Antibodies to heteromeric glycolipid complexes in multifocal motor neuropathy.

BACKGROUND: Measurement of anti-GM1 IgM antibodies in multifocal motor neuropathy (MMN) sera is confounded by relatively low sensitivity that limits clinical usefulness. Combinatorial assay methods, in which antibodies react to heteromeric complexes of two or more glycolipids, are being increasingly applied to this area of diagnostic testing. METHODS: A newly developed combinatorial glycoarray able to identify antibodies to 45 different heteromeric glycolipid complexes and their 10 individual glycolipid components was applied to a randomly selected population of 33 MMN cases and 57 normal or disease controls. Comparison with an enzyme-linked immunosorbent assay (ELISA) was conducted for selected single glycolipids and their complexes. RESULTS: By ELISA, 22/33 MMN cases had detectable anti-GM1 IgM antibodies, whereas 19/33 MMN samples were positive for anti-GM1 antibodies by glycoarray. Analysis of variance (anova) revealed that of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside (GM1:GalC) complex were most significantly associated with MMN, returning 33/33 MMN samples as positive by glycoarray and 29/33 positive by ELISA. Regression analysis revealed a high correlation in absolute values between ELISA and glycoarray. Receiver operator characteristic analysis revealed insignificantly different diagnostic performance between the two methods. However, the glycoarray appeared to offer slightly improved sensitivity by identifying antibodies in four ELISA-negative samples. CONCLUSIONS: The use of combinatorial glycoarray or ELISA increased the diagnostic sensitivity of anti-glycolipid antibody testing in this cohort of MMN cases, without significantly affecting specificity, and may be a useful assay modification for routine clinical screening.

LINC00641 regulates prostate cancer cell growth and apoptosis via the miR-365a-3p/VGLL4 axis.

OBJECTIVE: Long non-coding RNA (lncRNA) was frequently abnormally expressed in cancers. LINC00641 was reported to play crucial roles in regulating tumor progression. However, its role in prostate cancer (PCa) has not been fully explored. PATIENTS AND METHODS: In this work, proliferation, invasion and apoptosis assays were performed to detect the biological roles of LINC00641 in PCa. Bioinformatic analyses, Luciferase activity reporter assay, and rescue experiments were performed to investigate the potential mechanisms of LINC00641 in PCa. Expression levels of LINC00641, microRNA-365a-3p (miR-365a-3p), and vestigial like family member 4 (VGLL4) in PCa tissues and normal tissues were analyzed at ENCORI. RESULTS: We found LINC00641 and VGLL4 was reduced, while miR-365a-3p was elevated expression in PCa tissues compared with normal tissues. LINC00641 overexpression inhibited growth and invasion abilities of PCa cells in vitro. Functional assays revealed that miR-365a-3p/VGLL4 pair was the downstream targets of LINC00641. CONCLUSIONS: The findings of our work provided evidence that LINC00641 serves as a tumor suppressive lncRNA in PCa by regulating miR-365a-3p/VGLL4 axis.

Novel anti-idiotype antibody therapy for lipooligosaccharide-induced experimental autoimmune neuritis: use relevant to Guillain-Barré syndrome.

Campylobacteriosis is a frequent antecedent event in Guillain-Barré syndrome (GBS), inducing high-titer serum antibodies for ganglioside antigens in the peripheral nervous system (PNS). Molecular mimicry between the lipooligosaccharide (LOS) component of Campylobacter jejuni and human peripheral nerve gangliosides is believed to play an important role in the pathogenesis of GBS. Conventional treatment strategies for patients with GBS include plasmapheresis, intravenous immunoglobulin (IVIG), and immunosuppression, which are invasive or relatively ineffective. In this study, we used our animal model of GBS, in which Lewis rats were immunized with GD3-like LOS isolated from C.jejuni. The animals developed anti-GD3 ganglioside antibodies and manifested neuromuscular dysfunction. To develop novel therapeutic strategies, we treated the animals by intraperitoneal administration of an anti-GD3 antiidiotype monoclonal antibody (BEC2) that specifically interacts with the pathogenic antibody. The treated animals had a remarkable reduction of anti-GD3 antibody titers and improvement of motor nerve functions. The results suggest that ganglioside mimics, such as antiidiotype antibodies, may be powerful reagents for therapeutic intervention in GBS by neutralizing specific pathogenic antiganglioside antibodies.

