Diglyceride kinase from Escherichia coli: Modulation of enzyme activity by glycosphingolipids

Diglyceride kinase was purified from membranes of Escherichia coli K-12 using organic solvents. The enzyme apoprotein depended on lipids, such as cardiolipin (diphosphatidylglycerol), phosphatidylcholine or 1-monooleoylglycerol, for activity with 1,2-dipalmitoylglycerol. Mixed brain cerebrosides and gangliosides as well as defined ganglioside fractions and synthetic lactocerebroside were devoid of lipid cofactor activity. However, all these glycosphingolipids were strong inhibitors of activation by phosphatidylcholine. When cardiolipin was used as lipid activator with the detergent, Triton X-100, as solubilizing agent, the addition of mixed or purified gangliosides first (at about 0.4 mM) resulted in additional activation, but higher ganglioside concentrations were strongly inhibitory. Both effects were absolutely dependent on the presence of lipid-bound sialic acid and were not given by cerebrosides, by free sialic acid or by sialyl-lactose. The stimulating and inhibitory effects of glycosphingolipids could also be demonstrated when 1-monooleoylglycerol was used as substrate, lipid activator and solubilizing agent at the same time. The modulation of kinase activity by glycosphingolipids is discussed at the level of lipid/protein interactions.     

Influence of FADS polymorphisms on tracking of serum glycerophospholipid fatty acid concentrations and percentage composition in children

Background

Tracking of fatty acid (FA) contribution to plasma or serum lipids over time was shown in children and adults. However, the potential role of FADS gene variants has not been investigated.

Methods and Principal Findings

Serum GP FA composition of 331 children aged 2 and 6 years, participating in an ongoing birth cohort study, was analyzed. Correlation coefficients were estimated to describe FA tracking over 4 years and to assess the influence of FADS variants on tracking. We found low to moderate tracking (r = 0.12–0.49) of FA compositions and concentration between 2 and 6 years. Concentration changes of total monounsaturated FA and total saturated FA over time correlated closely (r = 0.79) but percentage values were unrelated (r = −0.02). Tracking for n-6 long chain polyunsaturated fatty acid (LC-PUFA) concentrations was lower in subjects homozygous for the major allele of FADS variants and higher in carriers of at least one minor allele, whereas for total n-3 LC-PUFA concentrations and compositions this was vice versa. For individual n-3 PUFA inconsistent results were found.

Conclusions and Significance

Serum GP FA composition shows low to moderate tracking over 4 years with a higher tracking for LC-PUFA metabolites than for their precursor FA. Serum PUFA levels and their tracking seem to be more influenced by lipid and lipoprotein metabolism than by FA specific pathways.     

Functional expression of the transient receptor potential channel TRPA1, a sensor for toxic lung inhalants,in pulmonary epithelial cells

The cation channel TRPA1 functions as a chemosensory protein and is directly activated by a number of noxious inhalants. A pulmonary expression of TRPA1 has been described in sensory nerve endings and its stimulation leads to the acceleration of inflammatory responses in the lung. Whereas the function of TRPA1 in neuronal cells is well defined, only few reports exist suggesting a role in epithelial cells. The aim of the present study was therefore (1) to evaluate the expression of TRPA1 in pulmonary epithelial cell lines, (2) to characterize TRPA1-promoted signaling in these cells, and (3) to study the extra-neuronal expression of this channel in lung tissue sections. Our results revealed that the widely used alveolar type II cell line A549 expresses TRPA1 at the mRNA and protein level. Furthermore, stimulating A549 cells with known TRPA1 activators (i.e., allyl isothiocyanate) led to an increase in intracellular calcium levels, which was sensitive to the TRPA1 blocker ruthenium red. Investigating TRPA1 coupled downstream signaling cascades it was found that TRPA1 activation elicited a stimulation of ERK1/2 whereas other MAP kinases were not affected. Finally, using epithelial as well as neuronal markers in immunohistochemical approaches, a non-neuronal TRPA1 protein expression was detected in distal parts of the porcine lung epithelium, which was also found examining human lung sections. TRPA1-positive staining co-localized with both epithelial and neuronal markers underlining the observed epithelial expression pattern. Our findings of a functional expression of TRPA1 in pulmonary epithelial cells provide causal evidence for a non-neuronal TRPA1-mediated control of inflammatory responses elicited upon TRPA1-mediated registration of toxic inhalants in vivo.     

Structural determinants of calmodulin binding to the intracellular C-terminal domain of the metabotropic glutamate receptor 7A

Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.     

Purification of a bovine liver S6 kinase

A bovine liver protein serine kinase that catalyzes the multisite phosphorylation of ribosomal protein S6 has been purified to near homogeneity. The enzyme has an Mr of 67,000 on SDS-polyacrylamide gel electrophoresis and an apparent molecular weight of 55,000 on glycerol gradient sedimentation. Its enzymic properties, substrate specificity, molecular size and chromatographic behaviour are similar to those of the principal growth factor--and phorbol 12-myristate 13-acetate-stimulated S6 kinase of cultured cells.     

