Hysteretic Behavior of Proprotein Convertase 1/3 (PC1/3)

Hysteretic Behavior of Proprotein Convertase 1/3 (PC1/3) Marcelo Y. Icimoto1, Nilana M. Barros2, Juliana C. Ferreira1, Marcelo F. Marcondes1, Douglas Andrade1, Mauricio F. Machado1, Maria A. Juliano1, Wagner A. Ju´ dice3, Luiz Juliano1, Vitor Oliveira1* 1 Departamento de Biofı´sica, Universidade Federal de Saõ Paulo, Saõ Paulo, Brazil, 2 Cieˆncias Exatas e da Terra, Universidade Federal de Saõ Paulo, Saõ Paulo, Brazil, 3 Centro Interdisciplinar de Investigaçaõ Bioquı´mica, Universidade de Mogi das Cruzes, Saõ Paulo, Brazil Abstract The proprotein convertases (PCs) are calcium-dependent proteases responsible for processing precursor proteins into their active forms in eukariotes. The PC1/3 is a pivotal enzyme of this family that participates in the proteolytic maturation of prohormones and neuropeptides inside the regulated secretory pathway. In this paper we demonstrate that mouse proprotein convertase 1/3 (mPC1/3) has a lag phase of activation by substrates that can be interpreted as a hysteretic behavior of the enzyme for their hydrolysis. This is an unprecedented observation in peptidases, but is frequent in regulatory enzymes with physiological relevance. The lag phase of mPC1/3 is dependent on substrate, calcium concentration and pH. This hysteretic behavior may have implications in the physiological processes in which PC1/3 participates and could be considered an additional control step in the peptide hormone maturation processes as for instance in the transformation of proinsulin to insulin. Citation: Icimoto MY, Barros NM, Ferreira JC, Marcondes MF, Andrade D, et al. (2011) Hysteretic Behavior of Proprotein Convertase 1/3 (PC1/3). PLoS ONE 6(9): e24545. doi:10.1371/journal.pone.0024545 Editor: Vladimir N. Uversky, University of South Florida College of Medicine, United States of America Received June 17, 2011; Accepted August 12, 2011; Published September 15, 2011 Copyright: ß 2011 Icimoto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Fundaçaõ de Amparo Pesquisa do Estado de Saõ Paulo (FAPESP), Coordenaçaõ de Aperfeiçoamento de Pessoal de Nı´vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) – Instituto Nacional de Fluidos complexos (INCT-Fx). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction environment [11] and along the secretory pathway PC1/3 finds an environment that pH decreases from 6.7 to 5.5 [12]. It also known Mammalian subtilases of yeast-kexin type are called proprotein that in the secretory granules calcium concentration can raise up convertases (PCs) or subtilisin-like proprotein convertases (SPCs) to milimolar concentrations [13], [14]; and the peptidase activities [1]. PCs are calcium-dependent proteases responsible for the of 87 and 66-kDa forms of PC1/3 are increased in calcium ion processing/maturation of precursors proenzymes, neural and concentrations range 1 to 20 mM [11]. PC1/3 was reported to hormonal peptides, serum proteins, growth factors and surface present complex enzymatic kinetics for the hydrolysis of substrates glycoproteins of pathogenic bacteria and viruses [2], [3]. This [15], and a lag phase (pre-steady-state) in the initial 8 to family of peptidases is composed by PC1/3, PC2, furin, PC4, 10 minutes followed by a linear phase with a constant velocity PACE4, PC5/6, PC7/8, SKI-1 and NARC-I [4]. Furin, PACE4, of hydrolysis (steady-state) was reported [11]. PC5-B, and PC7 are preferentially expressed in the constitutive The aim of the present paper was to explore the unusual lag secretory pathway, whereas PC1/3, PC2, and PC5-A are active in phase observed in the time course of activity of mPC1/3 as the secretory granules of the regulated secretory pathway of showed in Figure 1 for hydrolysis of the commercially available endocrine and neuroendocrine tissues [5]. fluorogenic substrate pERTKR-AMC. In addition to this peptide All PCs have in tandem disposition the following structures: N- we also examined the hydrolysis of fluorescence resonance energy terminal signal peptide followed by profragment region, conserved subtilisin-like catalytic domain, conserved P-domain and divergent transfer (FRET) peptides based on the PC1/3 natural consensus C-terminal tail [4]. PC1/3 initiates the formation of the active cleavage sites (indicated by the arrows): Abz-YTPKSRRQEVED- forms of neuropeptides by cleaving the precursor proteins after Q-EDDnp, (proinsulin: NP_032412), Abz-SPREGKRQSYSM- pairs of basic residues [6], the same general and conserved motif Q-EDDnp (POMC: EDL01384) and Abz-SKRSRRQSVSV-Q- recognized by the majority of PCs [2]. PC1/3 is synthesized as a EDDnp (Japanese encephalitis virus polyprotein: AAT00231) 753-amino acid zymogen [7], [8] and undergoes autocatalytic [Abz, ortho-aminobenzoic acid; Q-EDDnp, glutamyl-N-(2,4-dini- intramolecular processing of its N-terminal profragment in the