Factors that install activity and specificity in glycolipid biosynthesis pathways
Final Report Abstract
The big variety of carbohydrate structures proteins and lipids on the cell surface of human cells are made by several hundred different glycosyltransferases. In this project, we were most interested in how enzymes can specifically use lipids as acceptor structures for the transfer of sugars. Glycolipid biosynthesis is a potential target for treatment of several human diseases including lysosomal storage diseases, cancer, and chronic central nervous system inflammation. Indeed, a medicine is on the marked that inhibits the enzyme that transfers the first sugar to the lipid acceptor to make glucosylceramide. Reducing the production of glycolipids (substrate deprivation therapy) can prevent accumulation of compounds that are only slowly degraded in patients with glycolipid storage diseases. We work on the second enzyme that acts in the glycolipis biosynthetic pathway. This is a galactosyltransferase that uses glucosylceramide as substrate and makes lactosylceramide. Inhibition of this enzyme can be advantageous in many cases in which lack of degradation of glucosylceramide is not the primary defect. The two galactosyltransferases that can act on glucosylceramide are members of a larger family of glycosyltransferases that act on glycoproteins as well. It was not known what makes these two enzymes different from the others to act on lipids. We had discovered before that another glycosyltransferase in the fruit fly Drosophila melanogaster required domains outside of the catalytic domain on both the protein itself and on another protein to be able to use glycolipids as acceptor substrates. We now discovered that a similar mechanism is used by the human galactosyltransferases. No additional protein is required but amino acids outside of the catalytic domain were required for activity. Glycosyltransferases are build-up modularly, having a membrane anchor and a catalytic domain that is linked by a spacer that is called the stem region. Whereas in other related enzymes the catalytic domain alone is able to catalyse the transfer of sugars, this was not the case for β4galactosyltransferase5 (gene name B4GALT5), the enzyme that we studied. It requires specific amino acid at the border of the membrane anchor and stem for activity on its lipid linked substrate. Due to the fact that the substrate of the enzyme is localized directly on the membrane surface, it is most likely that this region of the protein is required to recognize the substrate to present it to the catalytic domain. To be able to study the activity of the the human galactosyltransferase we had to set up a new assay system. We were able to measure the activity of the enzyme directly in the cell by expression in insect cells that do not possess this enzymatic activity and co-expression of a second enzyme that puts another galactose on top of lactosylceramide to make the lipid linked trisaccharide that is called Gb3. This is the well-known substrate for shiga toxin and can easily be detected by specific antibodies. This allowed sensitive detection and distinction of transfer of galactose to glucosylceramide from transfer of galactose to other substrates. Our results show that it must be feasible to specifically inhibit transfer of galactose to the lipid acceptor by interfering with the lipid recognition and not with the catalytic domain that is much conserved in other galactosyltransferases.
Publications
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(2011)"Addon" domains of drosophila β1,4-N-acetylgalactosaminyltransferase B in the stem region and its pilot protein. Cellular and Molecular Life Sciences 68 p4091-4100
Kraft B, Johswich A, Kauczor G, Scharenberg M, Gerardy-Schahn R, Bakker H
