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Decreased Aggregation of Carboxypeptidase O upon Incorporation into Lipid Rafts
Author(s) -
Ezeribe Hazel O,
Burke Linnea C,
Barfi Gifty B,
Lyons Peter J
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.596.5
Subject(s) - lipid raft , chemistry , microbiology and biotechnology , carboxypeptidase , biochemistry , transfection , subcellular localization , extracellular , enzyme , biology , membrane , cytoplasm , gene
Carboxypeptidase O (CPO) is a member of the M14 family of proteolytic enzymes. Many members of this family are secreted from cells and are involved in degradation of extracellular peptides; other carboxypeptidases remain within the secretory pathway where they are involved in the maturation of bioactive peptides. CPO is a GPI‐anchored carboxypeptidase that is expressed in epithelial cells of the small intestine. To further understand the function of CPO, we investigated its subcellular localization. CPO was found to be present in detergent‐insoluble complexes, suggesting a lipid raft localization and function. Immunofluorescence analysis of CPO in stably‐transfected MDCK cells, however, indicated a punctate distribution of aggregates having little co‐localization with lipid raft proteins. Incubation with cholesterol dramatically decreased this aggregation and resulted in the localization of CPO to puncta of uniform size (< 300nm diameter) and distribution. Similar results were obtained when cells were incubated with oleic acid, which also led to increased GPI anchoring and membrane association. We reasoned that increased membrane cholesterol might mediate conformational changes in CPO, via its GPI anchor, that result in decreased aggregation. We found that loss of the GPI anchor by deletion of the C‐terminal GPI signal peptide, but not the N‐terminal partial prodomain, resulted in greatly reduced expression of CPO in Sf9 cells, suggestive of a conformational and/or folding defect. A decrease in expression of this C‐terminally deleted CPO was also observed in HEK293T cells, although further analysis showed no alteration in protease susceptibility of CPO that might suggest large changes in conformation or folding. Our results suggest that incorporation of CPO into cholesterol‐rich lipid rafts leads to subtle changes in CPO conformation that prevent aggregation. Support or Funding Information Supported by Faculty Research Grants to PJL from Andrews University