Glycosphingolipid antigens in cultured bovine brain microvascular endothelial cells: sulfoglucuronosyl paragloboside as a target of monoclonal IgM in demyelinative neuropathy [corrected].

Since a number of anti-glycosphingolipid (GSL) antibody activities have been demonstrated in patients with various neurological disorders, the presence of common antigens between brain microvascular endothelial cells (BMECs) and the nervous tissues presents a potential mechanism for the penetration of macromolecules from the circulation to the nervous system parenchyma. We first investigated GSL composition of cultured bovine BMECs. Bovine BMECs express GM3(NeuAc) and GM3(NeuGc) as the major gangliosides, and GM1, GD1a, GD1b, GT1b, as well as sialyl paragloboside and sialyl lactosaminylparagloboside as the minor species. Sulfoglucuronosyl paragloboside was also found to be a component of the BMEC acidic GSL fraction, but its concentration was lower in older cultures. On the other hand, the amounts of neutral GSLs were extremely low, consisting primarily of glucosylceramide. In addition, we analyzed the effect of anti-SGPG IgM antibody obtained from a patient of demyelinative polyneuropathy with macroglobulinemia against cultured BMECs. Permeability studies utilizing cocultured BMEC monolayers and rat astrocytes revealed that the antibody facilitated the leakage of [carboxy-14C]-inulin and 125I-labeled human IgM through BMEC monolayers. A direct cytotoxicity of this antibody against BMECs was also shown by a leakage study using [51Cr]-incorporated BMECs. This cytotoxicity depended on the concentration of the IgM antibody, and was almost completely blocked by preincubation with the pure antigen, sulfoglucuronosyl paragloboside. Our present study strongly supports the concept that immunological insults against BMECs induce the destruction or malfunction of the blood-nerve barrier, resulting in the penetration of the immunoglobulin molecule to attach peripheral nerve parenchyma.

Thyroid hormone influence on the susceptibility of mice to audiogenic seizures.

Serum thyroxine levels peak earlier and are significantly higher in audiogenic seizure-susceptible DBA/2J mice than in seizure-resistant C57BL/6J mice during early postnatal life. The seizure susceptibility of DBA/2J mice is suppressed by administration of an antithyroid drug or by radiothyroidectomy, while the seizure susceptibility of C57BL/6J mice is enhanced by treatment with excess thyroxine.

Glycosphingolipids in Bomirski transplantable melanomas in hamsters.

The glycosphingolipid compositions of Bomirski melanomas at different stages of differentiation, including Ab amelanotic melanoma (fast growing), Ma melanotic melanoma (slow growing), and MI hypomelanotic melanoma (slow growing), were studied. The total concentration of lipid-bound sialic acid in Ab amelanotic melanoma was found to be much lower than those in Ma and MI melanomas (0.8 micrograms versus 1.4 micrograms and 1.4 micrograms/mg of dry tissue, respectively). The ganglioside patterns in melanoma tissues were composed mainly of three components, which were confirmed as NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer (GM3), acetyl1-9-O-NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer (9-O-acetyl-GD3), and NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer(GD3) by structural analysis and monoclonal antibody detections. However, the relative ratios of these gangliosides expressed in the different types of melanomas were completely different. The MI melanoma tissues contained GM3 as the predominant species (greater than 90% of the total gangliosides) with very little of GD3 and 9-O-acetyl-GD3 gangliosides (less than 2% of the total gangliosides). In contrast, Ab amelanotic melanomas contained mainly 9-O-acetyl-GD3 (greater than 27%) and GD3 (greater than 51%) with lesser amounts of GM3. However, Ma melanoma had intermediate levels of GM3, GD3, and 9-O-acetyl GD3. The MI and Ma melanomas also contained monohexosylceramide (GL1) (about 60% as Gal beta 1-1'Cer and 40% as Glc beta 1-1'Cer in Ma and 30% as Gal beta 1-1'Cer and 70% as Glc beta 1-1'Cer in MI) and Gal beta 1-4Glc beta 1-1'Cer as the predominant neutral glycosphingolipid species. In contrast, Ab melanoma tissues contained more GalNAc beta 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (Gb5), Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (Gb3), and GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer (Gb4) than MI and Ma melanomas. Our data suggest that the expression of glycosphingolipids in hamster melanoma cells may be closely related to cell growth and the degree of differentiation, with slow growing, highly differentiated cells expressing GM3 and GL1, and fast growing, undifferentiating cells having a preponderance of GD3, 9-O-acetyl-GD3, Gb5, Gb3, and Gb4.

