The Spn4 gene of Drosophila encodes a potent furin-directed secretory pathway serpin.
Proprotein convertases (PCs) are an important class of host-cell serine endoproteases implicated in many physiological and pathological processes. Owing to their expanding roles in the proteolytic events required for generating infectious microbial pathogens and for tumor growth and invasiveness, there is increasing interest in identifying endogenous PC inhibitors. Here we report the identification of Spn4A, a previously uncharacterized secretory pathway serine protease inhibitor (serpin) from Drosophila melanogaster that contains a consensus furin cleavage site, -Arg(P4)-Arg-Lys-Arg(P1) downsream-, in its reactive site loop (RSL). Our biochemical and kinetics analysis revealed that recombinant Spn4A inhibits human furin (K(i), 13 pM; k(ass), 3.2 x 10(7) M(-1) x s(-1)) and Drosophila PC2 (K(i), 3.5 nM; k(ass), 9.2 x 10(4) M(-1) x s(-1)) by a slow-binding mechanism characteristic of serpin molecules and forms a kinetically trapped SDS-stable complex with each enzyme. For both PCs, the stoichiometry of inhibition by Spn4A is nearly 1, which is characteristic of known physiological serpin-protease interactions. Mass analysis of furin-Spn4A reaction products identified the actual reactive site center of Spn4A to be -Arg(P4)-Arg-Lys-Arg(P1)-downstream-. Moreover, we demonstrate that Spn4A's highly effective PC inhibition properties are critically dependent on the unusual length of its RSL, which is composed of 18 aa instead of the typical 17-residue RSL found in most other inhibitory serpins. The identification of Spn4A, the most potent and effective natural serpin of PCs identified to date, suggests that Spn4A could be a prototype of endogenous serpins involved in the precise regulation of PC-dependent proteolytic cleavage events in the secretory pathway of eukaryotic cells.
Richer
Martin J
MJ
Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada V6T 1Z3.
Keays
Clairessa A
CA
Waterhouse
Jennifer
J
Minhas
Jessey
J
Hashimoto
Carl
C
Jean
François
F
D000595:Amino Acid Sequence; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D045683:Furin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D043484:Proprotein Convertases / Q000037:antagonists & inhibitors / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D015843:Serpins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D029721:Drosophila Proteins; D015843:Serpins; C488721:Spn42Da protein, Drosophila; D043484:Proprotein Convertases; D045683:Furin