The molecular biology of the sphingolipid hydrolases

Our understanding of the sphingolipidoses has improved as a result of the investigation of molecular mechanisms causing clinical heterogeneity. This knowledge, derived from both the protein and gene structures, should bring therapy for these inherited disorders closer to a realistic possibility.     

Endosomes differ from plasma membranes in the phospholipid molecular species composition

125I-Labeled epidermal growth factor was incorporated into and highly concentrated in endosomes of Chinese hamster V79-UF cells during incubation at 37 degrees C for 8 min after binding to its receptors on the cell surface at 4 degrees C. From the labeled cells, endosomes were isolated by isopycnic centrifugation on a Percoll density gradient and then sucrose density gradients. The isolated endosomes were mostly free from contamination by Golgi, endoplasmic reticulum, lysosome, plasma membrane and mitochondria. Endosome membranes were found to differ from plasma membranes in the phospholipid composition. Sphingomyelin and phosphatidylserine were enriched in endosomes, compared with plasma membranes. Diacylphosphatidylcholine and diacylphosphatidylethanolamine were major phospholipids of the membranes in both organelles. The contents of molecular species of diacylphosphatidylcholine and diacylphosphatidylethanolamine with two monoenoic fatty acids were lower in endosomes than in plasma membranes. The differences in the polar head group and molecular species compositions of phospholipids between endosomes and plasma membranes did not change, regardless of whether or not the proportions of phospholipid molecular species in plasma membranes changed. The significance of the lipids in endosomes is discussed.     

Differential methylation patterns in molecular species of phosphatidylethanolamine derivatives in rat liver membranes

The appearance of individual molecular species of phospholipids in the complete sequence of the transmethylation of phosphatidylethanolamine (PE) was examined in rat liver microsomes incubated with S-adenosyl-L-[methyl-14C]methionine. Reverse-phase HPLC analysis of phosphatidylcholine (PC), phosphatidyl-N,N-dimethylethanolamine (dimethyl-PE), or phosphatidyl-N-monomethylethanolamine (monomethyl-PE) showed that radioactivity was present in the same six principal molecules; a first group is constituted by 16:0/22:6, 16:0/20:4 and 16:0/18:2 and a second one by the homologous molecules with 18:0 instead of 16:0 at the sn-1 position of glycerol. In PC, 16:0/22:6 (23% of total radioactivity) was preponderant, and 18:0/20:4 was the lowest. The ratios cpm in PC/nmol in PE were in the order: 16:0/22:6 greater than 16:0/18:2 greater than 16:0/20:4 followed by the corresponding 18:0 molecules. On the other hand, in intermediate phospholipids, incorporation of methyl groups was most marked in 18:0/20:4 (24-27% of total). 16:0/22:6 and 16:0/18:2 were low in comparison to their relative values in PC. The ratio (18:0/20:4)/(16:0/22:6) was 4.5-5.6-times higher in monomethyl-PE and dimethyl-PE than in PC. These differences were found consistently, regardless of incubation time of microsomes (2.5-60 min) and of S-adenosyl-L-methionine (AdoMet) concentration (3 or 100 microM). In liver membranes, it would therefore seem that there is a different selectivity in methyl group transfer, depending upon whether the first two steps or the third step of the reaction are considered. Side reactions, such as deacylation/reacylation, are unlikely to account for this difference, which could rather be related to the enzyme itself.     

