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beta-Hexosaminidases (EC 3.2.1.52) are lysosomal enzymes that remove terminal beta-glycosidically bound N-acetylglucosamine and N-acetylgalactosamine residues from a number of glycoconjugates. Reliable assay systems are particularly important for the diagnosis of a family of lysosomal storage disorders, the GM2 gangliosidoses that result from inherited beta-hexosaminidase deficiency. More recently, aberrant hexosaminidase levels have also been found to be associated with a variety of inflammatory diseases. Apart from patient testing and carrier screening, practical in vitro assays are indispensable for the characterization of knock-out mice with potentially altered hexosaminidase activities, for detailed structure-function studies aimed at elucidating the enzymatic mechanism, and to characterize newly described enzyme variants from other organisms. The purpose of this article is to discuss convenient hexosaminidase assay procedures for these and other applications, using fluorogenic or chromogenic artificial substrates as well as the physiological glycolipid substrate GM2. Attempts are also made to provide an overview of less commonly used alternative techniques and to introduce recent developments enabling high-throughput screening for enzyme inhibitors.

To elucidate the mechanism underlying the hydrolysis of the GalNAcbeta1-->4Gal linkage in ganglioside GM2 [GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbeta1-->1' Cer] by beta-hexosaminidase A (Hex A) with GM2 activator protein, we designed and synthesized two kinds of GM2 linkage analogues-6'-NeuAc-GM2 and alpha-GalNAc-GM2. In this paper, the efficient and systematic synthesis of these GM2 analogues was described. The highlight of our synthesis process is that the key intermediates, newly developed sialyllactose derivatives, were efficiently prepared in sufficient quantities; these derivatives directly served as highly reactive glycosyl acceptors and coupled with GalNTroc donors to furnish the assembly of GM2 tetrasaccharides in large quantities.

A series of GM2 analogs in which GM2 epitope was coupled to a variety of glycosyl lipids were designed and synthesized to investigate the mechanism of enzymatic hydrolysis of GM2 ganglioside. The coupling of N-Troc-protected sialic acid and p-methoxyphenyl galactoside acceptor gave the crystalline disaccharide, which was further coupled with galactosamine donor to give the desired GM2 epitope trisaccharide. After conversion into the corresponding glycosyl donor, the trisaccharide was coupled with galactose, glucose and artificial ceramide (B30) to give the final compounds. The result on hydrolysis of GM2 analogs indicates that GM2 activator protein requires one spacer sugar between GM2 epitope and the lipid moiety to assist the hydrolysis of the terminal GalNAc residue.

The human glioma D-54MG cell line grown in vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3'-isoLM1, an alteration that was dose dependent, with the highest dose (1 x 10(8)) resulting in a phenotype that was most like that of the inoculum. After one passage in vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grown in vitro than in those grown in vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors.

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.

Several properties of the exchangeable amide protons of the ganglioside GM2 were studied in detail by 1H-NMR spectroscopy in fully deuterated dimethylsulfoxide [2H6]DMSO/2% H2O, and compared with data obtained for the simpler constituent glycosphingolipids GA2 and GM3. In addition to chemical shifts, 3J2,HN coupling constants, and temperature shift coefficients, the kinetics of NH/2H chemical exchange were examined by following the disappearance of the amide resonances in [2H6]DMSO/2% 2H2O. The results included observation of an increase in half-life of the N-acetylgalactosamine acetamido HN by more than an order of magnitude in GM2 compared to GA2, attributable to the presence of the additional N-acetylneuraminic acid residue. Additional one-dimensional dipolar cross relaxation experiments were also performed on nonexchangeable protons of GM2. The results of all of these experiments support a three-dimensional model for the terminal trisaccharide in which a hydrogen bond is formed between the N-acetylgalactosamine acetamido NH and the N-acetylneuraminic acid carboxyl group. The interaction is proposed to be of the pi-acceptor type, a possibility which has not yet been explored in the literature on carbohydrates. The proposed model is discussed in comparison with that of Sabesan et al. (1984, Can J Chem 62:1034-45), and the models of GM1 proposed more recently by Acquotti et al. (1990, J Am Chem Soc 112:7772-8) and Scarsdale et al. (1990, Biochemistry 29:9843-55).