ER trophenyl) ethylenediamine]. The last FRET substrate was [9], [10]. The generated 87-kDa protein is targeted to the selected from a series of sixty substrates previously designed for regulated secretory pathway where it is further shortened by furin studies [16]. The influence of calcium ion concentration and removal of 135 amino acids of its C-terminal tail, resulting in the the pH on mPC1/3 activity lag phase were also examined. Finally, 66-kDa form [8]. This C-terminal cleavage occurs at the dibasic we verify if the mPC1/3 possesses the same uncomom kinetic Arg-Arg617–618 site, possibly by an autocatalytic event and this tail behaviour with the larger substrate human salivary histatin-3, a has been proposed to play a role in sorting of PC1/3 to the histidine-rich peptide with 32 amino that was reported to be a regulated secretory pathway [7]. The reported pH assays showed substrate for PC1/3 [17]. Histatin 3 was synthesized, and the that both 87 and 66-kDa forms have higher activity in acidic exclusive cleavage at the carboxyl side of R25 (DSHAKRHH- PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24545 Hysteretic Behavior of PC1/3 were produced by the Fmoc procedure in an automated bench top simultaneous multiple solid-phase peptide synthesizer (PSSM 8 system from Shimadzu, Tokyo, Japan). The final deprotected peptides were purified by HPLC (Shimadzu, Tokyo, Japan) in a semi-preparative column. The molecular mass and purity of the synthesized peptides were checked by Maldi-TOF and/or peptide sequencing with a PPSQ-23 protein sequencer (Shimadzu, Tokyo, Japan). The concentration of the Abz peptide solutions was determined by measuring the absorption of the 2,4-dinitrophenyl group at 365 nm (l = 17,300 M21 cm21). The pERTKR-MCA (pE = pyroglutamic acid) was purchased from Peptanova (Sand- hausen, Germany). Determination of hysteretic parameters mPC1/3 activities were monitored spectrofluorometrically in a Hitachi F-2500 spectrofluorometer using the FRET peptides as substrates, with wavelengths of excitation at 320 nm and emission at 420 nm for Abz, and excitation at 380 and emission at 460 nm Figure 1. Time course of pERTKR-MCA (pE = pyrogluthamyl and for MCA peptide. A standard cuvette (1 cm pathlength) MCA = 7-amino-4-methyl coumarin) hydrolysis by 66-kDa containing 0.5 mL of substrate solution was placed in a mPC1/3. The MCA release increases with time and the exponential thermostatically-controlled cell compartment for 5 min before transient phase (lag phase) precedes the linear steady-state phase. the addition of enzyme. The kinetic parameters of peptide Hydrolysis conditions: mPC1/3 = 20 nM, Buffer = 20 mM BisTris, 20 mM hydrolysis were determined in 20 mM BisTris pH 6.0 at 37uC, CaCl2, pH 6.0 at 37uC. A.U.F. = Arbitrary Units of Fluorescence. doi:10.1371/journal.pone.0024545.g001 any other condition is indicated in the text. The pH values were adjusted at 25uC and checked before assays at 37uC using a model GYKRKFHEKHHSHRGYR25QSNYLYDN) by mPC1/3 was 710A Orion pH meter with an automatic temperature compen- analyzed. sation (ATC) glass probe. The reactions were monitored continuously based on the Materials and Methods fluorescence of the released product. The time course of product generated by mPC1/3 cleavage was fitted to the exponential Enzyme preparation function Equation 1 using the software Grafit version 5.0 Mouse PC1 was produced by overexpression in CHO cells using (Erithacus Software, Horley, Surrey, U.K.), and the hysteretic the dihydrofolate reductase-coupled amplification method. The parameters (k, t,vss and vi) were obtained. expression and purification procedure were earlier reported [18], ÀÁ but several modifications were made. We used motionless T-75 {t:k ½Product ~vsst{ðÞvss{vi 1{e k ð1Þ bottles (Corning Life Sciences, Lowell, MA, USA) instead of roller bottles in order to reduce cell lysis during expression and thus the where vi is the initial velocity, vss is the steady-state velocity and k is contaminants. Each expression gives a different proportion of 87, 74 the apparent rate constant for the transition between vi and vss. and 66-kDa mPC1/3 forms, and many expressions were conducted The secondary constant induction time t (k21) can also be until the pool obtained had the form of 66-kDa as the major obtained. This parameter can be understood as the time required component of the medium, according to SDS-PAGE analysis. The to reach the steady state. The rate of increase in fluorescence was pooled conditioned medium was filtered through a 0.25 mmfilter converted into moles of substrate hydrolyzed per second based on (Millex), diluted 1:3 in buffer A (20 mM Bis-Tris, 2 mM CaCl2, the fluorescence curves of standard peptide solutions after total 0.02% NaN3, and 0.4 mM octylglucoside, pH 7.0) and then enzymatic hydrolysis.

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