Initial insight into the function of the lysosomal 66.3 kDa protein from mouse by means of X-ray crystallography

In order to gain insight into the function and the post-translational maturation process of the glycosylated 66.3 kDa protein, three crystal structures were determined that represent different maturation states. These structures demonstrate that the 28 kDa and 40 kDa fragment which have been derived by a proteolytic cleavage remain associated. Mass spectrometric analysis confirmed the subsequent trimming of the C-terminus of the 28 kDa fragment making a large pocket accessible, at the bottom of which the putative active site is located. The crystal structures reveal a significant similarity of the 66.3 kDa protein to several bacterial hydrolases. The core αββα sandwich fold and a cysteine residue at the N-terminus of the 40 kDa fragment (C249) classify the 66.3 kDa protein as a member of the structurally defined N-terminal nucleophile (Ntn) hydrolase superfamily.Due to the close resemblance of the 66.3 kDa protein to members of the Ntn hydrolase superfamily a hydrolytic activity on substrates containing a non-peptide amide bond seems reasonable. The structural homology which comprises both the overall fold and essential active site residues also implies an autocatalytic maturation process of the lysosomal 66.3 kDa protein. Upon the proteolytic cleavage between S248 and C249, a deep pocket becomes solvent accessible, which harbors the putative active site of the 66.3 kDa protein.In order to spatially separate the vast number of divergent reactions carried out by intracellular enzymes, eukaryotic cells are compartmentalized into several membrane-bound organelles. Among these organelles, the lysosomal compartment contains more than 50 hydrolases required for degradation of macromolecules or even whole organelles entering the lysosome by endocytotic or autophagic pathways [1,2] (reviewed in [3]).This degradation process and thus the hydrolases involved are essential for the cell as reflected by the manifestation of severe diseases which are characterized by the accumula