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Results for “protein folding”

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At least 631 records · Page 35

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NASA NTRS2005

Kinetic Studies of Iron Deposition Catalyzed by Recombinant Human Liver Heavy, and Light Ferritins and Azotobacter Vinelandii Bacterioferritin Using O2 and H2O2 as Oxidants

The discrepancy between predicted and measured H2O2 formation during iron deposition with recombinant heavy human liver ferritin (rHF) was attributed to reaction with the iron protein complex [Biochemistry 40 (2001) 10832-10838]. This proposal was examined by stopped-flow kinetic studies and analysis for H2O2 production using (1) rHF, and Azotobacter vinelandii bacterial ferritin (AvBF), each containing 24 identical subunits with ferroxidase centers; (2) site-altered rHF mutants with functional and dysfunctional ferroxidase centers; and (3) rccombinant human liver light ferritin (rLF), containing 110 ferroxidase center. For rHF, nearly identical pseudo-first-order rate constants of 0.18 per second at pH 7.5 were measured for Fe(2+) oxidation by both O2 and H2O2, but for rLF, the rate with O2 was 200-fold slower than that for H2O2 (k-0.22 per second). A Fe(2+)/O2 stoichiometry near 2.4 was measured for rHF and its site altered forms, suggesting formation of H2O2. Direct measurements revealed no H2O2 free in solution 0.5-10 min after all Fe(2+) was oxidized at pH 6.5 or 7.5. These results are consistent with initial H2O2 formation, which rapidly reacts in a secondary reaction with unidentified solution components. Using measured rate constants for rHF, simulations showed that steady-state H2O2 concentrations peaked at 14 pM at approx. 600 ms and decreased to zero at 10-30 s. rLF did not produce measurable H2O2 but apparently conducted the secondary reaction with H2O2. Fe(2+)/O2 values of 4.0 were measured for AvBF. Stopped-flow measurements with AvBF showed that both H2O2 and O2 react at the same rate (k=0.34 per second), that is faster than the reactions with rHF. Simulations suggest that AvBF reduces O2 directly to H2O without intermediate H2O2 formation.

Bunker, Jared↗

DOE OSTI2021

Identification and structural analysis of a thermophilic β-1,3-glucanase from compost

β-1,3-glucanase can specifically hydrolyze glucans to oligosaccharides and has potential applications in biotechnology. We used the metatranscriptomic technology to discover a thermophilic β-1,3-glucanase from compost. The phylogenetic study shows that it belongs to the family 16 glycoside hydrolase (GH16) and is most homologous with an enzyme from Streptomyces sioyaensis, an actinobacterium. It has the activity of 146.9 U/mg in the optimal reaction condition (75 °C and pH 5.5). Its catalytic domain was crystallized and diffracted to 1.14 Å resolution. The crystal structure shows a sandwich-like β-jelly-roll fold with two disulfide bonds. After analyzing the occurring frequencies of these cysteine residues, we designed two mutants (C160G and C180I) to study the role of these disulfide bonds. Both mutants have decreased their optimal temperature from 75 to 70 °C, which indicate that the disulfide bonds are important to maintain thermostability. Interestingly, the activity of C160G has increased ~ 17% to reach 171.4 U/mg. We speculate that the increased activity of C160G mutant is due to increased dynamics near the active site. Our studies give a good example of balancing the rigidity and flexibility for enzyme activity, which is helpful for protein engineering.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Supercharged cellulases show superior thermal stability and enhanced activity towards pretreated biomass and cellulose

