Biomedical subjects

P Brocca

Publications and source records attributed to P Brocca.

11 recordsLinked to original sources

PubMed2000

Modeling ganglioside headgroups by conformational analysis and molecular dynamics.

The conformations and dynamics of gangliosides GM1, GM2, 6'-GM2 and GM4 have been studied by computational means, and the results compared to NMR data. Unconstrained conformational searches were run using the AMBER* force field augmented by MNDO derived parameters for the Neu5Ac anomeric torsion, the GB/SA water solvation model, and the MC/EM alogorithm; extended (10-12 ns) dynamic simulations in GB/SA water were performed with the MC/SD protocol, and the stored structures were minimized. The overall mobility of the Neu5Ac alpha2,3Gal linkage and the position of its minimum energy conformation have been shown to depend mainly on the presence or the absence of a GalNAc residue at the adjacent position. The best quantitative agreement with the available NOE data was achieved after minimization of the structures stored during the MC/SD dynamic runs. The latter protocol appears to reproduce satisfactorily the available experimental data, and can be used with confidence to build three-dimensional models of ganglioside headgroups.

Algorithms↗

PubMed1999

Structural basis for the resistance of Tay-Sachs ganglioside GM2 to enzymatic degradation.

To understand the reason why, in the absence of GM2 activator protein, the GalNAc and the NeuAc in GM2 (GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer) are refractory to beta-hexosaminidase A and sialidase, respectively, we have recently synthesized a linkage analogue of GM2 named 6'GM2 (GalNAcbeta1-->6(NeuAcalpha2-->3)Galbeta1-->4Glcbet a1-1'Cer). While GM2 has GalNAcbeta1-->4Gal linkage, 6'-GM2 has GalNAcbeta1-->6Gal linkage (Ishida, H., Ito, Y., Tanahashi, E., Li, Y.-T., Kiso, M., and Hasegawa, A. (1997) Carbohydr. Res. 302, 223-227). We have studied the enzymatic susceptibilities of GM2 and 6'GM2, as well as that of the oligosaccharides derived from GM2, asialo-GM2 (GalNAcbeta1-->4Galbeta1--> 4Glcbeta1-1'Cer) and 6'GM2. In addition, the conformational properties of both GM2 and 6'GM2 were analyzed using NMR spectroscopy and molecular mechanics computation. In sharp contrast to GM2, the GalNAc and the Neu5Ac of 6'GM2 were readily hydrolyzed by beta-hexosaminidase A and sialidase, respectively, without GM2 activator. Among the oligosaccharides derived from GM2, asialo-GM2, and 6'GM2, only the oligosaccharide from GM2 was resistant to beta-hexosaminidase A. Conformational analyses revealed that while GM2 has a compact and rigid oligosaccharide head group, 6'GM2 has an open spatial arrangement of the sugar units, with the GalNAc and the Neu5Ac freely accessible to external interactions. These results strongly indicate that the resistance of GM2 to enzymatic hydrolysis is because of the specific rigid conformation of the GM2 oligosaccharide.

Acetylgalactosamine↗

PubMed1999

The structural basis for the susceptibility of gangliosides to enzymatic degradation.

The conformational properties of GM2, GalNacbeta-4(Neu5Acalpha-3) Galbeta-4Glcbeta-1Cer have been compared to those of 6'-GM2, in which the linkage between the GalNAc and Gal was altered from GalNacbeta-4Galbeta- to GalNacbeta-6Galbeta-, and to those of GD1a, Neu5Acalpha-3Galbeta-3GalNAcbeta-4(Neu5Acalpha-3 )Galbeta-4Glcbeta-1Cer, and GalNAc-GD1a. Our results revealed that unlike the compact and rigid oligosaccharide head group found in GM2, where the Neu5Ac and the GalNAc residues interact, the sugar chain of 6'-GM2 is in an open spatial arrangement, with the Neu5Ac no longer interacting with GalNAc, freely accessible to external interactions. The structure of GD1a can be regarded as that of GM2 with an extension of the terminal Neu5Acalpha-3Galbeta-disaccharide. The inner portion of GD1a is that of GM2 comprising the very rigid GalNAc-[Neu5Ac-]Gal trisaccharide. The terminal Neu5Ac-Gal linkage is flexible and fluctuates between two limiting conformations. In GalNAc-GD1a the outer sialic acid gains conformational rigidity due to the presence of the outer GalNAc in position 4 of galactose. This ganglioside has two 'core' GalNAc-[Neu5Ac-]Gal trisaccharide linked in tandem.

Carbohydrate Sequence↗

PubMed1998

Conformation of the oligosaccharide chain of G(M1) ganglioside in a carbohydrate-enriched surface.

