Characterization of Aspartylglucosaminidase Activation and Aspartylglucosaminuria Mutations

..........................................................................................................................8 REVIEW OF THE LITERATURE ..........................................................................................9 1. LYSOSOMES ....................................................................................................................9 2. PROTEIN FOLDING, MODIFICATION, AND DEGRADATION.......................................................9 2.1. Protein folding ......................................................................................................10 2.11. Chaperones .....................................................................................................10 2.2. Protein modification .............................................................................................12 2.3. Degradation of misfolded proteins .......................................................................13 3. INTRACELLULAR TRANSPORT OF SOLUBLE LYSOSOMAL PROTEINS.....................................14 4. ASPARTYLGLUCOSAMINIDASE (AGA) ..............................................................................17 4.1. Properties and purification of AGA ......................................................................17 4.2. The AGA gene .....................................................................................................18 4.3. Dimerization and autoproteolysis of AGA precursors..........................................19 4.4. Intracellular processing and transport of AGA.....................................................19 4.5. Structure and reaction mechanism of human AGA .............................................21 4.6. The prokaryotic AGA enzyme..............................................................................24 5. ASPARTYLGLUCOSAMINURIA (AGU)................................................................................25 5.1. Clinical features and diagnosis ............................................................................25 5.2. AGU mutations.....................................................................................................26 5.3. AGU mouse model...............................................................................................27 6. N-TERMINAL NUCLEOPHILE HYDROLASES ........................................................................27 6.1. Proteasome β-subunit (PRO) ..............................................................................28 6.2. Penicillin G acylase (PGA)...................................................................................29 6.3. Glutamine amidotransferase (GAT).....................................................................30 6.4. Other members of the superfamily ......................................................................30 6.5. Autoproteolytic activation of other proteins..........................................................31 AIMS OF THE PRESENT STUDY ......................................................................................32 MATERIALS AND METHODS............................................................................................33 7. STABLE EXPRESSION OF AGA IN CHO CELLS (I) .............................................................33 8. PRODUCTION OF THE YEAST CELL LINE FOR AGA PRECURSOR EXPRESSION .....................33 9. EXPRESSION, PURIFICATION, AND ANALYSES OF AGA PRECURSOR...................................34 RESULTS AND DISCUSSION............................................................................................35 10. ANALYSIS OF CONSERVED RESIDUES OF THE AGA MOLECULE........................................35 10.1. Dimerization of the precursor molecules (I, II) ...................................................36 10.2. Autoproteolysis of the precursor molecules (I, III) .............................................39 10.2.1. Residues participating in autocatalysis (I) ....................................................39 10.2.2. Human versus bacterial AGA precursor .......................................................40 10.2.3. Residues with a structural role (III) ...............................................................41 10.2.4. Peptide bond distortion (I).............................................................................44 10.2.5. Activation mechanism of human AGA (I, III).................................................45 10.2.6. Autocatalytic activation in Ntn-hydrolases ....................................................47 11. CHARACTERIZATION OF AGU MUTATIONS (II) ................................................................49 11.1. Consequences of AGU mutations .....................................................................50 11.1.1. Mutations with mild effects............................................................................50 11.1.2. Mutations with moderate effects...................................................................51 11.1.3. Mutations with severe effects .......................................................................52 12. EXPRESSION AND PURIFICATION OF RECOMBINANT HUMAN AGA PRECURSOR .................55 12.1. Expression of recombinant human AGA precursor ...........................................55 12.2. Purification and crystallization of human AGA precursor ..................................56 CONCLUDING REMARKS .................................................................................................59 ACKNOWLEDGEMENTS ...................................................................................................61 REFERENCES ....................................................................................................................63 LIST OF ORIGINAL PUBLICATIONS This thesis is based on the following original articles, which are referred to in the text by their Roman numerals: I. Saarela, J., Laine, M., Tikkanen, R., Oinonen, C., Jalanko, A., Rouvinen, J. and Peltonen, L. (1998) Activation and oligomerization of aspartylglucosaminidase. J. Biol. Chem., 273, 25320-8. * II. Saarela, J., Laine, M., Oinonen, C., von Schantz, C., Jalanko, A., Rouvinen, J. and Peltonen, L. (2001) Molecular pathogenesis of a disease: structural consequences of aspartylglucosaminuria mutations. Hum. Mol. Genet., 10, 983-95. III. Saarela, J., Oinonen, C., Jalanko, A., Rouvinen, J., and Peltonen, L. (2003) Autoproteolytic activation of human aspartylglucosaminidase. In press, The Biochemical Journal. In addition, some unpublished data are presented. * Appeared also in the thesis of Carita Oinonen (2000).