Non-productive binding of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Protein supercharging was previously employed as one of the strategies to reduce non-productive binding to biomass. However, various questions remain unanswered regarding the hydrolysis kinetics of supercharged enzymes towards pretreated biomass substrates and the role played by enzyme interactions with individual cell wall polymers such as cellulose and xylan. In this study, CBM2a (from Thermobifida fusca ) fused with endocellulase Cel5A (from T. fusca ) was used as the model wild-type enzyme and CBM2a was supercharged using Rosetta, to obtain eight variants with net charges spanning -14 to +6. These enzymes were recombinantly expressed in E. coli , purified from cell lysates, and their hydrolytic activities were tested against pretreated biomass substrates (AFEX and EA treated corn stover). Although the wild-type enzyme showed greater activity compared to both negatively and positively supercharged enzymes towards pretreated biomass, thermal denaturation assays identified two negatively supercharged constructs that perform better than the wild-type enzyme (~3 to 4-fold difference in activity) upon thermal deactivation at higher temperatures. To better understand the causal factor of reduced supercharged enzyme activity towards AFEX corn stover, we performed hydrolysis assays on cellulose-I/xylan/pNPC, lignin inhibition assays, and thermal stability assays. Altogether, these assays showed that the negatively supercharged mutants were highly impacted by reduced activity towards xylan whereas the positively supercharged mutants showed dramatically reduced activity towards cellulose and xylan. It was identified that a combination of impaired cellulose binding and lower thermal stability was the cause of reduced hydrolytic activity of positively supercharged enzyme sub-group. Overall, this study demonstrated a systematic approach to investigate the behavior of supercharged enzymes and identified supercharged enzyme constructs that show superior activity at elevated temperatures. Future work will address the impact of parameters such as pH, salt concentration, and assay temperature on the hydrolytic activity and thermal stability of supercharged enzymes.

09 BIOMASS FUELS↗

NASA NTRS1987

Spermine stimulation of a nuclear NII kinase from pea plumules and its role in the phosphorylation of a nuclear polypeptide

We have previously demonstrated that spermine stimulates the phosphorylation of a 47 kilodalton nuclear polypeptide from pea plumules (N Datta, LK Hardison, SJ Roux 1986 Plant Physiol 82: 681-684). In this paper we report that spermine stimulates the activity of a cyclic AMP independent casein kinase, partially purified from a chromatin fraction of pea plumule nuclei. This effect of spermine was substrate specific; i.e. with casein as substrate, spermine stimulated the kinase activity, and with phosvitin as substrate, spermine completely inhibited the activity. The stimulation by spermine of the casein kinase was, in part, due to the lowering of the Mg2+ requirement of the kinase. Heparin could partially inhibit this casein kinase activity and spermine completely overcame this inhibition. By further purification of the casein kinase extract on high performance liquid chromatography, we fractionated it into an NI and an NII kinase. Spermine stimulated the NII kinase by 5- to 6-fold but had no effect on the NI kinase. Using [gamma-32P]GTP, we have shown that spermine promotes the phosphorylation of the 47 kilodalton polypeptide(s) in isolated nuclei, at least in part by stimulating an NII kinase.

NASA Discipline Plant Biology↗

DOE OSTI2020

A Long Non-coding RNA, LOC157273, Is an Effector Transcript at the Chromosome 8p23.1-PPP1R3B Metabolic Traits and Type 2 Diabetes Risk Locus

Causal transcripts at genomic loci associated with type 2 diabetes (T2D) are mostly unknown. The chr8p23.1 variant rs4841132, associated with an insulin-resistant diabetes risk phenotype, lies in the second exon of a long non-coding RNA (lncRNA) gene, LOC157273, located 175 kilobases from PPP1R3B, which encodes a key protein regulating insulin-mediated hepatic glycogen storage in humans. We hypothesized that LOC157273 regulates expression of PPP1R3B in human hepatocytes. We tested our hypothesis using Stellaris fluorescent in situ hybridization to assess subcellular localization of LOC157273; small interfering RNA (siRNA) knockdown of LOC157273, followed by RT-PCR to quantify LOC157273 and PPP1R3B expression; RNA-seq to quantify the whole-transcriptome gene expression response to LOC157273 knockdown; and an insulin-stimulated assay to measure hepatocyte glycogen deposition before and after knockdown. We found that siRNA knockdown decreased LOC157273 transcript levels by approximately 80%, increased PPP1R3B mRNA levels by 1.7-fold, and increased glycogen deposition by >50% in primary human hepatocytes. An A/G heterozygous carrier (vs. three G/G carriers) had reduced LOC157273 abundance due to reduced transcription of the A allele and increased PPP1R3B expression and glycogen deposition. We show that the lncRNA LOC157273 is a negative regulator of PPP1R3B expression and glycogen deposition in human hepatocytes and a causal transcript at an insulin-resistant T2D risk locus.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