Suppression of ganglioside GD3 expression in a rat F-11 tumor cell line reduces tumor growth, angiogenesis, and vascular endothelial growth factor production.

Ganglioside GD3 is overexpressed in many types of tumors and may be associated with tumor progression and the development of metastatic potential. In our previous study (G. Zeng et al., Biochemistry, 38: 8762-8769, 1999), we established a subclone of the rat dorsal root ganglion-derived F-11 cells in which the expression of ganglioside GD3 was inhibited by stable transfection of the antisense vector against CMP-NeuAc: GM3 alpha2-8 sialyltransferase (GD3-synthase) gene. This cell line exhibits markedly reduced rate of tumor growth in vivo. Here, we further characterized the antisense-transfected cell line, and the results showed that these cells formed small, minimally vascularized tumors exhibiting extensive necrosis. In vivo Matrigel assay revealed reduced vascularization and low hemoglobin content in the antisense xenografts. Significantly fewer new vessels were found on the antisense xenografts and the skin around them than those on/around the xenografts formed by the sense-transfected and untransfected F-11 cells. The hemoglobin content of the antisense xenografts was much lower than that of the xenografts formed by the control cells. The reduced angiogenesis in the antisense xenografts was correlated with a decrease in vascular endothelial growth factor (VEGF) production. The expression of VEGF was suppressed in the antisense xenografts and the conditioned culture media of the antisense-transfected F-11 cells as determined by Western blotting analysis. This was further confirmed by immunohistochemistry of the tumors using antibodies against VEGF and platelet/endothelial cell adhesion molecule (PECAM-1). Therefore, our results demonstrate that reduced tumor growth in nude mice by suppression of GD3-synthase expression in F-11 cells results from minimal angiogenesis of the tumors through down-regulation of the VEGF expression, which indicates an important role for GD3 in tumor angiogenesis.

Ganglioside composition of an experimental mouse brain tumor.

The ganglioside composition of an experimental ependymoblastoma was examined in C57BL/6 mice. This tumor was produced by Dr. H. Zimmerman in 1949 from methylcholanthrene implantation in the brain and has been maintained in serial transplants through many generations. The influence of tumor environment on ganglioside composition was determined by studying the tumor growing intracerebrally and s.c. (over the skull and in the flank). The ganglioside composition of this tumor is markedly different from that of adult mouse brain. The total ganglioside sialic acid content (micrograms/100 mg dry weight) of the tumor growing in the cerebrum, s.c. over the skull, and in the flank was 70.4 +/- 3.8 (N = 3), 66.8 (N = 2), and 41.7 +/- 0.7 (N = 3), respectively. These values are about 10-fold lower than the ganglioside content of normal mouse cerebrum. This tumor contained a significant amount of N-glycolyneuraminic acid (NGNA). Histological analysis revealed two basically different cell types. The predominant cell type is densely packed and poorly defined in shape, whereas the minor cell type is less densely packed and fibroblastlike in shape. GM3, which migrates as double bands on thin-layer chromatography, is the predominant ganglioside of this tumor in all three regions of growth. Also present in all regions are gangliosides NGNA-GM3 and GM1. Significant amounts of GD1a, GD1b, GT1b, and GQ1b are present only in the cerebral tumor. These gangliosides therefore represent contaminants from normal brain tissue surrounding the tumor and are not native to the tumor. Ganglioside GD3, however, is a minor component of the tumor. Using a thin-layer chromatography-immunostaining method with anti-GA1 antibody, we found significant amounts of ganglioside with a GA1 oligosaccharide backbone migrating near GD3 and GD2. This tumor is similar to other neural tumors in having elevated amounts of GM3 and reduced amounts of total ganglioside and polysialogangliosides but is unique in having a high content of NGNA-containing gangliosides. The possible origin of the NGNA-containing gangliosides is discussed.

Changes in glycosphingolipids accompanying the differentiation of human squamous SQCC/Y1 cells.