The solution structure of ganglioside G(M1) carbohydrate moiety at the surface of a 102-kDa lipid-modified-G(M1) micelle is investigated by high-resolution 1H-NMR in H2O. The micellar surface can be considered a cluster-like lateral distribution of the gangliosides, each single monomer being anchored in a carbohydrate-enriched model membrane matrix. 1H NOESY measurements at short mixing times reveal a rigid trisaccharide core -beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal- and a more flexible beta-Gal-(1-3)-beta-GalNAc- terminal glycosidic bond. In the lipid-modified G(M1) ganglioside micellar system, there is no evidence that intermolecular side-by-side carbohydrate interactions modulate, or alter in any way, the head-group spatial arrangement. Possible intermonomer interactions at the level of the branched trisaccharide portion were further investigated on mixed micelles of natural N-glycolyl- and N-acetylneuraminic acid containing G(M1) in D2O, taking advantage of the different NMR features of N-glycolyl- and N-acetylneuraminic acids, which allow discrimination between sialic acid ring proton signals. Measurements of the water/ganglioside-OH proton chemical exchange rates suggest hydroxyl group involvement at position 8 of sialic acid in strong intramolecular interaction processes.

Animals↗

PubMed1997

Metabolic processing of gangliosides by human fibroblasts in culture--formation and recycling of separate pools of sphingosine.

Human cultured fibroblasts were fed with GM3 ganglioside species isotopically labeled at C3 of C18-sphingosine, or at C3 of C18-sphinganine, or at the sialic acid acetyl group, and with C18-sphingosine and C18-sphinganine, both labeled at C1. After a lipid pulse the cells were subjected until 7-day chase; measurements were then made of the radioactive products resulting from the administered long-chain base and ganglioside species catabolism and the salvage processes of catabolic fragments. From the data we drew the following conclusions. The GM3 species differing in the long-chain base structure were taken up by the cells and metabolized. About 80% of the total catabolic C18-sphingosine and C18-sphinganine were recycled for the biosynthesis of complex sphingolipids, the rest being degraded. Results obtained by administering ganglioside species of GM3 containing radioactive sphingosine or the free radioactive sphingosine to fibroblasts suggested the existence in the cells of two quite separate pools of sphingosine. One pool was the direct result of either the catabolism of radioactive GM3 high-density microdomains or the diffusion of exogenous sphingosine into the cell; this pool was mainly used for the biosynthesis of the GD3 species that contain palmitic and stearic acids. The other pool of sphingosine, the cell basal pool, came from the catabolism of radioactive sphingolipids in the recycling of sphingosine, and was used for the biosynthesis of the GD3 species that mainly contain very long fatty acid chains, the main fibroblast endogenous species of GD3. Administration of the ganglioside species of GM3 containing sphinganine or free sphinganine to fibroblasts yielded the GD3 species containing mainly very long-chain fatty acids and sphingosine. These results show the possible existence of a pool of ganglioside-derived sphingosine, quite separate from the cell basal pool of sphingosine, suggesting that sphingosine derived from sphingolipid catabolism is reduced to sphinganine before entering the sphingolipid biosynthetic pathway.

Autoradiography↗

PubMed1996

Nuclear Overhauser effect investigation on GM1 ganglioside containing N-glycolyl-neuraminic acid (II3Neu5GcGgOse4Cer).

The conformational properties of the oligosaccharide chain of GM1 ganglioside containing N-glycolyl-neuraminic acid, beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]-beta-Gal- (1-4)1-4)-beta-Glc-(1-1)-Cer, were studied through NMR nuclear Overhauser effect investigations on the monomeric ganglioside in dimethylsulfoxide, and on mixed micelles of ganglioside and dodecylphosphocholine in water. Several interresidual contacts for the trisaccharide core -beta- GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]-beta-Gal- were found to fix the relative orientation of the three saccharides, while the glycosidic linkage of the terminal beta-Gal- was found to be quite mobile as the beta-Gal-(1-3)-beta-GalNAc- disaccharide exists in different conformations. These results are similar to those found for two GM1 gangliosides containing N-acetyl-neuraminic acid and neuraminic acid [1].

Carbohydrate Conformation↗

PubMed1995

Isolation and structural characterization of N-acetyl- and N-glycolylneuraminic-acid-containing GalNAc-GD1a isomers, IV4GalNAcIV3Neu5AcII3Neu5GcGgOse4Cer and IV4GalNAcIV3Neu5GcII3Neu5AcGgOse4Cer, from bovine brain.