Cleavage at the nsp5–nsp6 site of SARS-CoV-2 main protease intermediate precursor is faster from a monomer than a dimer form

Our previous studies of severe acute respiratory syndrome coronavirus 2 main protease (MPro) precursor monomer indicate that the initial N-terminal nonstructural protein (nsp)4/nsp5 cleavage occurs intramolecularly, with a small fraction of the active site loop equilibrium being in the active state. To understand the influence of dimer formation of MPro upon N-terminal cleavage on the subsequent C-terminal nsp5/nsp6 intermolecular cleavage kinetics, the stepwise processing of a monomeric, inactive precursor containing the native terminal cleavage sites of MPro (MBP- (−6) MPro C145A(+3) -GB1-6H, 86.2 kDa) by mature WT MPro (MPro WT ) was investigated. Differential scanning fluorimetry and analytical ultracentrifugation measurements of various MPro constructs suggest that the C145A mutation decreases the dimer dissociation constant (K dimer ) by ∼26-fold, relative to WT C145 and H41A. The monomeric precursor’s nsp4–nsp5 site appears to saturate MProWT’s active sites and cleave faster, followed by a slower first-order cleavage at the C-terminal site. No detectable product resulting from the C-terminal cleavage is observed until most of the N-terminal cleavage is complete. The initial intermediate product (termed MPro C145A-IP ) is a homodimer with an estimated K dimer of <0.05 μM. In contrast, the first-order kinetics observed for the cleavage of the monomeric form of the intermediate product is at least 300 times faster than that of the dimer form. Room-temperature X-ray structure of the MPro C145A-IP –ensitrelvir complex is like that of the MPro WT –ensitrelvir complex and reveals a dynamic C-terminal region including MPro residues 302 to 306. These results are interpreted from the point of view of a mechanism in which nsp5–nsp6 cleavage may occur from a monomeric intermediate, and dimer formation restricts this cleavage.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2025

Leveraging structure-informed machine learning for fast steric zipper propensity prediction across whole proteomes

Predicting the amyloid fold and the propensity of peptide segments to adopt amyloid-like structures remain a challenge. However, recent progress has facilitated structure-based prediction of steric zipper propensity and the use of machine learning to accelerate the calculation of predictive models across many scientific areas. Leveraging these advances, we have developed a new approach for rapid proteome-wide assessment of zipper profiles that is informed by four million steric zipper predictions collected over ten years. This collection is used to build a machine learning model capable of rapidly predicting steric zipper propensity, and allowing for the assessment of zippers at both the protein and proteome level. Our predictions show enrichment for zipper forming segments in proteins involved in cell wall reorganization in yeast, highlighting a potential category of interest for experimental characterization. Overall, our predictive model allows for the exploration of amyloid formation across the tree of life and provides a tool for assessment of both novel and designed sequences for zipper density.

Biochemistry & Molecular Biology↗

DOE OSTI2024

p14 ARF forms meso-scale assemblies upon phase separation with NPM1

NPM1 is an abundant nucleolar chaperone that, in addition to facilitating ribosome biogenesis, contributes to nucleolar stress responses and tumor suppression through its regulation of the p14 Alternative Reading Frame tumor suppressor protein (p14 ARF ). Oncogenic stress induces p14 ARF to inhibit MDM2, stabilize p53 and arrest the cell cycle. Under non-stress conditions, NPM1 stabilizes p14 ARF in nucleoli, preventing its degradation and blocking p53 activation. However, the mechanisms underlying the regulation of p14 ARF by NPM1 are unclear because the structural features of the p14 ARF -NPM1 complex were elusive. Here we show that p14 ARF assembles into a gel-like meso-scale network upon phase separation with NPM1. This assembly is mediated by intermolecular contacts formed by hydrophobic residues in an α-helix and β-strands within a partially folded N-terminal portion of p14 ARF . These hydrophobic interactions promote phase separation with NPM1, enhance p14 ARF nucleolar partitioning, restrict NPM1 diffusion within condensates and nucleoli, and reduce cellular proliferation. Our structural analysis provides insights into the multifaceted chaperone function of NPM1 in nucleoli by mechanistically linking the nucleolar localization of p14 ARF to its partial folding and meso-scale assembly upon phase separation with NPM1.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

