Inactivation of the 7B2 inhibitory CT peptide depends on a functional furin cleavage site.
The eukaryotic subtilisin prohormone convertase 2 (PC2) is known to require in vivo exposure to the neuroendocrine protein 7B2 in order to produce an enzymatically active species capable of proteolytic action on prohormone substrates. In the present study, we examined the role of the pentabasic site within 27-kDa 7B2 in this process. We prepared two His-tagged recombinant 7B2s by overexpression in bacteria: 7B2-Ser-Ser (SS), with an inactivating mutation in the CT peptide from Lys171-Lys172 (KK) to SS, rendering the CT peptide non-inhibitory; blockade-SS, a double mutant of both the CT peptide as well as of the pentabasic furin cleavage site. These purified proteins were used in a cell-free proPC2 activation assay. Both 7B2-SS as well as blockade-SS were able to facilitate the activation of proPC2 (as judged by efficient production of enzyme activity), suggesting that cleavage at the furin site is not required for 7B2s lacking inhibitory CT peptides. Plasmids encoding proPC2 and various 7B2s were transiently transfected into human embryonic kidney (HEK293) cells and PC2 enzymatic activity and CT forms in each overnight conditioned medium were measured. Cells transfected with proPC2 and wild-type 7B2 secreted CT peptide cleavage products, but cells transfected with proPC2 and the blockade mutant overwhelmingly secreted intact, 27-kDa, blockaded 7B2. Medium obtained from HEK293 cells transfected with proPC2 and either wild-type 7B2, 7B2-SS, or blockade-SS exhibited PC2 activity, but medium from cells expressing the 7B2 blockade mutant did not. We conclude that cleavage at the 7B2 furin consensus site is required to produce PC2 capable of efficient proteolytic inactivation of the CT peptide.
Hwang
J R
JR
Department of Biochemistry, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA.
Lindberg
I
I
D000818:Animals; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation / Q000502:physiology; D045683:Furin; D006056:Golgi Apparatus / Q000201:enzymology; D006801:Humans; D009154:Mutation / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000502:physiology*; D051859:Neuroendocrine Secretory Protein 7B2; D010446:Peptide Fragments / Q000502:physiology; D010907:Pituitary Hormones / Q000235:genetics / Q000502:physiology*; D045706:Proprotein Convertase 2; D051381:Rats; D013381:Subtilisins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D013997:Time Factors
D009419:Nerve Tissue Proteins; D051859:Neuroendocrine Secretory Protein 7B2; D010446:Peptide Fragments; D010907:Pituitary Hormones; C497678:SCG5 protein, human; C497680:Scg5 protein, rat; D002268:Carboxypeptidases; D043423:Carboxypeptidase H; D013381:Subtilisins; D045683:Furin; D045706:Proprotein Convertase 2