Mining alpha-helix-forming molecular recognition features with cross species sequence alignments

Previously described algorithms for mining alpha-helix-forming molecular recognition elements (MoREs), described by Oldfield et al. (Oldfield, C. J., Cheng, Y., Cortese, M. S., Brown, C. J., Uversky, V. N., and Dunker, A. K. (2005) Comparing and combining predictors of mostly disordered proteins, Biochemistry 44, 1989-2000), also known as molecular recognition features (MoRFs) (Mohan, A., Oldfield, C. J., Radivojac, P., Vacic, V., Cortese, M. S., Dunker, A. K., and Uversky, V. N. (2006) Analysis of Molecular Recognition Features (MoRFs), J. Mol. Biol. 362, 1043-1059), revealed that regions undergoing disorder-to-order transition are involved in many molecular recognition events and are crucial for protein-protein interactions. However, these algorithms were developed using a training data set of a limited size. Here we propose to improve the prediction algorithms by (1) including additional alpha-MoRF examples and their cross species homologues in the positive training set, (2) carefully extracting monomer structure chains from the Protein Data Bank (PDB) as the negative training set, (3) including attributes from recently developed disorder predictors, secondary structure predictions, and amino acid indices, and (4) constructing neural network based predictors and performing validation. Over 50 regions which undergo disorder-to-order transition that were identified in the PDB together with a set of corresponding cross species homologues of each structure-based example were included in a new positive training set. Over 1500 attributes, including disorder predictions, secondary structure predictions, and amino acid indices, were evaluated by the conditional probability method. The top attributes, including VSL2 and VL3 disorder predictions and several physicochemical propensities of amino acid residues, were used to develop the feed forward neural networks. The sensitivity, specificity, and accuracy of the resulting predictor, alpha-MoRF-PredII, were 0.87 +/- 0.10, 0.87 +/- 0.11, and 0.87 +/- 0.08 over 10 cross validations, respectively. We present the results of these analyses and validation examples to discuss the potential improvement of the alpha-MoRF-PredII prediction accuracy.     

Structural determinants of sphingolipid recognition by commercially available anti-ceramide antibodies

The sphingolipid mediator ceramide is involved in cellular processes such as apoptosis, differentiation, responses to cytokines, and stress responses. Experimental evidence suggests that the intracellular location of ceramide may be a key factor in determining its ultimate cellular effects. One approach to ceramide localization is the use of recently developed anti-ceramide antibodies for immunocytochemical studies. Two such commercial preparations are now available; we sought to compare and contrast their specificity for ceramide and/or other cellular lipids. By using lipid overlay assays and a diverse panel of sphingolipids, we were able to delineate the specificity and thus, the utility of these reagents. Our results indicate that one of these anti-ceramide preparations is quite specific for ceramide and dihydroceramide, whereas the other preparation recognizes dihydroceramide, phosphatidylcholine, and sphingomyelin. Furthermore, through the use of chemically modified ceramides in similar assays, we were able to determine some structural determinants of lipid recognition by both of these reagents.     

Cold stress induces in situ phospholipid molecular species changes in cell surface membranes

The structural organization of Tetrahymena pyriformis is such that its cilia are remote from the main centers of lipid metabolism. As a result, the ciliary membrane lipid composition of cells exposed to low-temperature stress is initially unaffected by the significant metabolic changes induced in microsomal membranes. Nevertheless, changes in the ciliary membrane lipid composition can be detected during the first 4 h of cold exposure. A combination of in vivo and in vitro experiments has provided strong evidence for a substantial retailoring of ciliary phospholipid molecular species in situ in the absence of any importation of lipids from the cell interior or change in overall ciliary fatty acid composition. The mechanism responsible for the ciliary lipid changes is independent of the one(s) triggering internal acclimation responses. Our observations establish for the first time that chilling stress can simultaneously induce separate and distinctive lipid modification responses in different parts of a cell. This finding could be important in identifying the molecular ‘sensor’ capable of actuating stress-induced lipid changes.     

A role for molecular genetics in the recognition and conservation of endangered species

Taxonomies based on morphological traits alone sometimes provide inadequate or misleading guides to phylogenetic distinctions at the subspecies and species levels. Yet taxonomic assignments inevitably shape perceptions of biotic diversity, including recognition of endangered species. Case histories are discussed in which the data of molecular genetics revealed prior systematic errors of the two possible kinds: taxonomic recognition of groups showing little evolutionary differentiation, and lack of taxonomic recognition of phylogenetically distinct forms. In such cases, conservation efforts for 'endangered species' can be misdirected with respect to the goal of protecting biological diversity.     