From primordial clocks to circadian oscillators

Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator1. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides , which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1989

Latent nitrate reductase activity is associated with the plasma membrane of corn roots

Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.

NASA Discipline Number 61-10↗

DOE OSTI2020

In Situ Proteolysis Condition-Induced Crystallization of the XcpVWX Complex in Different Lattices

Although prevalent in the determination of protein structures; crystallography always has the bottleneck of obtaining high-quality protein crystals for characterizing a wide range of proteins; especially large protein complexes. Stable fragments or domains of proteins are more readily to crystallize; which prompts the use of in situ proteolysis to remove flexible or unstable structures for improving crystallization and crystal quality. In this work; we investigated the effects of in situ proteolysis by chymotrypsin on the crystallization of the XcpVWX complex from the Type II secretion system of Pseudomonas aeruginosa. Different proteolysis conditions were found to result in two distinct lattices in the same crystallization solution. With a shorter chymotrypsin digestion at a lower concentration; the crystals exhibited a P3 hexagonal lattice that accommodates three complex molecules in one asymmetric unit. By contrast; a longer digestion with chymotrypsin of a 10-fold higher concentration facilitated the formation of a compact P212121 orthorhombic lattice with only one complex molecule in each asymmetric unit. The molecules in the hexagonal lattice have shown high atomic displacement parameter values compared with the ones in the orthorhombic lattice. Taken together; our results clearly demonstrate that different proteolysis conditions can result in the generation of distinct lattices in the same crystallization solution; which can be exploited in order to obtain different crystal forms of a better quality

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2024

Mechanical forces orchestrate the metabolism of the developing oilseed rape embryo

The initial free expansion of the embryo within a seed is at some point inhibited by its contact with the testa, resulting in its formation of folds and borders. Although less obvious, mechanical forces appear to trigger and accelerate seed maturation. However, the mechanistic basis for this effect remains unclear. Manipulation of the mechanical constraints affecting either the in vivo or in vitro growth of oilseed rape embryos was combined with analytical approaches, including magnetic resonance imaging and computer graphic reconstruction, immunolabelling, flow cytometry, transcriptomic, proteomic, lipidomic and metabolomic profiling. Our data implied that, in vivo, the imposition of mechanical restraints impeded the expansion of testa and endosperm, resulting in the embryo's deformation. An acceleration in embryonic development was implied by the cessation of cell proliferation and the stimulation of lipid and protein storage, characteristic of embryo maturation. The underlying molecular signature included elements of cell cycle control, reactive oxygen species metabolism and transcriptional reprogramming, along with allosteric control of glycolytic flux. Constricting the space allowed for the expansion of in vitro grown embryos induced a similar response. The conclusion is that the imposition of mechanical constraints over the growth of the developing oilseed rape embryo provides an important trigger for its maturation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2026

The distinct trimeric structure of the immunodominant chlamydial antigen Major Outer Membrane Protein

The chlamydial Major Outer Membrane Protein (MOMP) is a promising subunit vaccine candidate due to its abundance in the outer membrane (OM), the presence of four surface-exposed variable domains (VDs) containing neutralizing and serotyping epitopes, and confirmed T-cell epitopes located in its constant domains (CDs). However, recombinant and denatured forms of MOMP have failed to elicit immune responses comparable to native preparations, indicating that conformation is critical for immunogenicity. Here, we present two cryo-EM structures of native Chlamydia muridarum MOMP, isolated from infectious elementary bodies (EBs). EB MOMP forms a distinct trimer with a stem of three narrow, non-permeable β-barrels and an extracellular, folded antigenic cap that displays VDs. In complex with a neutralizing Fab fragment of a conformational antibody, the cap undergoes structural reorganization that shows how epitope presentation is modulated by immune engagement. These structures reveal the molecular basis of MOMP’s serovar specificity and provide a template for structure-based vaccine design.

