Search

The appropriate regulation of thylakoid lipid synthesis is essential for the function of chloroplasts. In plant cells, membrane lipids synthesized in the ER are utilized as a precursor for the synthesis of chloroplast glycolipids. This pathway is thought to be mediated by the transport of glycerolipids synthesized in the ER into chloroplasts. However, we have little knowledge about the proteins involved in the lipid transfer between these organelles in plant cells. Here we show a protein, STAR2, containing the START (Steroidogenic acute regulatory protein-related lipid transfer) domain known to function as a lipid transporter, is involved in the incorporation of ER-derived fatty acids into chloroplast glycolipids in Marchantia polymorpha. We found that STAR2 localizes on the chloroplast envelope membrane as a punctuate structure and is required for the increase of C20 fatty acids, which are synthesized in the ER, in chloroplast glycolipids in response to phosphate deprivation. Our results indicate that STAR2 of M. polymorpha is likely to be involved in the lipid transfer from ER to chloroplast, presumably as a lipid transporter.

Muscle myogenesis is an essential step for muscle development and recovery. During muscle fusion, multiple molecules are thought to be necessary for the formation of normal myotubes. Milk fat globule-EGF factor 8 (MFG-E8) and Gas6 are phosphatidylserine-recognizing bridging molecules that are secreted mainly from immune cells. In this study, we confirmed that these molecules are expressed and secreted from C2C12 cells. Mouse muscle and satellite cells also expressed these molecules. MFG-E8 was highly expressed and secreted in both undifferentiated and differentiated C2C12 cells. We observed that MFG-E8 and Gas6 were bound to the surface of differentiated C2C12 cells more compared with undifferentiated cells. Additionally, the treatment of recombinant MFG-E8 upregulated expression of myogenic genes and suppressed apoptosis during myogenesis in C2C12 cells. In this paper, we discuss the presence of novel functional molecules expressed and secreted in the skeletal muscle. The results of this study suggest that bridging molecules are one of the determinants of myogenesis or other muscle responses.

Arabidopsis PATATIN-RELATED PHOSPHOLIPASE 2A (pPLA-IIα) participates in the responses to various growth conditions. The factors affecting pPLA-IIα gene expression and pPLA-IIα protein activity for gycerolipids have been studied thoroughly, but the role of pPLA-IIα during the reproductive phase remains unclear. The effect of pPLA-IIα on flowering time was therefore investigated. ppla-iiα mutants flowered later than wild-type plants under long day conditions. Expression of the floral stimulators FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) was downregulated in ppla-iiα mutants compared with their expression in wild-type plants, but expression of the floral repressor FLOWERING LOCUS C (FLC) was upregulated. In addition, expression levels of COLDAIR, a long intronic noncoding RNA, decreased in ppla-iiα mutants. Taken together, these data indicate that pPLA-IIα acts as a positive regulator of flowering time through repression of FLC expression.

MPIase is a glycolipid involved in protein integration in E. coli. Recently, we identified CdsA, a CDP-diacylglycerol (CDP-DAG) synthase, as a biosynthetic enzyme for MPIase. YnbB is a CdsA paralogue with a highly homologous C-terminal half. Under CdsA-depleted conditions, YnbB overproduction restored MPIase expression, but not phospholipid biosynthesis. YnbB complemented the growth defect of the cdsA knockout when Tam41p, a mitochondrial CDP-DAG synthase, was co-expressed, suggesting that YnbB possesses sufficient activity for MPIase biosynthesis, but not for phospholipid biosynthesis. Consistently, a chimera consisting of the CdsA N-terminal half and the YnbB C-terminal half (CdsA-N-YnbB-C) complemented the cdsA knockout by itself, but a chimera consisting of the YnbB N-terminal half and the CdsA C-terminal half (YnbB-N-CdsA-C) required co-expression of Tam41p for the complementation. The biosynthetic rate for CDP-DAG in CdsA and CdsA-N-YnbB-C was much faster than that in YnbB and YnbB-N-CdsA-C, indicating that the N-terminal half of CdsA accelerates CDP-DAG biosynthesis to give the fast cell growth. Therefore, the role of YnbB seems to be as a backup for MPIase biosynthesis, suggesting that YnbB is dedicated to MPIase biosynthesis. A mutant with a high pH-sensitive CdsA8 was unable to grow even under permissive conditions when the ynbB gene was deleted, supporting its auxiliary role in the CdsA function.

Sulfoquinovosyl diacylglycerol (SQDG) is present in the membranes of cyanobacteria or their descendants, plastids at species-dependent levels. We investigated the physiological significance of the intrinsic SQDG content in the cyanobacterium Synechococcus elongatus PCC 7942, with the use of its mutant, in which the genes for SQDG synthesis, sqdB and sqdX, were overexpressed. The mutant showed a 1.3-fold higher content of SQDG (23.6 mol% relative to total cellular lipids, cf., 17.1 mol% in the control strain) with much less remarkable effects on the other lipid classes. Simultaneously observed were 1.6- to 1.9-fold enhanced mRNA levels for the genes responsible for the synthesis of the lipids other than SQDG, as if to compensate for the SQDG overproduction. Meanwhile, the mutant showed no injury to cell growth, however, cell length was increased (6.1 ± 2.3, cf., 3.8 ± 0.8 μm in the control strain). Accordingly with this, a wide range of genes responsible for cell division were 1.6–2.4-fold more highly expressed in the mutant. These results suggested that a regulatory mechanism for lipid homeostasis functions in the mutant, and that SQDG has to be kept from surpassing the intrinsic content in S. elongatus for repression of the abnormal expression of cell division-related genes and, inevitably, for normal cell division.

