Biosynthesis, targeting, and processing of lysosomal Proteins: pulse-chase labeling and immune precipitation

TY - JOUR

T1 - Biosynthesis, targeting, and processing of lysosomal Proteins: pulse-chase labeling and immune precipitation

AU - Pohl, Sandra

AU - Hasilik, Andrej

N1 - Copyright © 2015 Elsevier Inc. All rights reserved.

PY - 2015

Y1 - 2015

N2 - 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.

AB - 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.

U2 - 10.1016/bs.mcb.2014.10.020

DO - 10.1016/bs.mcb.2014.10.020

M3 - SCORING: Journal article

C2 - 25665441

VL - 126

SP - 63

EP - 83

JO - METHODS CELL BIOL

JF - METHODS CELL BIOL

SN - 0091-679X

ER -