Pulse-chase analysis of procollagen biosynthesis by azidohomoalanine labeling

Abstract

Disruptions in procollagen synthesis, trafficking and secretion by cells occur in multiple connective tissue diseases. Traditionally, these disruptions are studied by pulse-chase labeling with radioisotopes. However, significant DNA damage, excessive accumulation of reactive oxygen species and formation of other free radicals have been well documented in the literature at typical radioisotope concentrations used for pulse-chase experiments. Therefore, it is important to keep in mind that the resulting cell stress response might affect interpretation of the data, particularly with respect to abnormal function of procollagen-producing cells. In this study, we describe an alternative method of pulse-chase procollagen labeling with azidohomoalanine, a noncanonical amino acid that replaces methionine in newly synthesized protein chains and can be detected via highly selective click chemistry reactions. At least in fibroblast culture, this approach is more efficient than traditional radioisotopes and has fewer, if any, unintended effects on cell function. To illustrate its applications, we demonstrate delayed procollagen folding and secretion by cells from an osteogenesis imperfecta patient with a Cys substitution for Gly766 in the triple helical region of the α1(I) chain of type I procollagen.

• Azidohomoalanine
• collagen
• metabolic labeling
• osteogenesis imperfecta
• pulse-chase

Acknowledgments

The authors thank Dr Dan L. Sackett for insightful comments and suggestions, Ms. Linda Powers for technical assistance as well as Drs Peter H. Byers and Joan C. Marini for generously providing cells used in this study.