Mutational analysis for enzyme activity of mouse Gal1,3GalNAc 2,3-sialyltransferase (mST3Gal I).

To determine which amino acid residues are essential for the catalytic activity of mouse Gal1,3GalNAc 2,3-sialyltransferase (mST3Gal I), chemical modification and site-directed mutagenesis were employed against tryptophan and cysteine residues located in the predicted catalytic domain. This enzyme was strongly inhibited by N-bromosuccinimide, a specific blocking reagent for tryptophan residues, and the enzyme activity was completely lost at 3 mM, suggesting the involvement of tryptophan residues in the catalytic activity of mST3Gal I. The N-ethylmaleimide, an irreversible reagent for sulfhydryl group, significantly inhibited the enzyme activity. Seven tryptophan and six cysteine residues conserved in the cloned Gal1,3GalNAc 2,3-sialyltransferases were separately substituted into phenylalanine and serine, respectively. The enzymatic activity assay for tryptophan mutants produced in COS cells showed a complete abolishment of the activity in all of the mutants, except that W70F and W97F retained about 60% and 40% activities of wild type, respectively. In the case of cysteine mutants, no enzyme activity was observed like tryptophan mutants, except for C139S. These results suggest that tryptophan and cysteine residues conserved in ST3Gal I are critical for its activity.

GD3 Accumulation in Cell Surface Lipid Rafts Prior to Mitochondrial Targeting Contributes to Amyloid-beta-induced Apoptosis

Neuronal apoptosis induced by amyloid beta-peptide (A beta) plays an important role in the pathophysiology of Alzheimer's disease (AD). However, the molecular mechanism underlying A beta-induced apoptosis remains undetermined. The disialoganglioside GD3 involves ceramide-, Fas- and TNF-alpha-mediated apoptosis in lymphoid cells and hepatocytes. Although the implication of GD3 has been suggested, the precise role of GD3 in A beta-induced apoptosis is still unclear. Here, we investsigated the changes of GD3 metabolism and characterized the distribution and trafficking of GD3 during A beta-induced apoptosis using human brain-derived TE671 cells. Extracellular A beta induced apoptosis in a mitochondrial-dependent manner. GD3 level was negligible in the basal condition. However, in response to extracellular A beta, both the expression of GD3 synthase mRNA and the intracellular GD3 level were dramatically increased. Neosynthesized GD3 rapidly accumulated in cell surface lipid microdomains, and was then translocated to mitochondria to execute the apoptosis. Disruption of membrane lipid microdomains with methyl-beta-cyclodextrin significantly prevented both GD3 accumulation in cell surface and A beta-induced apoptosis. Our data suggest that rapidly accumulated GD3 in plasma membrane lipid microdomains prior to mitochondrial translocation is one of the key events in A beta-induced apoptosis.

Sialylmotifs of sialyltransferases.

The sialyl moiety of sialylated glycoconjugates expressed on the cell surface are increasingly recognized as the key determinants of various biological recognition events. The transfer of sialic acid to these glycoconjugates are catalyzed by sialyltransferases, a group of 15 or more Golgi enzymes. Cloning of three sialyltransferases from this laboratory, indicated for the first time, that these enzymes are type II membrane proteins and share the topological features common to other glycosyltransferases. However, unlike the other members of the glycosyltransferase family, these enzymes showed the presence of two conserve protein domains, termed 'sialylmotifs'. This unique feature was subsequently found to be present in all the sialyltransferases cloned to-date. The larger 'L-sialylmotif' consisting of 48-49 amino acids is present in the middle of the luminal catalytic domain and has, eight invariant residues, while the 'S-sialylmotif' present closer to the C-terminal end of the enzyme has two invariants among a stretch of 23 amino acids. The other not-so-invariant amino acids are also conserved and their replacement is limited. The functional role of these two sialylmotifs were investigated by single-point site-directed mutagenesis using Gal beta 1, 4GlcNAc alpha 2,6-sialyltransferase (ST6Gal I) as a model. Detailed kinetic analysis of the mutants indicated that the 'L-sialylmotif' contributes to the binding of the common donor substrate CMP-NeuAc, while the 'S-sialylmotif' contributes to the binding of both the donor and acceptor substrates.