PEG and NaCl

The medium chain length polyhydroxyalkanoates (MCL-PHA) have attracted much attention from academic and industrial communities for their interesting applications in medical field. The aim of this study was to screen high MCL-PHA-producing fluorescent pseudomonads, and to compare the effect of osmotic stress generated by NaCl (ionic) and polyethylene glycol (PEG, non-ionic inert polymer) on PHA production. A total of 50 fluorescent pseudomonads isolated from rhizospheric soil were screened for PHA production by Sudan Black staining. Out of all the PHA-producing isolates only five were MCL-PHA producers as detected by MCL-PCR. Isolate Bar1 identified as Pseudomonas fluorescens by 16S rRNA gene sequencing was selected for further analysis due to its high MCL-PHA production ability. The iso-osmotic stress generated by NaCl and PEG-6000 showed 5.75- and 3.19-fold enhanced production of PHA at ?2 bar osmotic potential, over control (0 bar), respectively. There was 1.8-fold enhanced production of PHA at ?2 bar osmotic stress induced by NaCl over PEG. PEG reduces availability of water to microorganisms without reducing exogenously provided nutrients which appear to be responsible for its down performance over NaCl. The FTIR analysis of PHA sample purified from cells showed strong marker bands near 1742, 2870, 1170, 1099, and 2926 cm^?1, corresponding to MCL-PHA. The study reported that supplementation of NaCl (electrolyte) in growth media enhances the production of MCL-PHA which can be very useful for its industrial production.     

Estimates of (co)variance components and genetic parameters for body weights and first greasy fleece weight in Bharat Merino sheep

(Co)variance components and genetic parameters of weight at birth (BWT), weaning (3WT), 6, 9 and 12 months of age (6WT, 9WT and 12WT, respectively) and first greasy fleece weight (GFW) of Bharat Merino sheep, maintained at Central Sheep and Wool Research Institute, Avikanagar, Rajasthan, India, were estimated by restricted maximum likelihood, fitting six animal models with various combinations of direct and maternal effects. Data were collected over a period of 10 years (1998 to 2007). A log-likelihood ratio test was used to select the most appropriate univariate model for each trait, which was subsequently used in bivariate analysis. Heritability estimates for BWT, 3WT, 6WT, 9WT and 12WT and first GFW were 0.05 ± 0.03, 0.04 ± 0.02, 0.00, 0.03 ± 0.03, 0.09 ± 0.05 and 0.05 ± 0.03, respectively. There was no evidence for the maternal genetic effect on the traits under study. Maternal permanent environmental effect contributed 19% for BWT and 6% to 11% from 3WT to 9WT and 11% for first GFW. Maternal permanent environmental effect on the post-3WT was a carryover effect of maternal influences during pre-weaning age. A low rate of genetic progress seems possible in the flock through selection. Direct genetic correlations between body weight traits were positive and ranged from 0.36 between BWT and 6WT to 0.94 between 3WT and 6WT and between 6WT and 12WT. Genetic correlations of 3WT with 6WT, 9WT and 12WT were high and positive (0.94, 0.93 and 0.93, respectively), suggesting that genetic gain in post-3WT will be maintained if selection age is reduced to 3 months. The genetic correlations of GFW with live weights were 0.01, 0.16, 0.18, 0.40 and 0.32 for BWT, 3WT, 6WT, 9WT and 12WT, respectively. Correlations of permanent environmental effects of the dam across different traits were high and positive for all the traits (0.45 to 0.98).     

Molecular Cloning of the Gene Encoding Stearoyl-Acyl Carrier Protein Desaturase in Brassica juncea

Metabolic engineering of the pathways of lipid biosynthesis has generated transgenic oilseed crops with enhanced levels of specialty fatty acids of Industrial value. Stearic acid, a 18:0 saturated fatty acid, is one such important fatty acid. Stearoylacyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis and converts stearoyl-ACP to oleoyl-ACP. We have cloned the complete coding region of the gene for this enzyme in Brassica juncea. Based on the sequence information of the gene in B. napus, 27-mer forward and reverse primers were designed each of which incorporated a Sal I restriciton site at the end. The primers were used to fish out the desaturase gene from B. juncea genome by polymerase chain reaction (PCR). The PCR product conformed to the average size of the coding region of the gene in B. napus. The PCR product was cloned in the pGem-T vector. The cloning was reconfirmed by restriction enzyme analysis and by PCR of the recombinant plasmid. The potential use of this gene in molecular farming of designer oilseed brassicas is discussed.     

Role for Plasmacytoid Dendritic Cells in the Immune Control of Recurrent Human Herpes Simplex Virus Infection

Plasmacytoid dendritic cells (pDC) are an important component of the innate immune response, producing large amounts of alpha interferon in response to viral stimulation in vitro. Under noninflammatory conditions, pDC are not found in the skin and are restricted in location to the blood and lymph nodes. Therefore, their role in mucosal and cutaneous herpes simplex virus (HSV) infection has not been well-defined. In this study we show a role for human pDC in the immune response to HSV infection. First, by confocal microscopy we showed that pDC infiltrate the dermis of recurrent genital herpes simplex lesions at early and late phases, often at the dermo-epidermal junction. We then showed that pDC in vitro are resistant to HSV infection despite expressing the entry receptors CD111, CD112, and HVE-A. Within the lesions, pDC were found closely associated with CD3⁺ lymphocytes and NK cells, especially those which were activated (CD69⁺). Furthermore, these HSV-exposed pDC were able to stimulate virus-specific autologous T-lymphocyte proliferation. We conclude from this work that pDC may contribute to the immune control of recurrent herpes virus infection in vivo.