Mutations within the propeptide, the primary cleavage site or the catalytic site, or deletion of C-terminal sequences, prevents secretion of proPC2 from transfected COS-7 cells.
PC2 is a neuroendocrine endoprotease involved in the processing of prohormones and proneuropeptides. PC2 is synthesized as a proenzyme which undergoes proteolytic maturation within the cellular secretory apparatus. Cleavage occurs at specific sites to remove the N-terminal propeptide. The aim of the present study was to investigate structural requirements for the transfer of proPC2 through the secretory pathway. A series of mutant proPC2 constructs were transfected into COS-7 cells and the fate of the expressed proteins followed by pulse-chase analysis and immunocytochemistry. Human PC2 was secreted relatively slowly, and appeared in the medium primarily as proPC2 (75 kDa), together with much lower amounts of a processed intermediate (71 kDa) and mature PC2 (68 kDa). Mutations within the primary processing site or the catalytic triad caused the protein to accumulate intracellularly, whereas deletion of part of the propeptide, the P-domain or the C-terminal regions also prevented secretion. Immunocytochemistry showed that wild-type hPC2 was localized mainly in the Golgi, whereas two representative mutants showed a distribution typical of proteins resident in the endoplasmic reticulum. The results suggest that proenzyme processing is not essential for secretion of PC2, but peptides containing mutations that affect the ability of the propeptide (and cleavage sites) to fold within the catalytic pocket are not transferred beyond the early stages of the secretory pathway. C-terminal sequences may be involved in stabilizing such conformations.
Taylor
N A
NA
Department of Molecular and Cell Biology, University of Aberdeen, U.K.
Shennan
K I
KI
Cutler
D F
DF
Docherty
K
K
D000595:Amino Acid Sequence / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D019556:COS Cells; D002384:Catalysis; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics* / Q000502:physiology; D045706:Proprotein Convertase 2; D011498:Protein Precursors / Q000235:genetics* / Q000502:physiology; D017384:Sequence Deletion; D013381:Subtilisins / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D010446:Peptide Fragments; D011498:Protein Precursors; D013381:Subtilisins; D045706:Proprotein Convertase 2