DISSECTION OF THE MOLECULAR CONSEQUENCES OF A DOUBLE MUTATION CAUSINGA HUMAN LYSOSOMAL DISEASE

Aspartylglucosaminidase (AGA) is a lysosomal enzyme, the deficiency in which leads to human storage disease aspartylglucosaminuria (AGU). AG U(Fin) is the most common AGU mutation in the world and is found in 98 % of AGU alleles in Finland, where the population displays enrichment of the disease allele. The AGU(Fin) allele actually contains a double mutation, both individual mutations resulting in amino acid substituti ons: : Arg-161 --> Gln and Cys-163 --> Ser. The separate consequences of these two amino acid substitutions for the intracellular processing of the AGA polypeptides were analyzed using a stable expression of mu tant polypeptides in Chinese hamster ovary (CHO) cells. The synthesize d polypeptides were monitored by metabolic labeling, followed by immun oprecipitation, immunofluorescence, and immunoelectron microscopy. The Arg-161 --> Gln substitution did not affect the intracellular process ing or transport of AGA and the fully active enzyme was correctly targ eted to lysosomes. The Cys-163 --> Ser substitution prevented the earl y proteolytic cleavage required for the activation of the precursor AG A polypeptide and the inactive enzyme was accumulated in the endoplasm ic reticulum (ER). The precursors of the translation products of the A GU(Fin) double mutant and the Cys-163 --> Ser mutant were also observe d in the culture medium. When cells expressing the normal AGA or AGU(F in) double mutation were treated with DTT to prevent the formation of disulfide bonds, both normal and mutated AGA polypeptides remained in the inactive precursor form and were not secreted into the medium. The se results indicate that correct initial folding is essential for the proteolytic activation of AGA. They also suggest that a fraction of th e AGU(Fin) polypeptides remain in a conformation that is able to enter the secretory pathway, but in which the lysosomal targeting signals a re hidden.