Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation.
The recognition of lysosomal enzymes by UDP-GlcNAc: lysosomal-enzyme GlcNAc-1-phosphotransferase (phosphotransferase) is mediated by a protein structure on lysosomal enzymes. It has been previously demonstrated that lysine residues are required for phosphorylation of procathepsin L and are a common feature of the site on many lysosomal proteins. In this work, the procathepsin L recognition structure was further defined by identification of the region of the protein containing the structure and the critical lysine residues involved. Removal of the cathepsin L propeptide by low pH-induced autocatalytic processing abolished phosphorylation. The addition of either the purified propeptide or a glutathione S-transferase-propeptide fusion protein to the processed protein restored phosphorylation. Mutagenesis of individual lysine residues demonstrated that two propeptide lysine residues (Lys-54 and Lys-99) were required for efficient phosphorylation of procathepsin L. By comparison of the phosphorylation rates of procathepsin L, lysine-modified procathepsin L, and the procathepsin L oligosaccharide, lysine residues were shown to account for most, if not all, of the protein-dependent interaction. On this basis, it is concluded that the proregion lysine residues are the major elements of the procathepsin L recognition site. In addition, lysine residues in cathepsin D were shown to be as important for phosphorylation as those in procathepsin L, supporting a general model of the recognition site as a specific three-dimensional arrangement of lysine residues exposed on the surface of lysosomal proteins.
Cuozzo
J W
JW
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Tao
K
K
Wu
Q L
QL
Young
W
W
Sahagian
G G
GG
D016475:3T3 Cells; D000818:Animals; D002402:Cathepsin D / Q000737:chemistry; D056668:Cathepsin L; D002403:Cathepsins / Q000737:chemistry* / Q000378:metabolism; D004792:Enzyme Precursors / Q000737:chemistry* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000737:chemistry*; D008358:Mannose / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D010766:Phosphorylation
D004792:Enzyme Precursors; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L; D002402:Cathepsin D; D008239:Lysine; D008358:Mannose