Processing and trafficking of a prohormone convertase 2 active site mutant.
Processing of most PC zymogens is required for successful folding and/or passage through the secretory pathway; active site mutants are retained in the ER and degraded. We here report that the active site serine mutant of PC2 (PC2-S383A) was efficiently secreted as the intact zymogen in CHO-K1 cells, suggesting that its propeptide can productively insert into the mutated binding pocket without causing misfolding. In AtT-20 cells, PC2-S383A was cleaved at the secondary cleavage site within the propeptide; this cleavage event was pH-dependent and was inhibited by a proprotein convertase inhibitor. In vitro digestion of PC2-S383A with various convertases indicates that this site is accessible to in trans cleavage. Abundant immunoreactive S383A PC2 was found in secretory granules, supporting the idea that this protein is efficiently trafficked through the secretory pathway.
Lee
Sang-Nam
SN
Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center/Research Institute for Children, New Orleans, Louisiana 70118, USA.
Kacprzak
Magdalena M
MM
Day
Robert
R
Lindberg
Iris
I
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D045706:Proprotein Convertase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D021381:Protein Transport; D022142:Secretory Vesicles / Q000201:enzymology; D012694:Serine / Q000737:chemistry / Q000235:genetics
D012694:Serine; D045706:Proprotein Convertase 2