Yeast species recognition from gene sequence analyses and other molecular methods

This review discusses DNA-based methods used for identification of yeasts. Nuclear DNA reassociation was the first quantitative molecular method employed for recognition of yeast species and has provided a baseline for interpretation of other molecular comparisons. Among these, gene sequencing is the most definitive method, with ribosomal RNA gene sequences providing the preponderance of available data. Multigene analyses that include the sequences of protein encoding genes are being increasingly developed to provide a more definitive resolution of species. A number of rapid identification methods, such as denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and flow cytometry, which are based on species-specific gene sequences, are available for use in diagnostic laboratories.     

Cholesterol modulates maculosin's orientation in model systems of biological membranes. Relevance towards putative molecular recognition

Fluorescence techniques were used to study (1) the extent of insertion of the bioactive cyclic dipeptide cyclo(l-tyrosyl-l-prolyl), maculosin, in model systems of membranes of 1, 2-palmitoyl-sn-glycero-3-phosphatidyl choline (DPPC) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl choline (POPC), (2) its in-depth location in those lipidic membranes, and (3) the influence of cholesterol on the dipeptides's location and orientation. Partition into lipidic bilayers is extensive, mainly for liquid crystalline phase membranes (K(p)=1.3x10(4)). Maculosin locates at the lipid head groups level regardless of the membrane system. Nevertheless, its orientation is lipid phase dependent. When maculosin was inserted in liquid crystalline phase bilayers, its phenolic ring was perpendicular to the membrane surface, whereas it changed orientation when inserted in gel phase membranes. Cholesterol was able to reverse the lipid phase influence on maculosin's orientation.     

Oligosaccharides in molecular recognition

The development, organization and growth of complex organisms as well as their interactions with the environment involve an intricate array of molecular recognition events. There is an increased awareness of the involvement of oligosaccharides in many of these processes. In this article, studies of oligosaccharide antigenicity, and the way these have been interpreted with respect to oligosaccharide function will be discussed. In addition, examples of oligosaccharides as receptor, first, as receptors and determinants of susceptibility to an exogenous infective agent and secondly, as recognition structures possibly involved in endogenous interactions, will be described. This will be followed by a discussion of the recent hypothesis in which oligosaccharides are envisaged as recognition structures and integral components of cell growth-regulating networks. Finally, an outline of new strategies for decoding the information content in glycoprotein oligosaccharides will be given.     

Lipid metabolism in Chinese hamster V79-R membranes composed of unusual phospholipid molecular species

The relationship between the inhibition of cell growth and the changes in phospholipid metabolism in the presence of erucic acid was studied in Chinese hamster V79-R cells. 1. The addition of erucic acid to the medium inhibited cell growth. The degree of inhibition by erucic acid at a given concentration was dependent on cell density. 2. Exogenous erucic acid was incorporated into cellular phospholipids to form new phospholipid molecular species, which were identified to be the erucoyl/oleoyl, erucoyl/gondoyl and erucoyl/erucoyl species. 3. Synthesis of phosphatidylcholine and phosphatidylethanolamine in endoplasmic reticulum was reduced by erucic acid. Erucic acid had no effect on membrane flow of phospholipids from endoplasmic reticulum to plasma membrane. 4. The specific activity of sn-glycerol-3-phosphate acyltransferase in the membrane fraction from the cells supplemented with erucic acid was lower than that from the control cells. The reduction of phospholipid synthesis was attributed to the decrease in sn-glycerol-3-phosphate acyltransferase activity.     