The level of 8-hydroxy-2-deoxyguanosise (8-OHdG) is a marker of oxidative stress. The objective of this study was to evaluate the effect of enzyme replacement therapy (ERT) on the level of 8-OHdG in patients with Fabry cardiomyopathy and the clinical evolution of Fabry cardiomyopathy.We measured the serum levels of 8-OHdG in 20 healthy control and 22 patients with Fabry cardiomyopathy before and after ERT.The mean lysoGb3 and 8-OHdG levels was significantly increased in patients with Fabry cardiomyopathy compared with that of control subjects (lysoGb3, 3.6 ± 1.1 nM vs. 0.4 ± 0.1 nM, p 0.01; 8-OHdG, 4.5 ± 0.5 ng/mL vs. 3.4 ± 0.4 ng/mL, P 0.05). The mean lysoGb3 and 8-OHdG levels was significantly reduced after ERT for 14.2 months (lysoGb3, 3.6 ± 1.1 nM vs. 2.9 ± 1.1 nM, P 0.05; 8-OHdG, 4.5 ± 0.5 ng/mL to 4 ± 0.4 ng/mL, P 0.05). These changes were accompanied by decreases in LVM and LVMI.We demonstrated that the serum 8-OHdG levels is increased in patients with Fabry cardiomyopathy (FC) and that successful management of FC with ERT is associated with a decrease in this oxidative stress marker. Serum 8-OHdG levels can be used not only as a noninvasive biomarker of oxidative stress in patients with FC but also an objective and quantitative parameter in the follow-up of patients during ERT.

An ideal antibiofilm strategy is to control both in the quality and quantity of biofilm while maintaining the benefits derived from resident microflora. Vizantin, a recently developed immunostimulating compound, has also been found to have antibiofilm property. This study evaluated the influence on biofilm formation of Streptococcus mutans in the presence of sulfated vizantin and biofilm development following bacterial adhesion on a hydroxyapatite disc coated with sulfated vizantin. Supplementation with sulfated vizantin up to 50 μM did not affect either bacterial growth or biofilm formation, whereas 50 μM sulfated vizantin caused the biofilm to readily detach from the surface. Sulfated vizantin at the concentration of 50 μM upregulated the expression of the gtfB and gtfC genes, but downregulated the expression of the gtfD gene, suggesting altered architecture in the biofilm. Biofilm development on the surface coated with sulfated vizantin was inhibited depending on the concentration, suggesting prevention from bacterial adhesion. Among eight genes related to bacterial adherence in S. mutans, expression of gtfB and gtfC was significantly upregulated, whereas the expression of gtfD, GbpA and GbpC was downregulated according to the concentration of vizantin, especially with 50 μM vizantin by 0.8-, 0.4-, and 0.4-fold, respectively. These findings suggest that sulfated vizantin may cause structural degradation as a result of changing gene regulation related to bacterial adhesion and glucan production of S. mutans.

Sialyltransferases (STs) catalyze the addition of sialic acids to the non-reducing ends of glycoproteins and glycolipids. In this work, we examined the acceptor specificity of five human α(2,3)sialyltransferases, namely ST3Gal -I, -II, -III, -IV and -VI. KM values for each of these enzymes is presented using radioactivity for acceptors containing Type-I (Galβ1,3GlcNAc), Type-II (Galβ1,4GlcNAc), Type-III (Galβ1,3GalNAc) and Core-2 (Galβ1,3(GlcNAcβ1,6)GalNAc) reactive groups. Several variants of acceptors inhibited ST3Gal activity emphasizing structural role of acceptor in enzyme-catalyzed reactions. In some cases, mass spectrometry was performed for structural verification. The results demonstrate human ST3Gal-I catalysis towards Type-III and Core-2 acceptors with KM = 5-50 μM and high VMax values. The KM for ST3Gal-I and ST3Gal-II was 100 and 30-fold lower, respectively, for Type-III compared to Type-I acceptors. Variants of Type-I and Type-II structures characterized ST3Gal-III, -IV and -VI for their catalytic specificity. This manuscript also estimates KM for human ST3Gal-VI using Type-I and Type-II substrates. Together, these findings built a platform for designing inhibitors of STs having therapeutic potential.

To investigate effects of Ca level varying with feeding time daily in sows during late pregnancy on placental lipid metabolism and transport in pigs, sixty pregnant sows were assigned to 3 groups: the CON group was fed low-Ca diet with 11.25 g CaCO3 at 0600 h and 1500 h, H-L group was fed low-Ca diet with 22.5 g CaCO3 at 0600 h and low-Ca diet at 1500 h, and L-H group was fed low-Ca diet at 0600 h and low-Ca diet with 22.5 g CaCO3 at 1500 h, respectively. Serum from sows and umbilical cord and placenta were collected during delivery. Results showed that, compared with the CON group, H-L feeding significantly increased maternal serum total triglyceride (TG) and umbilical serum high-density lipoprotein (HDL) (P 0.05). The results showed that long chain fatty acid (FA) contents in placenta were significantly increased in H-L and L-H groups (P 0.05). Experiments on genes involved in glycolipid metabolism showed that H-L or L-H feeding inhibited mRNA expression of GLUT3, GLUT4, FAS, FABP1, FABPpm, FAT/CD36, while activated the mRNA expression of FASD1, FASD2 and SCD in placenta (P 0.05). In addition, experiments on genes involved in biological clock showed that L-H feeding sequence activated the mRNA expression of per1 and clock, while H-L and L-H feeding sequence inhibited mRNA expression of per2 in placenta (P 0.05). It is concluded that maternal supplementation with Ca varying with feeding time daily during late pregnancy affects placental lipid metabolism and transport in pigs by regulating the mRNA expression related to lipid metabolism and the circadian clock.

