Lipid Recognition Research Articles

Lipid A diversity and the innate host response to bacterial infection

Lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, is a potent immunostimulatory molecule which activates the innate host defense system. Over the past few years progress has been made in identifying the molecular mechanisms of host recognition of lipid A (a component of lipopolysaccharide), the identification of the genes requried for Escherichia coli lipid A biosynthesis, and the role of lipid A acylation when viable bacteria are presented to host cells. Recent data indicate that bacteria can regulate this molecule in response to different host microenvironments. Host factors that induce lipid A modifications and the resultant changes in host response remain to be determined.

Myelination: Coordinated regulation of many molecular elements

The formation of the myelin sheath requires the coordinated regulation of a number of gene products, both glial and axonal. These include the myelin structural genes, enzymes involved in lipid biosynthetic pathways, transcriptional regulators, trophic and recognition factors. Alterations in gene dosage or gene expression timing can have profoundly deleterious effects on either the establishment or maintenance of the myelin sheath. In this review, the major structural genes are described, as are the consequences of changes in gene dosage which occur as a result of either natural mutations or experimentally mediated events. While it is clear that our knowledge has expanded greatly with regard to these events, there continue to be large gaps, especially within the genetics of gene regulation and the mechanisms of glial axon interactions. MRDD Research Reviews 1998; 4:179–186 © 1998 Wiley-Liss, Inc.

Locating lipids in the CNS: an historical perspective.

Localization of lipids in the CNS is considered from an historical perspective. General consideration is given to the identification and separation of different parts of the CNS and to the recognition and detection of lipids. Problems associated with each of these aspects are noted. More treatment is given to the localization of gangliosides and the contributions of Leon Wolfe are highlighted.

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene. A possible link between infection and lupus.

The V H4-21 (V4-34) gene segment, a member of the V H4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of V n4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the Iii antigen, and also capable of encoding anti-DNA antibodies. Recognition of Iii antigen or DNA appears to be via two distinct sites on V H, with Iii binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with Iii, rendering a single sequence monospecific for Iii or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-Iii antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity. [Hum Antibod Hybridomas 1995: 6: 52-56]

Dual recognition of lipid A and DNA by human antibodies encoded by the VH4-21 gene: A possible link between infection and lupus

The VH4-21 (V4-34) gene segment, a member of the VH4 family, is expressed early in B-cell maturation and is utilized by approximately 6% of normal adult B lymphocytes. This prevalence indicates an importance of VH4-21 in the B-cell repertoire. The gene also encodes certain autoantibodies being mandatory for pathological IgM anti-red cell antibodies directed against the I/i antigen, and also capable of encoding anti-DNA antibodies. Recognition of I/i antigen or DNA appears to be via two distinct sites on VH, with I/i binding mediated by sequences in the framework region, and DNA binding correlating with the presence of positively charged amino acids in complementarity-determining region 3. However, these positively charged residues appear to suppress the ability of the framework region to interact with I/i, rendering a single sequence monospecific for I/i or DNA. The IgM anti-DNA antibodies also recognize bacterial lipid A, whereas the anti-I/i antibodies do not, indicating that CDR3 may be involved in binding the negatively charged lipid A. Structural similarities between the DNA backbone and lipid A provide a possible explanation for this cross-reactivity. This dual recognition of bacterial antigen and autoantigen provides a potential link between infection and autoimmunity.

Roles of lipid modifications of transducin subunits in their GDP-dependent association and membrane binding.