A ganglioside preparation containing two structurally related minor gangliosides (Gg 1 + 2) was isolated from bovine brain ganglioside mixture and characterized. Treatment of 50 g ganglioside mixture with Clostridium perfrigens sialidase, followed by chromatography on DEAE-Sepharose and silica gel columns, yielded 20 mg Gg 1 + 2. By chemical analyses, 1H- and 13C-NMR spectroscopy, enzymic hydrolyses using human beta-hexosaminidase A and clostridial sialidase, and TLC overlay with the conjugated cholera toxin B subunit, the two novel gangliosides Gg 1 and Gg 2 were identified to be: Gg 1, GalNAc-GD1a(Neu5Ac/Neu5Gc), beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta- Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]-beta-Gal-(1-4)-be ta- Glc-(1-1)-Cer; Gg 2, GalNAc-GD1a(Neu5Gc/Neu5Ac), beta-GalNAc-(1-4)-[alpha-Neu5Gc-(2-3)]- beta-Gal-(1-3)-beta-GalNAc-(1-4)-[alpha-Neu5Ac-(2-3)]-beta-Gal-(1- 4)-beta- Glc-(1-1)-Cer. These two gangliosides contain the identical pentasaccharide backbone except that the substitution of the two sialic acids, Neu5Ac and Neu5Gc, are in the reversed position of the external and the internal Gal residues. Our analyses showed that the content of Gg 1 and Gg 2 were approximately 0.12% and 0.08%, respectively, of the total brain ganglioside mixture.

Acetylgalactosamine↗

PubMed1995

Aggregation properties of semisynthetic GD1a ganglioside (IV3Neu5AcII3Neu5AcGgOse4Cer) containing an acetyl group as acyl moiety.

GD1a ganglioside containing an acetyl group as acyl moiety, GD1a(acetyl), was synthesized from natural GD1a. The aggregative properties in aqueous solution of GD1a(acetyl) have been studied by static and dynamic laser light-scattering measurements. GD1a(acetyl) spontaneously aggregates as small micelles showing a hydrodynamic radius and molecular mass of 33 A and 96 kDa, respectively. Vibrio cholerae sialidase showed a very high activity on the micelles of GD1a(acetyl), compared to GD1a. This has been explained as a consequence of the high surface curvature of the the small micelles. High resolution proton NMR spectra were recorded from micelles of GD1a(acetyl) in deuterated water. The low overall correlation time of the GD1a(acetyl) micelles was calculated to be about 2 x 10(-8)s, a value one order of magnitude lower than that determined for natural GD1a.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed1994

Some structural features of cluster-coordinating cysteines of Clostridium pasteurianum ferredoxin are revealed by 2D TOCSY 1H NMR on the oxidized protein.

Different sets of geminal J coupling constants for the eight beta-CH2 protons in the iron-coordinating cysteines in Clostridium pasteurianum ferredoxin were detected by 2D TOCSY 1H NMR experiments on the oxidized protein. Four resonances were characterized by quite similar high values of J, two more resonances had a J value about half of the former ones, while the last two had extremely low J values. These findings suggest that the cysteines required for cubane symmetry around the iron atoms are constrained into different geometries. The simplified model used for fine tuning of tau m in these TOCSY experiments is also presented and discussed.

Clostridium↗

PubMed1993

1H-NMR study on ganglioside amide protons: evidence that the deuterium exchange kinetics are affected by the preparation of samples.

The kinetics of H/2H chemical exchange of the amide proton has been suggested as one of the tools available for investigating hydrogenbond stabilizing interactions in gangliosides. The amide proton/deuterium (NH/2H) exchange rates in GM2 ganglioside were studied by 1H-NMR spectroscopy on 12 samples prepared following different procedures. In samples passed through a sodium salt Chelex-100 cation exchange resin column prior to being analysed the N-acetylneuraminic acid NH exchange occurred in less than 10 min and that of ceramide NH in 30 min. The N-acetylgalactosamine acetamido NH exchange was slower, the half-life of the signal ranging from 15 min to 3.5 h. Contact of the Chelex-treated GM2 samples with water, through a dialysis process, modified the NH/2H exchange rate values, the N-acetylgalactosamine acetamido NH exchange becoming faster than that of ceramide NH and similar to that of N-acetylneuraminic acid NH. Our results indicate that the deuterium/proton exchange rate strongly depends on sample preparation (ion content and minor contaminants present in water). The three-dimensional model involving the N-acetylgalactosamine acetamido NH and the N-acetylneuraminic acid carboxyl group hydrogen-bonding, which is supported by experimental evidence, cannot be confirmed by NH-exchange measurement.

Amides↗

PubMed1993

Influence of primary and secondary structures of oligosaccharide chain on the aggregative and geometrical properties of gangliosides.

The aggregative and geometrical properties of gangliosides have been compared and discussed. It is shown that, due to the steric packing features of gangliosides, significative considerations can be made on the primary and secondary structures of their head group, starting from their cooperative behaviour, supporting NMR direct observations.

Carbohydrate Conformation↗