7B2 prevents unfolding and aggregation of prohormone convertase 2.
Prohormone convertase 2 (PC2) requires interaction with the neuroendocrine protein 7B2 for the production of an activatable zymogen; the mechanism for this effect is unknown. 7B2 could act proactively to generate an activation-competent form of pro-PC2 during synthesis, or block spontaneous generation of activation-incompetent forms. We here demonstrate that addition of exogenous recombinant 7B2 to CHO cells expressing pro-PC2 prevented the unfolding and aggregation of secreted PC2 forms in a dose-dependent manner, as assessed by aggregation assays, activity assays, cross-linking experiments, and sucrose density gradients. Intracellular pro-PC2 was also found to exist in part as higher-order oligomers that were reduced in the presence of coexpressed 7B2. 7B2 addition did not result in the acquisition of enzymatic competence unless added before or very rapidly after pro-PC2 secretion, indicating that an activation-competent structure cannot be maintained in the absence of 7B2. Velocity sedimentation experiments showed that addition of extracellular 7B2 solubilized three different PC2 species from a precipitable aggregate: two activatable pro-PC2 species, the intact zymogen and a zymogen with a partially cleaved propeptide, and an inactive 66-kDa form. Our results suggest that 7B2 possesses chaperone activity that blocks partially unfolded pro-PC2 forms from losing catalytic competence and then aggregating. The loss of the catalytically competent conformer appears to represent the earliest indicator of pro-PC2 unfolding and is followed on a slower time scale by the appearance of aggregates. Because 7B2 expression is not confined to areas expressing pro-PC2, 7B2 may represent a general intracellular and extracellular secretory chaperone.
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.
Lindberg
Iris
I
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D011232:Chemical Precipitation; D006224:Cricetinae; D003412:Cricetulus; D019281:Dimerization; D004789:Enzyme Activation; D051379:Mice; D008954:Models, Biological; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism / Q000502:physiology; D008969:Molecular Sequence Data; D051859:Neuroendocrine Secretory Protein 7B2 / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D045706:Proprotein Convertase 2 / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000494:pharmacology; D013329:Structure-Activity Relationship
D018832:Molecular Chaperones; D051859:Neuroendocrine Secretory Protein 7B2; D011994:Recombinant Proteins; D045706:Proprotein Convertase 2