Yu-Teh Li

Identification of plasma membrane associated mature β‐hexosaminidase A, active towards GM2 ganglioside, in human fibroblasts

Mature β‐hexosaminidase A has been found associated to the external leaflet of plasma membrane of cultured fibroblasts. The plasma membrane association of β‐hexosaminidase A has been directly determined by cell surface biotinylation followed by affinity chromatography purification of the biotinylated proteins, and by immunocytochemistry. The immunological and biochemical characterization of biotinylated β‐hexosaminidase A revealed that the plasma membrane associated enzyme is fully processed, suggesting its lysosomal origin.

Specificity of mouse GM2 activator protein and beta-N-acetylhexosaminidases A and B. Similarities and differences with their human counterparts in the catabolism of GM2.

Tay-Sachs disease, an inborn lysosomal disease featuring a buildup of GM2 in the brain, is caused by a deficiency of β-hexosaminidase A (Hex A) or GM2activator. Of the two human lysosomal Hex isozymes, only Hex A, not Hex B, cleaves GM2 in the presence of GM2activator. In contrast, mouse Hex B has been reported to be more active than Hex A in cleaving GM2 (Burg, J., Banerjee, A., Conzelmann, E., and Sandhoff, K. (1983) Hoppe Seyler’s Z. Physiol. Chem. 364, 821–829). In two independent studies, mice with the targeted disruption of the Hexa gene did not display the severe buildup of brain GM2 or the concomitant abnormal behavioral manifestations seen in human Tay-Sachs patients. The results of these two studies were suggested to be attributed to the reported GM2 degrading activity of mouse Hex B. To clarify the specificity of mouse Hex A and Hex B and to better understand the observed results of the mouse model of Tay-Sachs disease, we have purified mouse liver Hex A and Hex B and also prepared the recombinant mouse GM2 activator. Contrary to the findings of Burget al., we found that the specificities of mouse Hex A and Hex B toward the catabolism of GM2 were not different from the corresponding human Hex isozymes. Mouse Hex A, but not Hex B, hydrolyzes GM2 in the presence of GM2activator, whereas GM2 is refractory to mouse Hex B with or without GM2 activator. Importantly, we found that, in contrast to human GM2 activator, mouse GM2activator could effectively stimulate the hydrolysis of GA2by mouse Hex A and to a much lesser extent also by Hex B. These results provide clear evidence on the existence of an alternative pathway for GM2 catabolism in mice by converting GM2 to GA2 and subsequently to lactosylceramide. They also provide the explanation for the lack of excessive GM2 accumulation in the Hexa gene-disrupted mice.

A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides

Abstract A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p -nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p -nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal β- N -acetylhexosaminidase using p -nitrophenyl-β-2-acetamido-2-deoxy- d -glucopyranoside (NPADG) at pH 5.0. A number of different wavelengths may be used for the assay depending upon the criterion of the user; maximum sensitivity at a selected pH, determination of enzyme pH optima with a pH-independent difference extinction coefficient, or the reduction of background absorbance for kinetic studies at high substrate concentrations.