Cholesterol is enriched in the sphingolipid patches on the substrate near nonpolarized MDCK cells,but not in the sphingolipid domains in their plasma membranes

Information about the distributions of cholesterol and sphingolipids within the plasma membranes of mammalian cells provides insight into the roles of these molecules in membrane function. In this report, high-resolution secondary ion mass spectrometry was used to image the distributions of metabolically incorporated rare isotope-labeled sphingolipids and cholesterol on the surfaces of nonpolarized epithelial cells. Sphingolipid domains that were not enriched with cholesterol were detected in the plasma membranes of subconfluent Madin-Darby canine kidney cells. Surprisingly, cholesterol-enriched sphingolipid patches were observed on the substrate adjacent to these cells. Based on the shapes of these cholesterol-enriched sphingolipid patches on the substrate and their proximity to cellular projections, we hypothesize that they are deposits of membranous particles released by the cell.     

Phospholipid metabolism in V79-R membranes composed of phospholipid molecular species containing trans-monoenoic fatty acids

The interrelationship between the inhibition of cell growth and changes in phospholipid molecular species was studied in the presence of elaidic, trans-11-eicosenoic, or brassidic acids in Chinese hamster V79-R cells. The addition of trans-monoenoic fatty acids to the medium inhibited cell growth and caused an increase in the total cellular content of phospholipids. However, there was no difference in the polar head group composition of these phospholipids among all the cells supplemented with trans-monoenoic fatty acids. Exogenous trans-monoenoic fatty acids were incorporated into cellular phospholipids to form novel phospholipid molecular species. Phospholipid synthesizing enzyme activities bound to the membranes composed of phospholipid molecular species of trans-monoenoic fatty acids were determined. Cholinephosphotransferase [EC 2.7.8.2] and ethanolaminephosphotransferase [EC 2.7.8.1] activities were decreased by trans-11-eicosenoic acid, but not changed by elaidic acid. Glycerophosphate acyltransferase [EC 2.3.1.15] activity was increased by elaidic acid and decreased by trans-11-eicosenoic acid. Cholinephosphate cytidylyltransferase [EC 2.7.7.15] activity was not changed by trans-monoenoic fatty acids.     

Phylogenetic species recognition and species concepts in fungi

The operational species concept, i.e., the one used to recognize species, is contrasted to the theoretical species concept. A phylogenetic approach to recognize fungal species based on concordance of multiple gene genealogies is compared to those based on morphology and reproductive behavior. Examples where Phylogenetic Species Recognition has been applied to fungi are reviewed and concerns regarding Phylogenetic Species Recognition are discussed.     

Target flexibility in molecular recognition

Induced-fit effects are well known in the binding of small molecules to proteins and other macromolecular targets. Among other targets, protein kinases are particularly flexible proteins, so that such effects should be considered in attempts at structure-based inhibitor design for kinase targets. This paper outlines some recent progress in methods for including target flexibility in computational studies of molecular recognition. A focus is the "relaxed complex method," in which ligands are docked to an ensemble of conformations of the target, and the best complexes are re-scored to provide predictions of optimal binding geometries. Early applications of this method have suggested a new approach to the development of inhibitors of HIV-1 Integrase.     

Screening of fungal species for fumonisin production andfumonisin-like disruption of sphingolipid biosynthesis

Fumonisins are mycotoxins produced by several species of Fusaria. They are found on corn and in corn-based products, can cause fatal illnesses in some animals and are suspected human esophageal carcinogens. Fumonisins are believed to cause toxicity by blocking ceramide synthase, a key enzyme in sphingolipid biochemistry which converts sphinganine (or sphingosine) and fatty acyl CoA to ceramide. Relatively fewfungal species have been evaluated for their ability to produce fumonisins. Fewer have been studied to determine if they produce ceramide synthase inhibitors, whether fumonisin-like structures or not, therefore potentially having toxicity similar to fumonisins. We analyzed corn cultures of 49 isolates representing 32 diversespecies of fungi for their ability to produce fumonisins. We also evaluated the culture extracts for ceramide synthase activity. Only cultures prepared with species reported previously to produce fumonisins – Fusarium moniliforme and F. proliferatum – tested positive for fumonisins. Extracts of these cultures inhibited ceramide synthase, as expected. None of the other fungal isolates we examined produced fumonisins or other compounds capable of inhibiting ceramide synthase. Although the fungi we selected for these studies represent only a few ofthe thousands of species that exist, they share the commonality that they are frequently associated with cereal grasses, including corn, either as pathogens or as asymptomatic endophytes. Thus,these results should be encouraging to those attempting to find ways to genetically manipulate fumonisin-producing fungi, tomake corn more resistant, or to develop biocontrol measures because it appears that only a relatively few fungal contaminants of corn can produce fumonisins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phosphatidylglucoside exists as a single molecular species with saturated fatty acyl chains in developing astroglial membranes