Recognition of 5-hydroxymethylcytosine by the Uhrf1 SRA domain

Recent discovery of 5-hydroxymethylcytosine (5hmC) in genomic DNA raises the question how this sixth base is recognized by cellular proteins. In contrast to the methyl-CpG binding domain (MBD) of MeCP2, we found that the SRA domain of Uhrf1, an essential factor in DNA maintenance methylation, binds 5hmC and 5-methylcytosine containing substrates with similar affinity. Based on the co-crystal structure, we performed molecular dynamics simulations of the SRA:DNA complex with the flipped cytosine base carrying either of these epigenetic modifications. Our data indicate that the SRA binding pocket can accommodate 5hmC and stabilizes the flipped base by hydrogen bond formation with the hydroxyl group.     

Binding of an antagonistic monoclonal antibody to an intact and fragmented EGF-receptor polypeptide

A murine monoclonal antibody (No. 425) raised against human A431 carcinoma cells specifically immunoprecipitates the 170,000 molecular weight epidermal growth factor (EGF)-receptor from extracts of A431 cells as well as from extracts of human placenta and cultured fibroblasts, but does not recognize the murine receptor. Binding to the external domain of the human EGF-receptor was indicated by indirect immunofluorescent staining of fixed nonpermeable cells. The antibody binds to both glyco- and aglycoreceptor forms, indicating that the epitope is a part of the polypeptide chain. Binding of the antibody to the receptor is conformation dependent; i.e., denatured receptors lacking EGF-binding activity are not recognized by the antibody. The results of antibody binding studies indicate that the epitope is closely linked to the EGF binding active site, and is common to both high- and low-affinity EGF-receptors. Interaction of this epitope with the antibody inhibits EGF binding and bioactivity, and triggers receptor down-regulation, but does not generate EGFlike kinase-stimulatory or mitogenic responses either in vitro or in vivo. The antibody was tested for its ability to bind to domain-sized fragments of the 170-kDa EGF-receptor. It can recognize both the proteolytically generated 110-kDa EGF binding peptide, and a soluble 100-kDa EGF-receptor secreted by A431 cells. This indicates that the epitope recognized this antibody retains its conformation after proteolytic separation of the EGF binding domain from the rest of the receptor molecule.     

Clearance of acute-phase plasma proteins with no, high-mannose-, hybrid-, or complex type oligosaccharide side chains by the isolated perfused rat liver

The clearance of the rat acute-phase proteins alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein with no, high-mannose, hybrid or complex type oligosaccharide side chains was determined in the isolated perfused rat liver. The differently glycosylated forms of the three proteins were obtained from rat hepatocyte primary cultures treated with different inhibitors of glycosylation. The complex type forms of the three proteins were essentially not cleared by the liver during 2 h of perfusion. Unglycosylated alpha 2-macroglobulin and alpha 1-acid glycoprotein decreased in the perfusate by about 50% after 2 h; unglycosylated alpha 1-proteinase inhibitor was not taken up by the liver. The high-mannose type forms of the three proteins were nearly totally cleared. After 2 h of perfusion 10%, 45% and 30% of the hybrid type forms of alpha 2-macroglobulin, alpha 1-proteinase inhibitor and alpha 1-acid glycoprotein, respectively, were cleared. The clearance rates of high-mannose and of hybrid type glycoproteins could be reduced to the rates of complex type glycoproteins by the addition of mannan to the perfusate. It is concluded that complex type glycosylation prevents the uptake of plasma glycoproteins by the liver.     

The efficiency of water use in water stressed plants is increased due to ABA induced stomatal closure

Gas exchange and abscisic acid content of Digitalis lanata EHRH. have been examined at different levels of plant water stress. Net photosynthesis, transpiration and conductance of attached leaves declined rapidly at first, then more slowly following the withholding of irrigation. The intercellular partial pressure of CO₂ decreased slightly. The concentration of 2-cis(S)ABA increased about eight-fold in the leaves of non-irrigated plants as compared with well-watered controls. A close linear correlation was found between the ABA content of the leaves and their conductance on a leaf area basis. In contrast, the plot of net assimilation versus ABA concentration was curvilinear, leading to an increased efficiency of water use during stress. After rewatering, photosynthesis reached control values earlier than transpiration, leaf conductance and ABA content. From these data it is concluded that transpiration through the stomata is directly controlled by the ABA content, whereas net photosynthesis is influenced additionally by other factors.Possible reasons for the responses of photosynthesis and water use efficiency to different stress and ABA levels are discussed.Abbreviations A net CO₂ assimilation - ABA abscisic acid - C_i intercellular CO₂ concentration - g stomatal conductance - T transpiration - WUE water use efficiency