Interaction of GM2Activator Protein with Glycosphingolipids

GM2 activator protein is a protein cofactor that has been shown to stimulate the enzymatic hydrolysis of both GalNAc and NeuAc from GM2 (Wu, Y. Y., Lockyer, J. M., Sugiyama, E., Pavlova, N.V., Li, Y.-T., and Li, S.-C. (1994) J. Biol. Chem. 269, 16276-16283). To understand the mechanism by which GM2 activator stimulates the hydrolysis of GM2, we examined the interaction of this activator protein with GM2 as well as with other glycosphingolipids by TLC overlay and Sephacryl S-200 gel filtration. The TLC overlay analysis unveiled the binding specificity of GM2 activator, which was not previously revealed. Under the conditions optimal for the activator protein to stimulate the hydrolysis of GM2 by β-hexosaminidase A, GM2 activator was found to bind avidly to acidic glycosphingolipids, including gangliosides and sulfated glycosphingolipids, but not to neutral glycosphingolipids. The gangliosides devoid of sialic acids, such as asialo-GM1 and asialo-GM2, and the GM2 derivatives whose carboxyl function in the NeuAc had been modified by methyl esterification or reduction, were only very weakly bound to GM2 activator. These results indicate that the negatively charged sugar residue or sulfate group in gangliosides is one of the important sites recognized by GM2 activator. For comparison, we also studied in parallel the complex formation between glycosphingolipids and saposin B, a separate activator protein with broad specificity to stimulate the hydrolysis of various glycosphingolipids. We found that saposin B bound to neutral glycosphingolipids and gangliosides equally well, and there was an exceptionally strong binding to sulfatide. In contrast to previous reports, we found that GM2 activator formed complexes with GM2 and other gangliosides in different proportions depending on the ratio between the activator protein and the ganglioside in the incubation mixture prior to gel filtration. We were not able to detect the specific binding of GM2 activator to GM2 when GM2 was mixed with GM1 or GM3. Thus, the specificity or the mode of action of GM2 activator cannot be simply explained by its interaction with glycosphingolipids based on complex formation. The binding of GM2 activator to a wide variety of negatively charged glycosphingolipids may indicate that this activator protein has functions other than assisting the enzymatic hydrolysis of GM2.

Structure of a new hexasaccharide from the coaggregation polysaccharide of Streptococcus sanguis 34

The major constituent of a coaggregation polysaccharide from Streptococcus sanguis 34 is a hexasaccharide, isolated as the alditol. The proposed structure is alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-beta-D-Glcp-(1----6) -beta-D-Galf- (1----6)-beta-D-GalpNAc-(1----3)-D-Galol, based upon g.l.c.-m.s. of alditol acetates and partially methylated alditol acetates, f.a.b.-m.s., 1H-n.m.r. spectroscopy, g.l.c.-m.s. of trimethylsilylated (+)- and (-)-2-butyl glycosides, and cleavage by alpha-N-acetylgalactosaminidase. The structural deduction was facilitated by cleavage of the hexasaccharide at the furanoside linkage by 48% hydrogen fluoride, and reduction of the product, to yield alpha-D-GalpNAc-(1----3)-beta-L-Rhap-(1----4)-beta-D-Glcp-(1----6) -D-Galol.

Isolation of three novel cholinergic neuron-specific gangliosides from bovine brain and theirin vitro syntheses

AbstractIn the present study, three extremely minor but novel Chol-1 antigens, termed X1, X2, and X3 have been isolated from bovine brain gangliosides. Based on the results of sialidase degradation, TLC-immunostaining with anti-Chol-1 antibody and fast atom bombardment mass spectrometry, their chemical structures were identified as:

A 1H NMR INVESTIGATION OF THE HYDROLYSIS OF A SYNTHETIC SUBSTRATE BY KDN-SIALIDASE FROM CRASSOSTREA VIRGINICA

\begin{gathered} III^6 NeuAc--GgOse4Cer (X1:GM1\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--GgOse4Cer (X2:GT1a\alpha ) \hfill \\ III^6 NeuAc,II^3 NeuAc--NeuGc--GgOse4Cer (X3:GT1b\alpha ) \hfill \\ \end{gathered}

[57] Endo-β-galactosidase from Escherichia freundii

The yields of GM1α, GD1aα, and GT1bα, were approximately 150, 20, and 10 µg, respectively, from 10 g of the bovine brain ganglioside mixture. In conjunction with our previous observations, all gangliosides with anti-Chol-1 reactivity were found to contain a common sialyl α2–6N-acetylgalactosamine residue, indicating that this unique sialyl linkage is the specific antigenic determinant. We subsequently examined the biosyntheses of the three novel Chol-1 gangliosides using rat liver Golgi fraction as an enzyme source. The results showed that GM1α, GD1aα, and GT1bα were synthesized from asialo-GM1, GM1a, and GD1b, respectively, by the action of a GalNAc α2-6sialyltransferase.