Regulatory roles of the P domain of the subtilisin-like prohormone convertases.
A unique feature of the eukaryotic subtilisin-like proprotein convertases (SPCs) is the presence of an additional highly conserved sequence of approximately 150 residues (P domain) located immediately downstream of the catalytic domain. To study the function of this region, which is required for the production of enzymatically active convertases, we have expressed and characterized various P domain-related mutants and chimeras in HEK293 cells and alpha-TC1-6 cells. In a series of C-terminal truncations of PC3 (also known as PC1 or SPC3), PC3-Thr594 was identified as the shortest active form, thereby defining the functional C-terminal boundary of the P domain. Substitutions at Thr594 and nearby sites indicated that residues 592-594 are crucial for activity. Chimeric SPC proteins with interchanged P domains demonstrated dramatic changes in several properties. Compared with truncated wild-type PC3 (PC3-Asp616), both PC3/PC2Pd and PC3/FurPd had elevated activity on several synthetic substrates as well as reduced calcium ion dependence, whereas Fur/PC2Pd was only slightly decreased in activity as compared with truncated furin (Fur-Glu583). Of the three active SPC chimeras tested, all had more alkaline pH optima. When PC3/PC2Pd was expressed in alpha-TC1-6 cells, it accelerated the processing of proglucagon into glicentin and major proglucagon fragment and cleaved major proglucagon fragment to release GLP-1 and tGLP-1, similar to wild-type PC3. Thus, P domain exchanges generated fully active chimeric proteases in several instances but not in all (e.g. PC2/PC3Pd was inactive). The observed property changes indicate a role for the P domain in regulating the stability, calcium dependence, and pH dependence of the convertases.
Zhou
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Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.
Martin
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Lipkind
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LaMendola
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J
Steiner
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DF
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D043484:Proprotein Convertases; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013381:Subtilisins / Q000737:chemistry / Q000378:metabolism*; D013912:Threonine / Q000737:chemistry / Q000378:metabolism
D011994:Recombinant Proteins; D013912:Threonine; D043484:Proprotein Convertases; D013381:Subtilisins; C473656:Pcsk1 protein, mouse; D045664:Proprotein Convertase 1; D045706:Proprotein Convertase 2; D016282:Aspartic Acid Endopeptidases