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P-27 A Possible Role for Carboxypeptidase O in the Regulation of Secretion through Cleavage of C-terminal ER Retention Signals
Celebration of Research and Creative Scholarship
  • Peter Lyons, Andrews University
  • Christian Bardan
  • Linnea C Burke
Presenter Status
Associate Professor, Biology
Second Presenter Status
Student, Biology
Third Presenter Status
Student, Biology
Preferred Session
Poster Session
Start Date
4-11-2016 2:00 PM
End Date
4-11-2016 3:00 PM
Presentation Abstract
Proteins containing C-terminal ER retention signals are sometimes secreted, possibly following proteolytic cleavage of the KDEL retention sequence. However, this cleavage has rarely been seen and only one responsible protease has previously been identified. Carboxypeptidase O (CPO) is a GPI-anchored proteolytic enzyme that cleaves acidic C-terminal amino acids from substrate proteins and peptides, with some ability to cleave hydrophobic amino acids. CPO lacks a prodomain and exhibits activity across a broad pH range, and thus is likely to have activity throughout its transit through the secretory system. To investigate a possible role in the cleavage of C-terminal ER retention signals, CPO expression, subcellular distribution, and function were examined. Western blotting analysis of T. striatus tissue lysates showed immunoreactive bands of the correct size in liver, kidney, and small intestine tissues. Immunofluorescence microscopy of transfected MDCK cells indicated membrane attached CPO was commonly found aggregated within the endomembrane system, particularly under cholesterol-depleted conditions. This distribution changed upon cholesterol enrichment, when CPO became more diffused throughout the endomembrane system. Cholesterol enrichment reduced the enzymatic activity of CPO, as measured with a chromogenic substrate containing C-terminal glutamate; no change in activity was observed with a substrate containing a C-terminal alanine. Depletion of cholesterol had the opposite effect – an increase in the enzymatic activity of CPO. To investigate the role of CPO within the secretory pathway, Gaussia Luciferase (GLuc) and variants with ER retention signals (GLuc-KDEL, GLuc-RTDL) were expressed in MDCK cells along with CPO. Increased secretion of GLuc-KDEL and GLuc-RTDL was observed from CPO-expressing cells, particularly when cholesterol-enriched. These results suggest secretion of some proteins may be regulated by cleavage of ER retention sequences by CPO, particularly when CPO is associated with cholesterol rich membrane domains.
Acknowledgments

Andrews University Faculty Research Grant

Citation Information
Peter Lyons, Christian Bardan and Linnea C Burke. "P-27 A Possible Role for Carboxypeptidase O in the Regulation of Secretion through Cleavage of C-terminal ER Retention Signals" (2016)
Available at: http://works.bepress.com/peter_lyons/13/