Endoplasmic reticulum (ER) is an indispensable organelle for secretory protein synthesis as well as metabolism of phospholipids and their derivatives in eukaryotic cells. Various external and internal factors may cause an accumulation of aberrant proteins in the ER, which causes ER stress and activates cellular ER stress responses to cope with the stress. In animal research, molecular mechanisms for protein quality control upon ER stress are well documented; however, how cells maintain lipid homeostasis under ER stress is an emerging issue. The ratio of phosphatidylcholine (PC) to phosphatidylethanolamine (PE), two major phospholipid classes, is important under ER stress in animal cells. However, in seed plants, no study has reported on the changes in membrane lipid content under ER stress, although a number of physiologically important environmental stresses, such as heat and salinity, induce ER stress. Here, we investigated membrane glycerolipid metabolism under ER stress in Arabidopsis. ER stress transcriptionally affected PC and PE biosynthesis pathways differentially, with no significant changes in membrane glycerolipid content. Our results suggest that higher plants maintain membrane lipid equilibrium during active transcription of phospholipid biosynthetic genes under ER stress.

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.

Recently, aqueous nanoparticles have been used in drug-delivery systems for new type medicines. In particular, milk-casein micelles have been used as drug nanocarriers for targeting cancer cells. Therefore, nanostructure observation of particles and micelles in their native liquid condition is indispensable for analysing their function and mechanisms. However, traditional optical and scanning electron microscopy have difficulty observing the nanostructures of aqueous micelles. Recently, we developed a novel imaging technique called scanning electron-assisted dielectric microscopy (SE-ADM) that enables observation of various biological specimens in water with very little radiation damage and high-contrast imaging without staining or fixation at an 8-nm spatial resolution. In this study, for the first time, we show that the SE-ADM system is capable of high-resolution observation of whole-milk specimens in their natural state. Moreover, we successfully observe the casein micelles and milk-fat globules in an intact liquid condition. Our SE-ADM system can be applied to various biological particles and micelles in a native liquid state.

Heterocyst glycolipid synthase (HglT) catalyzes the final step of heterocyst glycolipid (Hgl) biosynthesis, in which a glucose is transferred to the aglycone (fatty alcohol). Here we describe the isolation of hglT null mutants. These mutants lacked Hgls under nitrogen-starved conditions and instead accumulated fatty alcohols. Differentiated heterocyst cells in the mutants were morphologically indistinguishable from those of the wild-type cells. Interestingly, the mutants grew under nitrogen starvation but fixed nitrogen with lower nitrogenase activity than did the wild-type. The mutants had a pale green phenotype with a decreased chlorophyll content, especially under nitrogen-starved conditions. These results suggest that the glucose moiety of the Hgls may be necessary for optimal protection against oxygen influx but is not essential and that aglycones can function as barriers against oxygen influx in the heterocyst cells.

Trehalose 6,6′-dimycolate (TDM) is a major glycolipid of the cell wall of mycobacteria with remarkable adjuvant functions. To avoid detection by the host innate immune system, invading mycobacteria down-regulate the expression of TDM by utilizing host-derived glucose as a competitive substrate for their mycolyltransferases; however, this enzymatic reaction results in the concomitant biosynthesis of glucose monomycolate (GMM) which is recognized by the acquired immune system. GMM-specific, CD1-restricted T cell responses have been detected in the peripheral blood of infected human subjects and monkeys as well as in secondary lymphoid organs of small animals, such as guinea pigs and human CD1-transgenic mice. Nevertheless, it remains to be determined how tissues respond at the site where GMM is produced. Here we found that rhesus macaques vaccinated with Mycobacterium bovis bacillus Calmette–Guerin mounted a chemokine response in GMM-challenged skin that was favorable for recruiting T helper (Th)1 T cells. Indeed, the expression of interferon-γ, but not Th2 or Th17 cytokines, was prominent in the GMM-injected tissue. The GMM-elicited tissue response was also associated with the expression of monocyte/macrophage-attracting CC chemokines, such as CCL2, CCL4 and CCL8. Furthermore, the skin response to GMM involved the up-regulated expression of granulysin and perforin. Given that GMM is produced primarily by pathogenic mycobacteria proliferating within the host, the Th1-skewed tissue response to GMM may function efficiently at the site of infection.

Sulfoquinovosyl diacylglycerol, which mainly comprises thylakoid membranes in oxygenic photosynthetic organisms, plays species-dependent roles in freshwater microbes. In this study, a sulfoquinovosyl-diacylglycerol deficient mutant was generated in a cyanobacterium, Synechococcus sp. PCC 7002, for the first time among marine microbes to gain more insight into its physiological significance. The mutation had little deleterious impact on photoautotrophic cell growth, and functional and structural properties of the photosystem II complex. These findings were similar to previous observations for a freshwater cyanobacterium, Synechococcus elongatus PCC 7942, but were distinct from those for another freshwater cyanobacterium, Synechocystis sp. PCC 6803, and a green alga, Chlamydomonas reinhardtii, both of which require sulfoquinovosyl diacylglycerol for cell growth and/or photosystem II. Therefore, the functionality of PSII to dispense with sulfoquinovosyl diacylglycerol in Synechococcus sp. PCC 7002, similar to that in Synechococcus elongatus PCC 7942, seemed to have been excluded from the evolution of the PSII complex from cyanobacteria to green algal chloroplasts. Meanwhile, sulfoquinovosyl diacylglycerol was found to contribute to photoheterotrophic growth of Synechococcus sp. PCC 7002, which revealed a novel species-dependent strategy for utilizing SQDG in physiological processes.

The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulation of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2β (PI3KC2β) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2β-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2β activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease.

The term human epithelial carcinoma antigen (HCA) has been applied collectively to mucin-type high molecular weight (>1000 kDa) glycoproteins that are over-expressed in epithelial cancers. Since the 1990s, over 40 monoclonal antibodies have been raised that recognize HCA. There has been evidence that the antigenic determinants are mostly carbohydrates, but details have been elusive. Here we have carried out carbohydrate microarray in analyses of one of the monoclonal antibodies, AE3, that has been regarded the ‘most carcinoma specific’ in respect to its ability to detect HCA in sera of patients with epithelial cancers. The microarrays encompassed a series of 492 sequence-defined glycan probes in the form of glycolipids and neoglycolipids. We have thus established that the antigen recognized by antibody AE3 is a carbohydrate sequence distinct from the A, B, H, Lewisa/b, Lewisx/y and T antigens, but that it is strongly expressed on the monosulfated tetra-glycosyl ceramide, SM1a, Galβ1-3GalNAcβ1-4(3-O-sulfate)Galβ1-4GlcCer. This is the first report of an anti-HCA to be characterized with respect to its recognition sequence and of the occurrence of the antigen on a glycolipid as well as on glycoproteins. Knowledge of a discrete glycan sequence as target antigen now opens the way to its exploration as a serologic cancer biomarker, namely to determine if the antigen elicits an autoantibody response in early non-metastatic cancer, or if it is shed and immunochemically detectable in more advanced disease.