SqCC/Y1 cells grow as a monolayer in culture and differentiate when maintained in the plateau phase; in the absence of serum these cells differentiate more rapidly. The differentiation is characterized by the stratification of the culture to form a structure consisting of several cellular layers, synthesis of specific keratins, and the attainment of the capacity to form a cornified cell membrane. The stratification process is indicative of the importance of cell-cell interactions during maturation. To study the relationship between membrane glycosphingolipids (GSLs) and the state of differentiation of SqCC/Y1 cells, GSLs were measured in cultures grown in the presence or absence of fetal calf serum. Glycolipids were isolated by diethylaminoethyl-Sephadex and Iatrobeads column chromatographies, and their distributions were determined by high-performance thin-layer chromatography. GM3 was the major ganglioside present in these cells. Other ganglioside components were tentatively identified as GM2, GM1, and GD3. Differences in ganglioside patterns were observed in differentiated cultures; the major changes were accumulation of GD3 and depletion of GM1. The predominant neutral GSLs in SqCC/Y1 cells were identified as Glc beta 1-1Cer, Gal beta 1-4Glc beta 1-1Cer, Gal beta 1-4Gal alpha 1-4Glc beta 1-1Cer, Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer, and three unknown complex GSLs. Differentiated cultures, however, showed variations in banding patterns, which include an increase in Glc beta 1-1Cer and Gal beta 1-4Glc beta 1-1Cer and a decrease in Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer and Gal NAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta-1Cer. These changes, however, were not observed when the cells were grown in the presence of epidermal growth factor or retinoic acid, factors which inhibit the differentiation process. The findings demonstrate significant changes in glycolipid composition of differentiated SqCC/Y1 cells grown in the absence of serum, suggesting that these lipids may be important to the differentiated state.

GD3 vaccines for melanoma: superior immunogenicity of keyhole limpet hemocyanin conjugate vaccines.

Cell surface gangliosides show altered patterns of expression as a consequence of malignant transformation and have therefore been of interest as potential targets for immunotherapy, including vaccine construction. One obstacle has been that some of the gangliosides that are overexpressed in human cancers are poorly immunogenic in humans. A case in point is GD3, a prominent ganglioside of human malignant melanoma. Using an approach that has been effective in the construction of bacterial carbohydrate vaccines, we have succeeded in increasing the immunogenicity of GD3 in the mouse by conjugating the ganglioside with immunogenic carriers. Several conjugation methods were used. The optimal procedure involved ozone cleavage of the double bond of GD3 in the ceramide backbone, introducing an aldehyde group, and coupling to aminolysyl groups of proteins by reductive amination. Conjugates were constructed with a synthetic multiple antigenic peptide expressing repeats of a malarial T-cell epitope, outer membrane proteins of Neisseria meningitidis, cationized bovine serum albumin, keyhole limpet hemocyanin, and polylysine. Mice immunized with these conjugates showed a stronger antibody response to GD3 than mice immunized with unconjugated GD3. The strongest response was observed in mice immunized with the keyhole limpet hemocyanin conjugate of the GD3 aldehyde derivative and the adjuvant QS-21. These mice showed not only a long-lasting high-titer IgM response but also a consistent high-titer IgG response (predominantly IgG1), indicating recruitment of T-cell help, although the titers of IgM and IgG antibodies following booster immunizations were not as high as they are in the response to classical T-cell-dependent antigens. This method is applicable to other gangliosides, and it may be useful in the construction of immunogenic ganglioside vaccines for the immunotherapy of human cancers expressing gangliosides on their cell surface.

Biochemical and serological characteristics of natural 9-O-acetyl GD3 from human melanoma and bovine buttermilk and chemically O-acetylated GD3.

Because its expression appears to be largely restricted to human melanomas, 9-O-acetyl-GD3 is a candidate antigen for vaccine construction. Searching for potential sources, we compared chemically O-acetylated calf brain GD3 and 9-O-acetyl-GD3 extracted from bovine buttermilk with 9-O-acetyl-GD3 from human melanoma. Three fractions (F1-F3) of chemically O-acetylated GD3 differed in the number and position of O-acetyl groups. O-Acetylation sites were the lactose portion in F1 and lactose as well as sialic acid in F2 and F3. Natural (melanoma- or buttermilk-derived) 9-O-acetyl-GD3 was O-acetylated solely on the sialic acid moiety. While F1 was not reactive with monoclonal antibodies against 9-O-acetyl-GD3, F2 and F3 were as reactive as the natural products. Immunization with the natural products induced high-titer antibodies against natural 9-O-acetyl-GD3 as well as F2 and F3. In contrast, mice immunized with the synthetic fractions produced antibodies only against the immunogen but not against natural 9-O-acetyl-GD3. Only immunization with the natural products induced production of antibodies reactive with surface antigens of melanoma cells expressing 9-O-acetyl-GD3. The findings suggest (a) that C-9 of the subterminal sialic acid is the site of chemical O-acetylation in F2 and F3, as opposed to C-9 of the terminal sialic acid in the natural products; (b) that O-acetylation of both the terminal and subterminal sialic acid moieties of GD3 results in recognition by three murine monoclonal antibodies (D1.1, ME 311, and Jones) reactive with human melanoma cells; (c) that O-acetylation of the terminal sialic acid is critical, on the other hand, for inducing an immune response against melanoma 9-O-acetyl-GD3; and (d) that O-acetyl GD3 from bovine buttermilk can substitute as immunogen for inducing an immune response against human melanoma cell surface antigens in the mouse.