Guo, Yirui↗

DOE OSTI2022

Computational design and engineering of an Escherichia coli strain producing the nonstandard amino acid para -aminophenylalanine

Introducing heterologous pathways into host cells constitutes a promising strategy for synthesizing nonstandard amino acids (nsAAs) to enable the production of proteins with expanded chemistries. However, this strategy has proven challenging, as the expression of heterologous pathways can disrupt cellular homeostasis of the host cell. Here, we sought to optimize the heterologous production of the nsAA para -aminophenylalanine (pAF) in Escherichia coli . First, we incorporated a heterologous pAF biosynthesis pathway into a genome-scale model of E. coli metabolism and computationally identified metabolic interventions in the host’s native metabolism to improve pAF production. Next, we explored different approaches of imposing these flux interventions experimentally and found that the upregulation of flux in the chorismate biosynthesis pathway through the elimination of feedback inhibition mechanisms could significantly raise pAF titers (~20-fold) while maintaining a reasonable pAF production-growth rate trade-off. Overall, this study provides a promising strategy for the biosynthesis of nsAAs in engineered cells.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Engineered molecular sensors for quantifying cell surface crowding

Cells mediate interactions with the extracellular environment through a crowded assembly of transmembrane proteins, glycoproteins and glycolipids on their plasma membrane. The extent to which surface crowding modulates the biophysical interactions of ligands, receptors, and other macromolecules is poorly understood due to the lack of methods to quantify surface crowding on native cell membranes. In this work, we demonstrate that physical crowding on reconstituted membranes and live cell surfaces attenuates the effective binding affinity of macromolecules such as IgG antibodies in a surface crowding-dependent manner. We combine experiment and simulation to design a crowding sensor based on this principle that provides a quantitative readout of cell surface crowding. Our measurements reveal that surface crowding decreases IgG antibody binding by 2 to 20 fold in live cells compared to a bare membrane surface. Our sensors show that sialic acid, a negatively charged monosaccharide, contributes disproportionately to red blood cell surface crowding via electrostatic repulsion, despite occupying only ~1% of the total cell membrane by mass. We also observe significant differences in surface crowding for different cell types and find that expression of single oncogenes can both increase and decrease crowding, suggesting that surface crowding may be an indicator of both cell type and state. Our high-throughput, single-cell measurement of cell surface crowding may be combined with functional assays to enable further biophysical dissection of the cell surfaceome.

60 APPLIED LIFE SCIENCES↗

DOE OSTI2022

Cbp1, a fungal virulence factor under positive selection, forms an effector complex that drives macrophage lysis

Intracellular pathogens secrete effectors to manipulate their host cells. Histoplasma capsulatum (Hc) is a fungal intracellular pathogen of humans that grows in a yeast form in the host. Hc yeasts are phagocytosed by macrophages, where fungal intracellular replication precedes macrophage lysis. The most abundant virulence factor secreted by Hc yeast cells is Calcium Binding Protein 1 (Cbp1), which is absolutely required for macrophage lysis. Here we take an evolutionary, structural, and cell biological approach to understand Cbp1 function. We find that Cbp1 is present only in the genomes of closely related dimorphic fungal species of the Ajellomycetaceae family that lead primarily intracellular lifestyles in their mammalian hosts (Histoplasma, Paracoccidioides, and Emergomyces), but not conserved in the extracellular fungal pathogen Blastomyces dermatitidis. We observe a high rate of fixation of non-synonymous substitutions in the Cbp1 coding sequences, indicating that Cbp1 is under positive selection. We determine the de novo structures of Hc H88 Cbp1 and the Paracoccidioides americana (Pb03) Cbp1, revealing a novel “binocular” fold consisting of a helical dimer arrangement wherein two helices from each monomer contribute to a four-helix bundle. In contrast to Pb03 Cbp1, we show that Emergomyces Cbp1 orthologs are unable to stimulate macrophage lysis when expressed in the Hc cbp1 mutant. Consistent with this result, we find that wild-type Emergomyces africanus yeast are able to grow within primary macrophages but are incapable of lysing them. Finally, we use subcellular fractionation of infected macrophages and indirect immunofluorescence to show that Cbp1 localizes to the macrophage cytosol during Hc infection, making this the first instance of a phagosomal human fungal pathogen directing an effector into the cytosol of the host cell. We additionally show that Cbp1 forms a complex with Yps-3, another known Hc virulence factor that accesses the cytosol. Taken together, these data imply that Cbp1 is a fungal virulence factor under positive selection that localizes to the cytosol to trigger host cell lysis.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