We have previously shown that liposomes coated with a neoglycolipid constructed from mannotriose and dipalmitoylphosphatidylethanolamine (Man3-DPPE) activate peritoneal macrophages to induce enhanced expression of co-stimulatory molecules and MHC class II. In this study, we investigated the signaling pathways activated by the Man3-DPPE-coated liposomes (OMLs) in a murine macrophage cell line, J774A.1. In response to OML stimulation, ERK among MAPKs was clearly and transiently phosphorylated in J774 cells. ERK phosphorylation was also induced by treatment of the cells with Man3-DPPE and Man3-BSA, but not by uncoated liposomes. In addition, rapid and transient phosphorylation of Akt and Src family kinases (SFKs) was observed in response to OMLs. OML-induced ERK phosphorylation was inhibited by specific inhibitors of PI3K and SFKs, and OML-induced Akt phosphorylation was inhibited by a inhibitor of SFKs. Therefore, OMLs may activate the PI3K/Akt pathway through phosphorylation of Src family kinases to induce ERK activation.

Mycoplasma fermentans has been suspected as one of the causative pathogenic microorganisms of rheumatoid arthritis (RA) however, the pathogenic mechanism is still unclear. We, previously, reported that glycolipid-antigens (GGPL-I and III) are the major antigens of M. fermentans. Monoclonal antibody against the GGPL-III could detect the existence of the GGPL-III antigens in synovial tissues from RA patients. GGPL-III antigens were detected in 38.1% (32/84) of RA patient's tissues, but not in osteoarthritis (OA) and normal synovial tissues. Immunoelectron microscopy revealed that a part of GGPL-III antigens are located at endoplasmic reticulum. GGPL-III significantly induced TNF-alpha and IL-6 production from peripheral blood mononulear cells, and also proliferation of synovial fibroblasts. Further study is necessary to prove that M. fermentans is a causative microorganism of RA; however, the new mechanisms of disease pathogenesis provides hope for the development of effective and safe immunotherapeutic strategies based on the lipid-antigen, GGPL-III, in the near future.

We demonstrated that human serum albumin (HSA) patterns constructed in a solid substrate by using micro-contact printing (muCP) technique supported the deposition of phospholipid bilayer containing glycolipid, 10-tetradecyloxymethy-3,6,9,12-tetraoxahexacosyl 2-acetamido-2-deoxy-beta-D-glucopyranoside (PB1124). It is observed by confocal laser scanning microscopy (CLSM) that the obtained glycolipid patterns are well-defined, stable and can be used to recognize and immobilize Escherichia coli (E. coli). This strategy is promising to perform bacterial detection through solid surface recognition in a way of biosensors.

This is the first report on the antifungal effects of the new glycolipopeptide hassallidin A. Due to related molecular structure moieties between hassallidin A and the established antifungal drug caspofungin we assumed parallels in the effects on cell viability. Therefore we compared hassallidin A with caspofungin by antifungal susceptibility testing and by analysing the genome-wide transcriptional profile of Candida albicans. Furthermore, we examined modifications in ultracellular structure due to hassallidin A treatment by electron microscopy. Hassallidin A was found to be fungicidal against all tested Candida species and Cryptococcus neoformans isolates. MICs ranged from 4 to 8 microg/ml, independently from the species. Electron microscopy revealed noticeable ultrastructural changes in C. albicans cells exposed to hassallidin A. Comparing the transcriptional profile of C. albicans cells treated with hassallidin A to that of cells exposed to caspofungin, only 20 genes were found to be similarly up- or down-regulated in both assays, while 227 genes were up- or down-regulated induced by hassallidin A specifically. Genes up-regulated in cells exposed to hassallidin A included metabolic and mitotic genes, while genes involved in DNA repair, vesicle docking, and membrane fusion were down-regulated. In summary, our data suggest that, although hassallidin A and caspofungin have similar structures, however, the effects on susceptibility and transcriptional response to yeasts seem to be different.

Mycolic acids are long chain fatty acids that constitute the lipid-rich cell wall framework of mycobacteria. Upon infection, mycobacteria begin to synthesize glucose monomycolate (GMM), a glucosylated species of mycolic acids, by utilizing host-derived glucose as sugar source. Accordingly, GMM production serves as a good indicator for local invasion of mycobacteria, and its detection by the host immune system would favor efficient monitoring of mycobacterial infection. Here, we found that GMM was produced abundantly at 30 degrees C rather than at 37 degrees C and recognized by a GMM-specific, CD1-restricted T cell line that was isolated from mycobacteria-infected human skin. Since the common portal sites for mycobacterial infection include ventilating alveoli of the lung and the externally exposed skin that often render invading microbes survive at reduced temperature, sampling GMM by CD1 lipid antigen-presenting molecules may allow the host to detect mycobacterial infection at its early phases.

The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains d -glycero- d -talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy- d -manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe2+/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVA in vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis.

Flowers have distinct glycerolipid composition, yet its floral organ-specific profile remains elusive in Arabidopsis whose flowers are too tiny to dissect different floral organs. Here, we employed known floral homeotic mutants agamous-1 (ag-1) and apetala3-3 (ap3-3) to facilitate sample preparation enriched in different floral organs. The result of analysis on different polar glycerolipid classes and their fatty acid composition demonstrated that flowers of ap3-3 and ag-1 have distinct glycerolipid composition from that of wild type. Moreover, distinct set of glycerolipid biosynthetic genes is expressed in these mutants by qRT-PCR gene expression analysis. These data suggest that glycerolipid profile is distinct among different floral organs of Arabidopsis thaliana.