Modulation by glycosphingolipids of membrane-membrane interactions induced by myelin basic protein and melittin.

The effect of glycosphingolipids (GSLs) with oligosaccharide chains of different length and charge on membrane-membrane interactions induced by myelin basic protein (MBP) or melittin (Mel) was comparatively investigated with small unilamellar vesicles. MBP induces a fast vesicle aggregation and close membrane apposition. Merging of lipid bilayers and vesicle fusion induced by MBP are slower and less extensive processes compared to membrane apposition. The changes of membrane permeability concomitant to these phenomena are small. The Trp region of MBP remains in a rather polar environment when interacting with vesicles; its accessibility to NO3- or acrylamide quenching depends on the type of GSLs in the membrane. The Trp region of Mel is inserted more deeply into the lipid bilayer and its accessibility to the aqueous quenchers is less dependent on variations of the oligosaccharide chain of the GSLs. Mel induces a faster and more extensive membrane apposition and bilayer merging than does MBP. Extensive vesicle disruption occurs in the presence of Mel. Negatively charged GSLs facilitate membrane proximity and vesicle aggregation but an increase of the oligosaccharide chain length of either neutral or acidic GSLs decreases the interaction among vesicles that are induced by either protein. This effect is independent of the different mode of insertion of MBP and Mel into the membrane. Our results suggest that the modulation by the oligosaccharide chain on the protein-induced interactions between bilayers containing GSLs is probably exerted beyond the level of local molecular interactions between the basic proteins and the lipids.

Multi-enzyme kinetic analysis of glycolipid biosynthesis.

Gangliosides are acidic glycosphingolipids synthesized sequentially by a series of glycosyltransferases acting in parallel biosynthetic pathways. While most glycosyltransferases are highly specific, some, however, may catalyze equivalent steps in each pathway using different gangliosides as substrates (e.g. N-acetylgalactosaminyltransferase, sialyltransferase-IV). A multi-enzyme kinetic analysis was developed on the condition that serial enzymatic reactions operate below substrate saturation. A multi-enzyme kinetic analysis enabled a simultaneous calculation of the Vmax/Km value of each enzyme derived from the equilibrium concentration of the respective substrate. Substrate concentrations [S] were determined by radioactive labelling of gangliosides in intact cells with the precursor sugars [14C]galactose and [14C]glucosamine, followed by high-performance thin-layer chromatography and autoradiography of the radiolabelled glycolipids. On the basis of Michaelis-Menten kinetics, Vmax/Km values were derived from [S] by a system of linear equations. The procedure was used to analyze the development of the glycolipid composition during differentiation of rat gliomaxmurine neuroblastoma (NG108-15) cells. The Vmax/Km values calculated by multi-enzyme kinetic analysis were consistent with the kinetic data obtained with solubilized enzymes. Application of multi-enzyme kinetic analysis to published data on the correlation of enzyme activities with ganglioside levels in various cell lines and tissues indicated the validity of this method for analysis of the glycolipid biosynthesis, in particular, of its initial steps. On the basis of the kinetic analysis, it is suggested that the cell lines can be divided into two groups with respect to the substrate pools of GM3 used by sialyltransferase-II and N-acetylgalactosaminyltransferase-I. The first group encompasses the majority of the neuroblastoma cell lines and the embryonic rat brain where the two enzymes share a common pool of GM3. In the second group, the two enzymes do not compete for the same pool of GM3, indicating a different subcellular localization of CMP-NeuAc:GM3 alpha2-8-sialyltransferase and UDP-N-acetylgalactosaminyl:GM3 N-acetylgalactosaminyltransferase. In this study, the theory of a multi-enzyme kinetic analysis is discussed and its application to analysis of the glycolipid biosynthesis in neuroblastoma cells is demonstrated. A multi-enzyme kinetic analysis can be applied to other biosynthetic pathways and provides the advantage of analyzing kinetic data with intact cells or tissue samples.