Genome editing with sequence-specific nucleases, such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), is revolutionizing crop improvement. Developing efficient genome-editing protocols for highly polyploid crops, including sugarcane ( x = 10–13), remains challenging due to the high level of genetic redundancy in these plants. Here, we report the efficient multiallelic editing of magnesium chelatase subunit I ( MgCh ) in sugarcane. Magnesium chelatase is a key enzyme for chlorophyll biosynthesis. CRISPR/Cas9-mediated targeted co-mutagenesis of 49 copies/alleles of magnesium chelatase was confirmed via Sanger sequencing of cloned PCR amplicons. This resulted in severely reduced chlorophyll contents, which was scorable at the time of plant regeneration in the tissue culture. Heat treatment following the delivery of genome editing reagents elevated the editing frequency 2-fold and drastically promoted co-editing of multiple alleles, which proved necessary to create a phenotype that was visibly distinguishable from the wild type. Despite their yellow leaf color, the edited plants were established well in the soil and did not show noticeable growth retardation. This approach will facilitate the establishment of genome editing protocols for recalcitrant crops and support further optimization, including the evaluation of alternative RNA-guided nucleases to overcome the limitations of the protospacer adjacent motif (PAM) site or to develop novel delivery strategies for genome editing reagents.

59 BASIC BIOLOGICAL SCIENCES↗

NASA NTRS1995

Lipopolysaccharide modulation of a CD14-like molecule on porcine alveolar macrophages

Cluster of differentiation antigen 14 (CD14) functions as a receptor for lipopolysaccharide (LPS) LPS-binding protein (LBP) complexes. Because LPS has varying effects on CD14 expression in vitro, we evaluated CD14 expression in response to LPS with a fully differentiated macrophage phenotype, the alveolar macrophage. By using flow microfluorometric analysis and a radioimmunoassay with an anti-human CD14 monoclonal antibody (My4) that cross-reacts with porcine CD14, we found that macrophages stimulated with LPS for 24 h exhibited a two- to fivefold increase in CD14-like antigen compared with unstimulated cells. At low concentrations of LPS, up-regulation of the CD14-like antigen was dependent on serum; at higher concentrations of LPS, serum was not required. In the absence of serum a 10-fold higher dose of LPS (10 ng/ml) was required to increase CD14-like expression. In addition, LPS-induced CD14-like up-regulation correlated with secretion of tumor necrosis factor-alpha, regardless of serum concentration. Blockade with My4 antibody significantly inhibited LPS-induced tumor necrosis factor-alpha secretion at 1 ng/ml of LPS. However, inhibition decreased as we increased the LPS concentration, suggesting the existence of CD14-independent pathways of macrophage activation in response to LPS. Alternatively, My4 may have a lower affinity for the porcine CD14 antigen than LPS, which may have only partially blocked the LPS-LBP binding site at high concentrations of LPS. Therefore, these data suggest that LPS activation of porcine alveolar macrophages for 24 h increased CD14-like receptor expression. The degree of CD14-like up-regulation was related to LPS concentration, however, activation did not require the presence of serum at high concentrations of LPS.

Non-NASA Center↗