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.

Endogenous GM3 synthesis and full N-glycosylation in membrane receptors occurred in "4-epimerase-less" ldlD (Krieger's CHO mutant) cells cultured in Gal-containing medium, whereby components of detergent-insoluble, low-density, buoyant membrane fraction, termed "glycolipid-enriched microdomain (GEM)," varied significantly by translocation into or out of GEM. Integrins alpha3 and alpha5 were translocated into GEM in the presence of 0.5 or 0.25% Triton X-100, particularly in the absence of Gal, whereby integrins are underglycosylated and GlcCer is the major glycolipid component in GEM. Src family kinase was translocated into and enriched in GEM fractions when prepared in 0.5 or 0.25% Triton X-100 from cells grown in Gal-containing medium, whereby GM3 synthesis is induced. In contrast, caveolin is highly enriched in GEM when GM3 synthesis does not occur, and is translocated into high-density membrane fraction when GM3 synthesis occurs. The results suggest that levels of key molecules controlling cell adhesion and signaling are defined by translocation into or out of GEM, which depends on glycosylation state.

The globotriaosylceramide (Gb 3 ) verotoxin (VT) interaction is one of several examples of glycolipid receptors where the ceramide (or lipid) free oligosaccharides fail to show the expected binding parameters. We present a novel, yet simple strategy to synthesize monovalent, water soluble glycosphingolipid mimics which retain receptor function. Replacing the fatty acid chain with rigid, three dimensional hydrocarbon frames, such as adamantane, gives a novel class of neohydrocarbon glycoconjugates. Such adamantyl conjugates derived from Gb 3 showed significantly enhanced solubility in water compared to natural Gb 3 . Adamantyl-Gb 3 showed a thousand fold enhanced inhibitory activity (IC 50 = 1 μM) for VT-Gb 3 binding as compared to a lipid free Gb 3 oligosaccharide derivative, αGal1-4βGal1-4βGlc1-O-CH 2 CH(CH 2 SO 2 C 4 H 9 ) 2 (IC 50 > 2 mM). This represents a new approach to the generation of antagonists of glycolipid receptors.

An acidic glycolipid (Band 1), purified from P. brasiliensis by a combination of ion exchange chromatography, HPLC, and HPTLC, was found to be reactive with sera of all patients with paracoccidioidomycosis (PCM). Monosaccharide analysis of Band 1 yielded mannose and galactose in a 2:1 ratio, while mild acid hydrolysis and mild periodate oxidation/NaB3H4 reduction indicated the presence of a terminal galactofuranose. Preliminary analysis of 1H-NMR and MS data suggests that the structure of the glycan is Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins (Ins = myo-inositol). Removal of the galacto-furanose decreased by 60-80% the reactivity of sera from PCM patients with Band 1, suggesting that this residue is immunodominant. With the presumed absence of galactofuranose in mammalian hosts, compounds containing this residue may be useful targets for therapy of several parasitic and fungal diseases.

In search of endogenous oligosaccharide ligands for the endothelial adhesion molecule E-selectin in mouse, glycolipids from tissues and the neutrophilic cell line 32D c13 were tested for E-selectin binding. Kidneys of BALB/c and NMRI mice (but not CBA) and the 32D c13 cells were found to contain minor glycolipid populations that support strongly the binding of murine E-selectin. By chromatogram binding experiments and in situ liquid secondary ion mass spectrometry (LSIMS) with neoglycolipids derived from their endoglycoceramidase-released oligosaccharides, in conjunction with compositional and linkage analyses, one of the glycolipid ligands in kidney was identified as the Le x -active extended globo-glycolipid: Galβ1-4GlcNAcβ1-6GalNAcβ1-3Galα1-4Galβ1-4Glcβ1-Cer;|||Fucα1,3||Ga1β1,3|. Neoglycolipids enriched for the ligand structures were obtained from oligosaccharides released by endo-β-galactosidase from the 32D c13 cells. By TLC-LSIMS and antibody binding, the main E-selectin binding determinant on these was identified as sialyl-Le a .

The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications.

Specific sugar ligands which bind to rat L-selectin (LECAM-1, lymphocyte homing receptor)-IgG chimera were investigated by thin-layer chromatography-binding assay and ELISA. Rat L-selectin-IgG bound to native sulfated glycolipids such as sulfatide (I 3 SO 3 -GalCer), seminolipid, SM3(II 3 SO 3 -LacCer), SB2(III 3 SO 3 ,II 3 SO 3 -Gg3Cer), SB1a (IV 3 SO 3 ,II 3 SO 3 -Gg4Cer). The binding activity of the SM2 (II 3 SO 3 -Gg3Cer) carrying the internal sulfated Gal was very low, indicating that non-reducing terminal SO 3 H-3Galβ1- structure is essential for the binding. The reactivity of sulfatide was higher than that of sialyl Le x [Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glcβ1-Ceramide] either in the presence of 1 mM Ca 2+ or 1 mM Ca 2+ and 5 mM EGTA. All the non-fucosylatedgangliosides including ganglio-series and lacto-series type I and II chains tested gave a negative reaction. Neutral glycosphingolipids tested were all negative. Binding assay using synthetic sulfatide analogs indicated that the L-selectin-binding to its sulfated sugar ligands is position specific and depends on the number of the sulfate group, i.e., (SO 3 ) 3 -3,4,6Gal- > (SO 3 ) 2 -3,6Gal- > SO 3 -3Gal- > SO 3 -3Glc- > SO 3 -6Gal- > SO 3 -2Gal-. Lower reactivity of the chimera to SO 3 -3Glc- rather than SO 3 -3Gal- indicates that the chimera recognizes the configuration of hydroxyl group linked to the 4 carbon atom of the pyranose ring in galactose.