Effect of calcium ions on the thermotropic behaviour of neutral and anionic glycosphingolipids.

In the concentration range of 10(-5) to 10(-1) M Ca2+ modulates the thermotropic properties of several neutral and anionic glycosphingolipids (galactosylceramide, asialo-GM1, sulfatide, GM1, GD1a, GT1b) and of their mixtures with dipalmitoylphosphatidylcholine. The transition temperature of gangliosides is not appreciably changed while the transition enthalpy increases by 20% in the presence of Ca2+. The more marked effect of Ca2+ is on the thermotropic behavior of systems containing sulfatide. Increasing concentrations of Ca2+ between 10(-5) and 10(-3) M (up to a molar ratio of Ca2+/sulfatide 1:2) induce a progressive increase of both the transition temperature and enthalpy. Further increases up to 10(-1) M Ca2+ induce a new phase transition at a lower temperature. No evidence is found for induction of phase separation of pure glycosphingolipid-Ca2+ domains in mixtures of any of the glycosphingolipids with dipalmitoylphosphatidylcholine. The modification of the phase behavior of anionic glycosphingolipids by Ca2+ does not involve detectable variations of the intermolecular packing but is accompanied by marked modifications of the dipolar properties of the polar head group region.

Thermodynamic-geometric correlations for the morphology of self-assembled structures of glycosphingolipids and their mixtures with dipalmitoylphosphatidylcholine.

The morphology of aqueous dispersions of five neutral glycosphingolipids (GalCer, GlcCer, LacCer, asialo-GM2, asialo-GM1), sulfatide, and five gangliosides (GM3, GM2, GM1, GD1a and GT1b) and their mixtures with dipalmitoylphosphatidylcholine was studied by negative staining electron microscopy. The morphological features are interpreted on the basis of thermodynamic and geometric constraints previously studied in these systems (Maggio, B (1985) Biochim. Biophys. Acta 815, 245-258). The correlation between the theoretical predictions and the experimental findings are in reasonable agreement. Small changes in the molecular parameters of the individual glycosphingolipids or in their proportion in mixtures with dipalmitoylphosphatidylcholine bring about remarkable variations on the type of structure formed, its radius of curvature and thermodynamic stability.

Isolation and functional analysis of the promoter of the rat CMP-NeuAc:GM3 alpha2,8 sialyltransferase gene 1.

A 2.1-kb 5'-flanking fragment of the rat CMP-NeuAc:GM3 alpha2,8 sialyltransferase (GD3-synthase) gene was cloned by the genomic walking procedure. The promoter activity of the fragment was assessed in F-11 cells by transient transfection and the locations for the basal and maximal promoter activities were defined. Primer extension analysis identified a transcription start site approximately 98 bp upstream of the ATG start codon. DNA sequence analysis of the promoter revealed a number of consensus binding sites for known transcription factors such as SP1, AP1, NFkappaB, C/EBP and TFIID, and a repeat GC-GT sequence motif seen for the formation of Z-type DNA. Both TATA and CCAAT boxes were not found in the promoter. Our results from deletion constructs suggested that both positive and negative cis-acting regulatory regions were present in this TATA-less promoter of the rat GD3-synthase gene.

Modulation of the activity of Clostridium perfringens neuraminidase by the molecular organization of gangliosides in monolayers.

