Previously we reported that serum leucine-rich alpha-2-glycoprotein-1 (LRG) binds cytochrome c (Cyt c; Cummings et al., Apoptosis 11:1121-1129, 2009). Here we show that LRG binding to Cyt c is similar to that of Apaf-1. LRG and Apaf-1 share partial amino acid sequences, compete for binding Cyt c, and are inhibited by modification at lysine 72 in Cyt c. However, in contrast to Apaf-1, LRG acts as a survival factor in vitro rather than a pro-apoptotic factor. By depleting LRG from culture medium we found that LRG protects against a toxic effect of exogenous Cyt c on lymphocytes that would otherwise result in an apoptotic phenotype. LRG, as well as antibodies specific for Cyt c, increased cell viability in the absence of added Cyt c indicating that Cyt c released by dying cells in the cultures is itself toxic. Protection from extracellular Cyt c-induced lymphotoxicity appears to involve an active mechanism rather than steric hindrance of Cyt c. Thus, serum LRG when bound to extracellular Cyt c that is released from apoptotic cells acts as a survival factor for lymphocytes and possibly other cells that are susceptible to the toxic effect of extracellular Cyt c.
Bibliographical noteFunding Information:
Acknowledgments The authors dedicate this work to the memory of Professor Emanuel Margoliash whose pioneering studies of Cyt c, in particular his determination of the complete amino acid sequence of horse Cyt c, as well as the sequences of many other Cyts c, supported groundbreaking research in several areas including molecular evolution, structure–function relations in proteins, antigen recognition in the immune system, and apoptosis. The authors thank Chris Pennell for assistance with the BIAcore, Yuk Sham for the molecular graphics of Cyt c, Tim Leonard for help in the preparation of the figures, Bogdan Polevoda and Fred Sherman for yeast Cyts c as well as yeast expressing recombinant pigeon Cyt c, Emanuel Margoliash for recombinant rat Cyt c as well as several other Cyts c, Jeanne Min-nerath for purification of recombinant pigeon Cyt c, Gabriel Núñez for recombinant Apaf-1, and Julia Molony for assistance with the statistical analyses. This work was supported by grants from the Minnesota Medical Foundation and the University of Minnesota Graduate School (to R.J.) and by contributions from R.J. Potential conflict of interest: R.J. holds a U.S. patent for the indirect ELISA employed in this study.
Copyright 2010 Elsevier B.V., All rights reserved.
- Cytochrome c
- Leucine-rich alpha-2-glycoprotein-1
- Survival factor