Fabry disease is a genetic disease caused by a deficiency of alpha-galactosidase A (GLA), which leads to systemic accumulation of glycolipids, predominantly globotriaosylceramide (Gb3). With the introduction and spread of enzyme replacement therapy (ERT) with recombinant GLAs for this disease, a useful biomarker for assessing the response to ERT is strongly required. We measured the tissue level of lyso-globotriaosylsphingosine (lyso-Gb3) in Fabry mice by means of high performance liquid chromatography, and compared it with the Gb3 level. The results revealed a marked increase in the lyso-Gb3 level in most tissues of Fabry mice, and which decreased after the administration of a recombinant GLA as in the case of Gb3, which is usually used as a biomarker of Fabry disease. The response was more impressive for lyso-Gb3 compared with for Gb3, especially in kidney tissues, in which a defect significantly influences the morbidity and mortality in patients with this disease. The plasma level of lyso-Gb3 also decreased after the injection of the enzyme, and it was well related to the degradation of tissue lyso-Gb3. Thus, lyso-Gb3 is expected to be a useful new biomarker for assessing the response to ERT for Fabry disease.

Several proteins including bovine erythrocyte acetylcholinesterase are anchored in the membrane through glycoinositol phospholipids containing an alkyl linkage at the sn -1 position of the glycerol. However, the existence of 1-alkyl-2-acyl- sn -glycero-3-phosphoinositol (alkylacyl-GPI) in biological systems has not been demonstrated. In this study, we identified the presence of alkylacyl-GPI in bovine erythrocytes by the following criteria: (1) TLC-Rf value, (2) radyllyso-GPI was produced after phospholipase A2 treatment of the diradyl-GPI, and (3) benzoate derivatives of alkylacylglycerols produced by phospholipase C hydrolysis of diradyl-GPI had the same retention time as that of authentic alkylacylglycerobenzoates on normal-phase HPLC. Diradyl-GPI consisted of 5–10% alkylacyl-GPI. Reverse-phase HPLC analysis of alkylacylglycerobenzoates derived from bovine erythrocyte alkylacyl-GPI showed a multiplicity of species with 18:0–20:4 (11.7%), 16:0–18:1 + 18:0–18:2 (34.9%), and 18:0–18:1 (19.4%) being the major components. Composition of alkyl chains of alkylacyl-GPI from bovine erythrocytes was similar to the reported value for alkylacylglycerols isolated from the glycoinositol phospholipid anchor of bovine erythrocyte acetylcholinesterase. Based on these results, we suggest that alkylacyl-GPI serves as a precursor for the glycoinositol phospholipid of the anchored proteins.

Amyloid {beta}-protein (A{beta}) has been reported to interact with a variety of lipid species, although the thermodynamic driving force remains unclear. We investigated the binding of A{beta}s labeled with the dye diethylaminocoumarin (DAC-A{beta}s) to lipid bilayers under various conditions. DAC-A{beta}-(1-40) electrostatically bound to anionic and cationic lipids at acidic and alkaline interfacial pH, respectively. However, at neutral pH, electroneutral A{beta} did not bind to these lipids, indicating little hydrophobic interaction between A{beta}-(1-40) and the acyl chains of lipids. In contrast, DAC-A{beta} associated with glycolipids even under electroneutral conditions. These results suggested that hydrogen-bonding as well as hydrophobic interactions with sugar groups of glycolipids drive the membrane binding of A{beta}-(1-40)

A new property of a heat-inducible heat shock protein (Hsp) 70.1 that it forms a complex with acidic lipids was first demonstrated. Based on the behaviors of the complexes on the native PAGE, the acidic lipid/Hsp70.1 complexes are categorized into two groups. The first group is the sulfatide-induced large-sized complex, which stays on the gel top on the native PAGE. Only the N-terminal ATPase domain is responsible for the complex formation. The second group is the ganglioside-induced complex, which is diffused in the resolution gel on the native PAGE. Both the N-terminal ATPase and the C-terminal peptide-binding domains are involved in the complex formation. No complex is formed by neutral glyco- and phospholipids. The complex formation with the acidic glyco- and phospholipids implicates the various functions of Hsp70 on the membrane surfaces.

The disaccharide Gal(alpha)1-3Gal is found on more than 45 different molecules on the endothelium of porcine cells and has recently attracted considerable interest, being the major target recognized by xenoreactive antibodies. In this study, the distribution and topology of Gal(alpha)1-3Gal on porcine endothelial cells was examined to access whether some Gal(alpha)1-3Gal-containing molecules might be preferentially recognized by antibodies binding to Gal(alpha)1-3Gal. Thirteen percent of the Gal(alpha)1-3Gal was found on glycolipid and 87% on glycoproteins. Of all the glycoproteins and glycolipids containing Gal(alpha)1-3Gal, two molecules, fibronectin and the integrin beta1 subunit, were most intensely labeled by galactose oxidase, suggesting that these molecules may be preferentially exposed on the apical surface of the endothelium. Binding of anti-Gal(alpha)1-3Gal antibodies to endothelial cell surfaces significantly diminished labeling of fibronectin and the integrin beta1 subunit by galactose oxidase, indicating that these glycoproteins are targets for the antibodies when binding to intact porcine cells.

Many microorganisms growing on water-insoluble substrates have been known to produce surface-active compounds called biosurfactants. Although biosurfactants have received increasing attention due to their special properties, there has been no information available until now of a role for them with regard to gene transfection. Thus, we studied here the effects of biosurfactants on gene transfection by cationic liposomes with a cationic cholesterol derivative. Our results showed clearly that a biosurfactant of mannosylerythritol lipid A (MEL-A) increased dramatically the efficiency of gene transfection mediated by cationic liposomes with a cationic cholesterol derivative. Among them, the liposomes with a cationic cholesterol derivative, cholesteryl-3 beta-carboxyamindoethylene-N-hydroxyethylamine (I), were much more effective for gene transfection than the liposomes with DC-Chol (cholesteryl-3 beta-oxycarboxyamidoethylenedimethylamine) or liposomes without MEL-A in various cultured cells. This demonstrates that this new finding has great potential in the experiment of gene transfection and gene therapy mediated by nonviral vectors such as cationic liposomes.

