title: Biosynthesis, targeting, and processing of lysosomal proteins: pulse-chase labeling and immune precipitation.
abstract: Incorporation of radioactive precursors of amino acids and/or modifier groups into proteins, isolation and sizing of polypeptide species of interest, and finally their detection and characterization provide a robust handle to examine the life cycle and varied modifications of any protein. A prerequisite in application of these techniques to lysosomal enzymes is the availability of an avid and specific antibody, because lysosomal proteins represent a very minor fraction of the cellular protein and must be purified without a significant loss many 1000-fold as conveniently as possible. Pulse-chase labeling and good knowledge on organelle-specific modifications of lysosomal proteins may enhance the information that can be obtained from such experiments. We describe procedures for pulse-chase labeling experiments that have proven to work with a commercially available antibody against a mouse and a human lysosomal protease and can be used as a reference in establishing the technique in any laboratory that has an access to a certified isotope facility and the knowledge to handle radioactivity safely. We discuss the crucial steps and refer to alternatives described in the literature. The present model protein cathepsin Z is synthesized as a larger proenzyme that contains two N-linked oligosaccharides and matures to a shorter single chain enzyme retaining the processed oligosaccharides. A pulse-chase experiment demonstrates the conversion of the precursor into the mature form. In addition, results on deglycosylation of metabolically labeled cathepsin Z are shown and the alterations in the apparent size of the glycopeptides are explained.
authors: Section Biochemistry, Children's Hospital, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors: Sandra
authors: S
authors: Pohl
authors: Institute of Physiological Chemistry, Philipps-University Marburg, Marburg, Germany.
authors: Andrej
authors: A
authors: Hasilik
mesh_terms: D000818:Animals; D056663:Cathepsin Z / Q000302:isolation & purification / Q000378:metabolism; D002478:Cells, Cultured; D006801:Humans; D047468:Immunoprecipitation; D008247:Lysosomes / Q000378:metabolism*; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D011506:Proteins; C018589:lysosomal proteins; C534182:CTSZ protein, human; D056663:Cathepsin Z
keywords: Biosynthesis; Immune precipitation; Lysosomal enzyme; Metabolic labeling; Processing; Targeting