The activity of Clostridium perfringens neuraminidase against gangliosides GM3, GD1a and GM1 was studied in lipid monolayers at the air-buffer solution interface. The enzyme activity assay against pure ganglioside monolayers is based on the markedly different molecular packing areas of the substrate gangliosides and the resulting product glycosphingolipids. This allows to control and monitor the surface pressure and the ganglioside intermolecular organization (cross-sectional packing areas and dipole potentials) in a continuous manner during the catalytic process. It was found that the rate and the extent of the enzymatic reaction depended markedly on the lateral surface pressure. In general, the activity of neuraminidase against GM3 and GD1a was higher at lower surface pressure. This corresponded to larger intermolecular spacings among the ganglioside molecules. Both the activity and the extent of the reaction against GM3 were higher than toward GD1a. GM1 could not be degraded by the enzyme, irrespective of the surface pressure but the enzyme could interact with this ganglioside. A latency period, longer for GM3 than for GD1a, was observed prior to the onset of rapid degradation; this indicates that pre-catalytic steps are occurring at the interface before effective ganglioside degradation takes place. The latency period, the total amount of ganglioside degraded, and the velocity of the reaction varied with the surface pressure in different manners. Our data indicate that the different steps of the catalytic reaction occurring at the surface (i.e., substrate recognition and interfacial adsorption, catalysis, maximum extent of substrate conversion) are independently regulated by the molecular organization of the substrate gangliosides.

Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter.

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.

Thermotropic behavior of binary mixtures of dipalmitoylphosphatidylcholine and glycosphingolipids in aqueous dispersions.

The thermotropic behavior of mixtures of dipalmitoylphosphatidylcholine (DPPC) with natural glycosphingolipids (galactosylceramide, phrenosine, kerasine, glucosylceramide, lactosylceramide, asialo-GM1, sulfatide, GM3, GM1, GD1a, GT1b) in dilute aqueous dispersions were studied by high sensitivity differential scanning calorimetry over the entire composition range. The pretransition of DPPC is abolished and the cooperativity of the main transition decreases sharply at mole fractions of glycosphingolipids below 0.2. All systems exhibit non-ideal temperature-composition phase diagrams. The mono- and di-hexosylceramides are easily miscible with DPPC when the proportion of glycosphingolipids in the system is high. A limited quantity (1-6 molecules of DPPC per molecule of glycosphingolipid (GSL) can be incorporated into a homogeneously mixed lipid phase. Domains of DPPC, immiscible with the rest of a mixed GSL-DPPC phase that shows no cooperative phase transition, are established as DPPC exceeds a certain proportion in the system. One negative charge (sulfatide) or four neutral carbohydrate residues (asialo-GM1) in the oligosaccharide chain of the glycosphingolipids results in phase diagrams exhibiting coexistence of gel and liquid phases over a broad temperature-composition range. Systems containing gangliosides show complex phase diagrams, with more than one phase transition. However, no evidence for phase-separated domains of pure ganglioside species is found. The thermotropic behavior of systems containing DPPC and glycosphingolipids correlates well with their interactions in mixed monolayers at the air/water interface.

Regulation by gangliosides and sulfatides of phospholipase A2 activity against dipalmitoyl- and dilauroylphosphatidylcholine in small unilamellar bilayer vesicles and mixed monolayers.

The modulation by gangliosides GM1 and GD1a, and sulfatide (Sulf) of the activity of porcine pancreatic phospholipase A2 was studied with small unilamellar vesicles of dipalmitoylphosphatidylcholine (L-dpPC) and lipid monolayers of dilauroylphosphatidylcholine (L-dlPC). The presence of Sulf always led to an increase of the maximum rate of the enzymatic reaction, irrespective on whether the vesicles were above, in the range of, or below the bilayer transition temperature. Sulf did not modify the latency period for the reaction that is observed at the bilayer transition temperature. Gangliosides inhibited the maximum rate of enzymatic activity bilayer vesicles in the gel phase but the effect was complex. When the reaction was carried out at a temperature within the range of the bilayer phase transition, the gangliosides inhibited the maximal rate of the reaction in proportion to their content in the bilayer. However, at the same time the latency period observed with vesicles of pure phospholipid at this temperature was shortened in proportion to the mole fraction of gangliosides in the bilayer. At temperatures above the bilayer phase transition, gangliosides stimulated the activity of PLA2. Preincubation of the enzyme with Sulf or gangliosides did not affect the activity against bilayer vesicles of pure substrate. These glycosphingolipids did not modify the rate or extent of desorption of the enzyme from the interface, nor the pre-catalytic steps for the interfacial activation of PLA2, or the enzyme affinity for the phospholipid substrate. Also, the activity of the enzyme was not altered irreversibly by glycosphingolipids. Our results indicate that Sulf and gangliosides modulate the catalytic activity of PLA2 at the interface itself, beyond the initial steps of enzyme adsorption and activation, probably through modifications of the intermolecular organization and surface electrostatics of the phospholipid substrate.