We previously found that phosphatidylglucoside (PtdGlc), a novel glycolipid expressed in HL60 cells, plays a role in forming signaling microdomains involved in cellular differentiation. Because cells contain minute levels of PtdGlc, pure PtdGlc is very difficult to isolate. Thus, its complete structure has never been assessed. To aid in analyzing PtdGlc, we generated a PtdGlc-specific monoclonal antibody, DIM21, by immunizing mice with detergent-insoluble membranes isolated from HL60 cells [Yamazaki, Y., et al. (2006) J. Immunol. Methods 311, 106-116]. DIM21 immunostaining of murine CNS tissues revealed stage- and cell type-specific localization of the DIM21 antigen during development, with especially high levels of expression in radial glia/astroglia. DIM21 immunostained cultured hippocampal astroglia in a punctate fashion. To characterize the structure of PtdGlc, we isolated DIM21 antigen from fetal brains. Using successive column chromatography, we purified two previously unrecognized glycolipids, PGX-1 and PGX-2, from embryonic day 21 rat brains. DIM21 reacted more strongly to PGX-2 than to PGX-1. Structural analyses with 600 MHz (1)H NMR, FT-ICR mass spectrometry, and GC revealed that PGX-1 is phosphatidyl beta-d-(6-O-acetyl)glucopyranoside and PGX-2 is phosphatidyl beta-d-glucopyranoside. The yields of PGX-1 and PGX-2 were approximately 250 +/- 150 and 440 +/- 270 nmol/g of dried brains, respectively. Surprisingly, both glycolipids were composed exclusively of C18:0 at the C1 position and C20:0 at the C2 position of the glycerol backbone. This saturated fatty acyl chain composition comprising a single molecular species rarely occurs in known mammalian lipids and provides a molecular basis for why PtdGlc resides in raftlike lipid microdomains.     

A single-channel sensor based on gramicidin controlled by molecular recognition at bilayer lipid membranes containing receptor

A novel ion-channel sensor based on a membrane bound receptor and a single gramicidin channel is described, in which the binding of an analyte to the membrane bound receptor modulates the single-channel activity of gramicidin. The sensor is composed of a planar bilayer lipid membrane (BLM) containing biotin-labeled phosphatidylethanolamine as receptor for avidin and gramicidin as signal transducer. When the receptor catches an analyte (avidin or ferritin-labeled avidin (FA)) at the membrane surface, the bilayer structure is locally distorted and the gramicidin monomer/dimer kinetics is modulated in a manner that the fraction of channel opening with a short lifetime ( < or = 100 ms) to the total opening events increases. The fraction was found to increase with the concentration of avidin from 1.0 x 10(-9) to 1.0 x 10(-6) M and of FA from 1.0 x 10(-9) to 1.0 x 10(-8) M. With dinitrophenyl-labeled PE embedded as receptor in the BLM for monoclonal anti-dinitrophenyl antibody (anti-DNP), the fraction of channel openings ( < or = 100 ms) increased with the concentration of anti-DNP from 2.0 x 10(-9) to 2.0 x 10(-7) g/ml. Bovine serum albumin (BSA) and anti-BSA antibody caused no changes in the channel opening. The possible mechanism of analyte-induced modulation of single-channel activity of gramicidin is also discussed.