Abstract
In Fabry’s Disease the defective -galcotosidase A (GALA) is not able to break down the GB3
which results in lysosomal GB3 accumulation. In current therapies the GALA is intravenous
infused but this therapy is not efficient enough. Therefore we did a pilot study to make
lactosylceramide 4-alpha-galactosyltransferase (A4GALT) which is the enzyme that produces
the GB3. In the future inhibitors can be found with the aid of the 3D structure of A4GALT,
those inhibitors are supposed to prevent the GB3 accumulation.
The A4GALT protein is a type II transmembrane protein (N-terminal transmembrane domain
and luminal catalytic C-terminal domain. The A4GALT must be soluble to improve the
conditions to generate crystals for structure analysis. In order to make the A4GALT soluble, a
signal peptide from chitotriosidase is placed instead of the A4GALTs transmembrane
domain, this signal peptide transfers the enzyme from the Golgi to the outside of the cell.
Two isoforms of this soluble enzyme are made, one with a myc-his tag (pRGB217) and the
other with a chitin binding domain (pRGB218). Those enzymes are tested for their binding
capacity towards Ni-NTA beads and Co-magnetic beads (for pRGB217) and chitin magnetic
beads (for pRGB218). We tested the activity and followed the enzymes by western blot.
pRGB218 bound really good at the chitin magnetic beads but we found aspecific binding as
well. We did not succeed in make it soluble again with thrombin cleavage. This is probably
due to the buffers used. We were not able to follow the pRGB218 because the commercial
anti-body anti-A4GALT did not work. The pRGB217 had an additional myc-his tag and the
anti-body anti-myc worked really well thus we were able to follow the enzyme during the
experiments. This enzyme binds to the Ni-NTA and the Co-magnetic beads used without a lot
of aspecific binding. We started a pilot study to purify pRGB217 with affinity
chromatography using a Ni-column but more experiments are needed before this can be
used to purify the protein at larger scales.
In the future the expression can be scaled up. To scale up there is a stable cell-line or a
better transient expression system needed. CHO-DG44 is promising because it can be used
for large scale transient protein expression. Another possibility are BHK cells which already
has been used for large scale production of human chitotriosidase.