Event Title

Activity of Carboxypeptidase O in the Secretory Pathway of MDCK Cells

Presenter Information

Linnea C. Burke, Andrews University

Start Date

3-3-2017 2:30 PM

End Date

3-3-2017 4:00 PM

Description

Carboxypeptidase O (CPO), a member of the M14 family of proteolytic enzymes, preferentially cleaves C-terminal acidic amino acids, with weak affinity towards hydrophobic amino acids. We investigated the subcellular localization of CPO, and after immunofluorescent analysis of stably-transfected MDCK cells, we found that CPO co-localized with calnexin, an ER marker. To determine what CPO does in the ER, MDCK cells were transfected with plasmids expressing Gaussia Luciferase (GLuc) containing a C-terminal ER retention signal (KDEL). Previous experiments suggested that CPO cleaves the KDEL sequence of GLuc, causing its secretion. In ongoing experiments, plasmids expressing GLuc tagged with modified KDEL sequences (KDELD, KDELE, and KDELEL) will be transfected, and the intracellular activity of CPO against these substrates will be assessed.

Acknowledgments

Dr. Peter J. Lyons

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Mar 3rd, 2:30 PM Mar 3rd, 4:00 PM

Activity of Carboxypeptidase O in the Secretory Pathway of MDCK Cells

Carboxypeptidase O (CPO), a member of the M14 family of proteolytic enzymes, preferentially cleaves C-terminal acidic amino acids, with weak affinity towards hydrophobic amino acids. We investigated the subcellular localization of CPO, and after immunofluorescent analysis of stably-transfected MDCK cells, we found that CPO co-localized with calnexin, an ER marker. To determine what CPO does in the ER, MDCK cells were transfected with plasmids expressing Gaussia Luciferase (GLuc) containing a C-terminal ER retention signal (KDEL). Previous experiments suggested that CPO cleaves the KDEL sequence of GLuc, causing its secretion. In ongoing experiments, plasmids expressing GLuc tagged with modified KDEL sequences (KDELD, KDELE, and KDELEL) will be transfected, and the intracellular activity of CPO against these substrates will be assessed.