Transducin is an unusually soluble and dissociable heterotrimeric G-protein, although its T alpha and T beta gamma subunits are N-acylated and farnesylated, respectively. These lipid modifications have been suggested to contribute directly to the GDP-dependent T alpha-T beta gamma association, through specific lipid recognition sites on both protein subunits. We studied the dependence of subunit association on their bound lipids and on the presence of different lipidic environments. Association of native N-acylated (nT alpha) or acyl-free recombinant (rT alpha) T alpha with farnesylated and carboxymethylated (fcT beta gamma), farnesylated (fT beta gamma), or farnesyl-free (dfT beta gamma) T beta gamma was analyzed by gradient centrifugation and gel filtration in the presence of detergent or phospholipid-cholate micelles and by cosedimentation with phospholipid vesicles. Without detergent, nT alpha GDP and fcT beta gamma associate only weakly in solution. The loss of T alpha acyl or T beta gamma farnesyl residues induces total dissociation. With detergent or lipids, isolated fcT beta gamma binds tightly to micelles or vesicles, while dfT beta gamma does not; nT alpha GDP binds weakly, while deacylated rT alpha GDP does not bind at all; and nT alpha GDP binds cooperatively with fcT beta gamma, while rT alpha GDP does not. Thus (i) the T alpha acyl chain binds weakly, whereas the T beta gamma farnesyl chain binds strongly to membrane lipids; (ii) there is no evidence for binding of the T alpha acyl chain to a polypeptide site in T beta gamma, nor for binding of the T beta gamma farnesyl chain to a polypeptidic site in T alpha, but the T alpha acyl chain seems to bind cooperatively with the T beta gamma farnesyl chain in the membrane lipids; (iii) the insertion of the two protein-attached lipids into the same membrane could contribute to the association of both subunits by favoring collision coupling of the properly oriented protein moieties on the membrane surface.

Human antilipid A monoclonal antibodies bind to human B cells and the i antigen on cord red blood cells.

We describe two independently derived human mAb, A6(H4C5) and 216, initially selected for their reactivity to the lipid A domain of bacterial LPS, which also react with the following Ag: the i Ag present on cord RBC, a ligand on human B lymphocytes, and to certain autoantigens, defining these mAb as polyreactive. Both mAb have specific affinity for a carbohydrate epitope consisting minimally of a disaccharide with an acyl substitution at the 2-carbon position. Structural examination of the diverse Ag recognized by the two antibodies reveals the presence of this carbohydrate structure required for antibody binding. A6(H4C5) and 216 are IgM in isotype, but differ in their L chain expression. Molecular analysis shows that both the mAb are encoded by a highly conserved VH4 gene, designated VH4-21. This gene encodes a number of autoantibodies, particularly cold agglutinins. Specific recognition of lipid A and of a carbohydrate epitope on B lymphocytes by the two human mAb suggests a dual function for the highly conserved VH4-21 gene in antibacterial response and in B cell development and regulation.

Lipopolysaccharide epitope specificity and binding cross reactivity of the human IgM anti-lipid a monoclonal antibody SdJ5-1.17.15

The binding properties and specificity of the SdJ5-1.17.15 (SdJ5) human IgM monoclonal antibody (mAb), prepared against S. minnesota R595 heat killed whole organisms, were assessed by dot blot assay in vitro. In that assay, lipopolysaccharide (LPS) related antigens, immobilized on nitrocellulose membrane, were reacted with the test and control human IgM antibodies. Results indicated the SdJ5 mAb reacted specifically with lipid A, a component of LPS, from a variety of bacterial species, but not with the whole LPS molecule. The inability of the mAb to react with whole LPS in dot blot assay was attributed to the possible effect of the solid phase on epitope exposure or structure. This hypothesis was tested by inhibition studies which indicated that liquid phase adsorption with LPS abolished or greatly reduced the specific recognition of solid phase lipid A by the mAb. These results indicated that LPS-associated antigens were recognized by the SdJ5 anti-lipid A mAb in liquid, but not solid, phase. In an attempt to identify the SdJ5-specific epitope on lipid A, the pattern of reactivities of different lipid A analogues with the mAb were examined by dot blot assay. Results indicated that a combination of the fatty acid side chains and the phosphate groups of lipid A (both groups being implicated as important for sepsis mediation) were either directly involved in the epitope structure or affected the exposure of epitope in solid phase. Because SdJ5 recognizes an LPS epitope on lipid A closely associated with endotoxin activity, this mAb could potentially be a useful therapeutic agent against septic shock.

Formation of Molecular Strands by Hydrogen Bonds at the Gas–Water Interface: Molecular Recognition and Quantitative Hydrolysis of Barbituric Acid Lipids

Reminiscent of an enzyme reaction is the proposed mechanism for the hydrolysis of the barbituric acid lipid 1, R = C16H33, when it forms a strand structure with 2,4,6-triaminopyrimidine (TAP) at the gas–water interface (2). The TAP-catalyzed hydrolysis at pH 6.5 is 14 times faster than the simple addition of 0H to the CC double bond at pH 10.