Some sulfoquinovosylacylglycerols (SQAG) have been shown to be potent DNA polymerase inhibitors, and to have strong antitumor activity in vivo. In this study, we investigated the mode of action of SQAG with regard to the interaction with the tumor cell surface. Of the SQAG used, the monoacyl forms (SQMG) with C18-, C18:1- or C16-fatty acids (SQMG-alphaC18, -alphaC18:1 or -alphaC16) effectively inhibited cell proliferation of a human adenocarcinoma cell line, DLD-1, but SQMG-alphaC14 and the diacyl forms (SQDG) did not. Analysis of the interaction of SQMG-alphaC18 and -alphaC18:1 on three oligosaccharides of cell surface, sLe(A), Le(X), and SM3, by flow cytometry demonstrated that the most effective interaction was observed on sLe(A). DLD-1 cells bound to SQMG-alphaC18:1-coated plates, and this binding was inhibited by monoclonal antibody against sLe(A) or SM3. However, these cells did not bind to SQMG-alphaC14-coated plates. Moreover the cytotoxic effects of SQMG-alphaC18, -alphaC18:1 on DLD-1 cells was inhibited by monoclonal antibodies against sLe(A) or SM3. Our results suggested that the interaction of SQMGs and sLe(A) plays an important role in suppression of the DLD-1 cell proliferation.

Previously, we showed that several minor macromolecular glycolipids accounting for less than 5% of the lipoteichoic acid (LTA) fraction from Enterococcus hirae ATCC 9790 possess cytokine-inducing activity, whereas the purified LTA does not. In other words, the immunobiological activity of the LTA fraction reported in the 1980s was not attributable to LTA itself, but to other glycolipids coexisting in the fraction. In the present study, we improved the procedure of separation of the active glycolipids and evaluated their effects on cellular activation. The immunobiologically active glycolipids were separated from the crude glycolipid fraction obtained by hot phenol-water extraction of the cells. The total yield of active glycolipids was about fivefold higher than that separated by the previous method. Interleukin-6-inducing activities of the active glycolipids from 1,25-dihydroxy vitamin D 3 -differentiated human monocytic leukemia cells, THP-1, were inhibited by anti-CD14 mAbs in a dose-dependent manner. Macrophages from Toll-like receptor (TLR)-2-deficient or -4-deficient mice completely lacked the ability to produce tumor necrosis factor-α on stimulation with active glycolipids. These observations indicated that the cellular activation by the active glycolipids from E. hirae is mediated by CD14 and by both TLR2 and TLR4.

Using a subcellular-specific proteomic approach, we have identified by protein microsequencing, a putative 35-kDa annexin from among the chloroplast envelope polypeptides. To confirm this identification, we demonstrate that (a) a 35-kDa protein, identified as annexin by antibody cross-reactivity, co-purifies with Percoll-purified chloroplasts and their envelope membranes when extracted in the presence of Ca2+ and (b) the native spinach annexin protein binds to chloroplast-specific lipids in a Ca2+-dependent manner. The binding of the spinach annexin to these glycerolipids occurs at similar Ca2+ concentrations as those, which promote the interaction of annexins to phospholipids in other membranes. Among chloroplast glycerolipids known to be accessible on the cytosolic face (outer leaflet) of the outer envelope membrane, sulfolipid, and probably phosphatidylinositol, would be the sole candidates for a putative Ca2+-dependent interaction of annexin with the chloroplast surface.

The vesicles composed of synthetic phytanyl-chained glycolipid and natural sulfoquinovosyldiacylglycerol at 9:1 molar ratio were successfully applied to functional reconstitution of photosystem II complex (PS II) from a thermophilic cyanobacterium. The synthetic glycolipid employed was one of our model archaeal diether lipids, 1,3-di- O -phytanyl-2- O -(β-D-maltotriosyl)glycerol. The light-induced oxygen-evolving activity of PS II reconstituted in the glycolipid vesicles was approximately 6-fold higher than that reconstituted in several phosphatidylcholine vesicles. The present results reveal the first evidence that a well-designed synthetic glycolipid is effective for the functional reconstitution of complicated and labile membrane protein complexes, such as PS II.

The MAL proteolipid, an integral membrane protein with selective residence in glycolipid- and cholesterol-enriched membrane (GEM) microdomains, has recently been identified as being an element of the integral protein machinery necessary for apical transport in MDCK cells. With the use of a recombinant baculovirus, we have expressed and purified polyhistidine-tagged MAL to determine whether MAL has special lipid requirements for becoming incorporated into membranes. In contrast with caveolin-1, a component of GEMs that requires cholesterol for its integration into artificial membranes, MAL incorporation took place with dimyristoylphosphatidylcholine as the only lipid component. The presence of cholesterol, sphingomyelin, or galactocerebrosides did not affect the efficiency of this process. These results indicated that MAL is compatible with membranes containing either only phospholipids or also glycolipids and cholesterol and are consistent with the reported requirement of a sorting event for the specific targeting of MAL to GEM microdomains.

Bacterial lipopolysaccharide (LPS) induces the production of various inflammatory cytokines and the inducibility is considered attributable to the glycolipid part of LPS called lipid A. We report an in vitro model in which lipid A is not necessarily a minimal structure for the LPS activity. Vitamin D3-differentiated THP-1 cells, cultured human monocytic leukemia cells, produced a high level of interleukin-6 (IL-6) by stimulating LPS from Escherichia coli O111:B4, but not by stimulating synthetic E. coli-type lipid A (compound 506), E. coli Re mutant LPS (ReLPS), or alkali-treated LPS. The induction by LPS was inhibited by the anti-CD14 antibodies or by the synthetic lipid A precursor (compound 406). An alkali-treated LPS or compound 506 partially inhibited the LPS-induced IL-6 production. These facts suggest that lipid A alone is not sufficient for the IL-6-inducing activity, but the polysaccharide part in LPS contributes or acts as a co-factor for activation of differentiated THP-1 cells.

