Graz University of Technology Austria Institute of Biochemistry ...

sensitive to stabilization by iminosugars, which bind at the active site to provide the proper
conformation. Thus the stabilized protein may escape from degradation processes, and reach
the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET).
In this work the influence of novel derivatives of 1-deoxygalactonojirimycin (DGJ)
on the β-Gal activity of cultured GM1 and MBD skin fibroblasts was examined. Furthermore,
the effect of selected compounds on natural substrate degradation in GM1 and MBD cells was
determined. Several novel iminosugars acting as pharmacological chaperones of β-Gal in
specific GM1 fibroblasts were discovered and described in this work. One specific compound,
DLHex-DGJ, proved to be a potent competitive inhibitor of β-Gal in vitro, and this work
describes its effects on activity, protein expression, maturation and intracellular transport in
vivo in 13 fibroblast lines with GLB1 mutations. DLHex-DGJ significantly increased the
catalytic activity in six GM1 cell lines, and normalization of transport and lysosomal
processing of β-Gal precursors was demonstrated for selected cell lines. Furthermore,
DLHex-DGJ and another, similar compound successfully reduced the level of internalized
radiolabeled G M1 -gangliosides in a specific GM1 cell line, suggesting that reduction of stored
material is possible under certain conditions.
Specific antibodies, directed against human β-Gal, were developed with the aid of
previously published protocols, and novel approaches to obtain large amounts of the purified
human enzyme were tested. Two novel polyclonal anti-β-Gal peptide antibodies were
produced and expression of human β-Gal in E. coli cells may provide the basis for further
development of antibodies directed against the human enzyme. Large parts of this thesis were
carried out at the University hospital under the guidance and supervision of Prof. Pascke.
International cooperations
Maria Abramic, Ruder Boskovic Institute Zagreb, Croatia
Steve Ealick, Cornell University, Ithaca, U.S.A.
Toni Kutchan, Donald Danforth Plant Science Center, St. Louis, U.S.A.
Shwu Liaw, National Yang-Ming University, Taipei, Taiwan
Matthias Mack, Hochschule Mannheim, Germany
Bruce Palfey, University of Michigan, Ann Arbor, U.S.A.
Research projects
FWF P22361: “Mechanism of redox controlled protein degradation”
FWF P19858: “Enzymes of nikkomycin biosynthesis”
FWF-Doktoratskolleg “Molecular Enzymology”
WTZ Austria-Croatia “Structure-function relationships in metallopeptidases of the M49
family”
Publications
1) Durchschein, K., Ferreira-da Silva, B., Wallner, S., Macheroux, P., Kroutil, W., Glueck,
S. M., Faber, K.: The flavoprotein-catalyzed reduction of aliphatic nitro-compounds
represents a biocatalytic equivalent to the Nef-reaction, Green Chemistry, 2010, 12:616-
619.
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