Sequence determination and characterization of a phospholipase A2 isozyme from Trimeresurus gramineus (green habu snake) venom

In addition to phospholipase A2-I (PLA2-I) reported previously (Odaet al., 1991, Toxicon29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA2-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-I, PLA2-II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLA2-I and PLA2-II for Ca2+. Secondary structure compositions of PLA2-II were similar to those of PLA2-I and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLA2-I and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA2-I and Asn for PLA2-II).

CDNA Molecular Cloning of Geotrichum candidum Lipase

The cDNA clone of Geotrichum candidum (Geo.) lipase was isolated from the Geo. cDNA library by colony hybridization using 32P-labeled oligonucleotides corresponding to a partial amino acid sequence of this enzyme. The nucleotide sequence of the cDNA determined by the dideoxy chain terminating method included some partial amino acid sequences determined by Edman degradation, and the overall amino acid composition deduced from the cDNA coincided with that from amino acid analysis of this protein. The cloned cDNA coded a protein of 554 amino acids and a hydrophobic signal sequence of 19 amino acids. Geo. lipase contained the -Gly-X-Ser-X-Gly- sequence which is believed to form part of the interfacial lipid recognition site.

The relation between the Nissl and Sudan black pictures of the motor neurones of the spinal cord of the adult horse, with special reference to the cytoplasmic inclusions

ABSTRACT The motor neurones of the spinal cord of the horse, fixed in neutral formaldehydecalcium, embedded in paraffin, and sectioned, are easily coloured with Sudan black dissolved in propylene glycol or in tertiary butanol. No procedure for ‘unmasking’ lipids is necessary. Any effect due to electropolar bonds are excluded by blocking and extraction procedures. The true lysochrome effect is confirmed by the removal of the colour by lipid solvents and the absence of stable sudanophilia and of background colouring. No brown colour suggestive of decomposition products is seen, and this is further confirmed by absorption spectra of the Sudan black solutions. The structures revealed are in the main identical with those of ordinary Nissl preparations made, for instance, by Einarson’s chromalum/gallocyanin stain. In addition, however, structures closely similar to ordinary Nissl bodies are seen in the axon hillock and in the axon. This suggests that in these situations Sudan black colours corpuscles that are lacking in ribonucleic acid (presumably endoplasmic reticulum devoid of ribosomes). The nature of the lipids revealed by Sudan black in these cells has not been determined histochemically. Extraction in pyridine either before or after fixation had no definite effect on the colouring of the Nissl bodies or of the nucleus. The histochemical recognition of lipids in the cytoplasmic network of neurones has recently been reported by David and Brown (1961). The present paper supports the findings of these authors. Different material and methods were used, but the colouring agents were the same.

The Histochemical Recognition of Lipid in the Cytoplasmic Network of Neurones of Vertebrates

1. A method for the histochemical demonstration of the lipid of the cytoplasmic network (‘Golgi apparatus’, ‘Nissl complex’) of the neurones of vertebrates was accidentally discovered. 2. Very small pieces of tissue are fixed in Lewitsky/calcium or Flemming/calcium, postchromed, and embedded in gelatine. Frozen sections are cut at 0.5 to 5 µ, and coloured in Sudan black after bleaching. 3. The intensity of the colouring of lipid by Sudan black can be increased by centrifuging the tissue before fixation. 4. The method described in this paper demonstrates lipid in the strands and bodies of the cytoplasmic network of vertebrate neurones. It was shown, in sections subsequently decolorized and then stained in a basic dye, that the sudanophil reticular objects are the Nissl complex.

Chloroplast Lipids as Carbohydrate Reservoirs.

While the diurnal deposition of starch within the chloroplast has long been recognized, little has been known of the photosynthate reservoirs more intimately associated with the photochemical apparatus. This paper reports recognition of chloroplast and chromatophore lipids containing significant concentrations of available carbohydrate. A high degree of orientation of lipids within the chloroplast has been revealed by observation of positive dichroism (1, 2) when dye molecules were adsorbed in the lipid regions. Positional relationships of the photoacceptors, chlorophylls and carotenoids (3), and the chloroplast lipids almost certainly exist. Most concepts of chloroplast structure embrace these observations. It now appears that these lipids may participate in carbohydrate metabolism as well. They consist largely of galactosyl monoglycerides (4) and phosphatidyl glycerols (5). Both have the requisite long chain fatty acids and hydrophylic groups essential for orientation at lipid-pigment-protein interfaces. Their remarkable metabolic activity (6) suggests their auxiliary role as energy storage reservoirs.