The MAL proteolipid is a nonglycosylated polytopic membrane protein with specific residence in glycolipid-enriched membrane (GEM) microdomains. MAL has been proposed as an element of the machinery for apical transport in polarized epithelial cells. Previous work demonstrated that MAL requires its four carboxyl-terminal amino acids to be targeted to GEMs. In the present work, we have engineered MAL with N-glycosylation consensus sequences to delimit the site at which commitment of MAL to access into GEMs takes place. Comparison of engineered MAL proteins bearing either an intact or a truncated carboxyl terminus revealed that whereas the former acquired endo H-sensitive and endo H-resistant mature glycosylation, the protein with a deleted carboxyl terminus did not. These results indicate that although MAL incorporation into GEMs takes place mainly in the Golgi, commitment of MAL to enter GEMs is a pre-Golgi event.

JT95 is a monoclonal antibody (MAb) raised against the membrane fraction of differentiated thyroid carcinoma. JT95 specifically reacts to differentiated thyroid carcinoma, but not to benign thyroid tumor, normal tissue or other cancers. In this study, we purified the antigen by affinity-chromatography using MAb JT95 from spent culture medium of cell line SW1736, which was derived from a human thyroid carcinoma. The antigen was a glycoprotein having a molecular weight of 250,000 and its partial sequences obtained by peptide sequencing coincided with fibronectin. However, MAb JT95 reacted to the fibronectin purified from SW1736 but not to normal fibronectin, indicating modification of fibronectin was responsible for its antigenicity. Furthermore, immunochemical analysis of glycolipids extracted from SW1736 cells demonstrated that a second antigen was displayed on ganglioside. These results show that MAb JT95 recognized a novel antigenic epitope on a glycochain carried by both fibronectin and by a ganglioside produced by thyroid carcinoma cells.

We examined the role of target membrane glycolipids in CD4-mediated HIV-1 fusion by altering the glycolipid levels in CD4 + cells. CD4 + human cells exhibited 50% reduction in extent of fusion with gp120-gp41 expressing cells (TF228) when grown in the presence of a glycolipid synthesis inhibitor PPMP. We added erythrocyte glycolipids (GL) to fusion-incompetent CD4 + non-human cells by influenza-hemagglutinin-mediated fusion between GL-containing liposomes and target cells. Human erythrocyte GL (HuGL)-modified CD4 + non-human cells became susceptible to fusion with TF228 cells. Transfer of bovine erythrocyte glycolipids (BoGL) to CD4 + non-human cells under similar conditions did not complement HIV-1 fusion. Furthermore, addition of HuGL, but not BoGL, to PPMP-inhibited cells rescued fusion to the original levels. Our observations demonstrate that human erythrocyte glycolipids promote CD4-mediated HIV-1 fusion and certain glycolipid(s) from human erythrocytes may serve as alternative and/or additional cofactors in HIV-1 entry.

Mycoplasma fermentans has unique glycoglycerophospholipids (GGPLs: GGPL-I and GGPL-III). Previously, the structure of these lipids was determined as phosphocholine-6′-α-glucopyranosyl-(1′-3)-1, 2-diacyl-glycerol (GGPL-I) and 1″-phosphcholine-2″-aminodihydroxypropane-3″-phospho-6′-α-glucopyranosyl (1′-3)-1, 2-diacyl-glycerol (GGPL-III). Thin-layer chromatography (TLC) immunostaining showed that the GGPLs were main lipid-antigens of the M. fermentans species. Anti- M. fermentans serum stained mainly the GGPLs, but the other anti-mycoplasma sera (anti- M. arginini, anti- M. hyorhinis, anti- M. pneumonia, anti- M. primatum, and anti- Acholeplasma laidlawii, anti- M. hominis, anti- M. orale, and M. salivarium ) stained neither GGPL-I nor GGPL-III. The TLC analysis of glycolipids and phospholipids of various human related mycoplasmas showed clearly that GGPLs are specifically expressed in M. fermentans species. GGPL-I and GGPL-III ranged from 1.6 to 28% and from an undetectable level to 35% of total phospholipids, respectively. Although there was heterogeneity among the amounts of GGPL-I or GGPL-III of M. fermentans strains, all of the M. fermentans strains had GGPL-I and/or GGPL-III. These observations showed that GGPL structures are species-specific immunodeterminants of M. fermentans. The fact that the GGPLs are main phospholipid components of the M. fermentans species means the M. fermentans has a unique choline metabolic pathway. This observation may raise phylogenetic interest.

A human Gal beta(1-3/1-4)GlcNAc alpha 2,3-sialyltransferase, called ST-4, is a sialyltransferase involved in the in vivo biosynthesis of sialyl Lewis X (NeuNAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc) determinant. The ST-4 enzyme could utilize nLc4Cer (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) containing type 2 sugar chain, Lc4Cer (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) containing type 1 sugar chain, Gg4Cer (Gal beta 1-3GalNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), and LacCer as glycolipid acceptor substrates, but not other neutral glycolipids (GalCer, GlcCer, Gb3Cer, Gg3Cer, Gb4Cer) and gangliosides (GM1a, GM2, GM3, GD1a, GD1b, and GT1b) as substrates. The order of sialic acid incorporation into glycolipids for the enzyme was nLc4CerGg4CerLc4CerLacCer. The apparent Km values of ST-4 for nLc4Cer and Gg4Cer were 0.47 and 2.5 mM, respectively. Thus, the ST-4 could efficiently utilize both nLc4Cer and Gg4Cer as glycolipid acceptor substrates in vitro, suggesting that the substrate specificity of the enzyme may be similar to that of a glycolipid sialyltransferase (SAT-3), which is defined as the enzyme that uses both nLc4Cer and Gg4Cer as glycolipid acceptor substrates.