Cloning, Expression and Characterization of Insulin-Degrading Enzyme from Tomato (Solanum Lycopersicum)

Article in press - uncorrected proof Biol. Chem., Vol. 389, pp. 91–98, January 2008 • Copyright ᮊ by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2008.006 Cloning, expression and characterization of insulin-degrading enzyme from tomato (Solanum lycopersicum) Yoann Huet1,a, Jochen Strassner2 and Andreas named inverzincins (Ding et al., 1992). It was demonstrat- Schaller1,* ed by site-directed mutagenesis that the two histidine residues are involved in the coordination of the zinc ion, 1 Institute of Plant Physiology and Biotechnology, while the glutamate is required for catalysis (Perlman et University of Hohenheim, D-70593 Stuttgart, Germany al., 1993; Becker and Roth, 1995). 2 Altana Pharma AG, D-78467 Konstanz, Germany Inverzincins generally accept a wide variety of peptide * Corresponding author substrates and appear to lack selectivity for specific e-mail: [email protected] sequence motives in the primary structure of their substrates. Indeed, IDE was shown to cleave many different peptides, including insulin and glucagon (Kirschner and Abstract Goldberg, 1983), natriuretic peptides (Muller et al., 1992), transforming growth factor a (Garcia et al., 1989; Gehm A cDNA encoding insulin-degrading enzyme (IDE) was and Rosner, 1991), b-endorphin and dynorphins (Safavi cloned from tomato (Solanum lycopersicum) and et al., 1996), growth hormone releasing factor (Garcia expressed in Escherichia coli in N-terminal fusion with et al., 1989), amylin (Bennett et al., 2000) and amyloid glutathione S-transferase. GST-SlIDE was characterized b peptide (Qiu et al., 1998; Vekrellis et al., 2000) at unre- as a neutral thiol-dependent metallopeptidase with insu- lated sites. However, despite the degeneracy of cleavage linase activity: the recombinant enzyme cleaved the oxi- sites, these peptide substrates are recognized by IDE dized insulin B chain at eight peptide bonds, six of which and cleaved with high affinity, while other biologically are also targets of human IDE. Despite a certain prefer- important peptides are not cleaved at all. IDE can there- ence for proline in the vicinity of the cleavage site, syn- fore not be considered as a general peptidase for non- thetic peptides were cleaved at apparently stochastic specific peptide turnover (Duckworth, 1990; Safavi et al., positions indicating that SlIDE, similar to IDEs from other 1996). Similarly, mitochondrial processing peptidase organisms, does not recognize any particular amino acid (MPP) and plant stromal processing peptidase (SPP) are motif in the primary structure of its substrates. Under specific inverzincins, which process mitochondrial and " steady-state conditions, an apparent Km of 62 7 mM and plastid precursor proteins to release the N-terminal tar- " -1 -1 a catalytic efficiency (kcat/Km)of62 15 mM s were geting peptides after import into the respective organelle determined for Abz-SKRDPPKMQTDLY(NO3)-NH2 as the (Vander Vere et al., 1995; Richter and Lamppa, 1998; substrate. GST-SlIDE was effectively inhibited by ATP Gakh et al., 2002). The targeting peptides are variable in at physiological concentrations, suggesting regulation of length and primary structure, and there is little sequence its activity in response to the energy status of the cell. conservation at MPP cleavage sites in yeast and plant While mammalian and plant IDEs share many of their bio- systems (Branda and Isaya, 1995; Glaser et al., 1998). chemical properties, this similarity does not extend to Pre-sequence proteases (PrePs), which also belong to their function in vivo, because insulin and the b-amyloid the M16 family and lack specificity towards particular peptide, well-established substrates of mammalian IDEs, peptide bonds are responsible for the further degradation as well as insulin-related signaling appear to be absent of targeting peptides after release from protein precur- from plant systems. sors (Sta˚ hl et al., 2002; Bhushan et al., 2003; Moberg et al., 2003). A recent report on the structure of human Keywords: insulinase; insulysin; metalloprotease; plant; IDE in complex with several peptide substrates (Shen et proteolysis. al., 2006) sheds light on the mechanisms underlying substrate recognition by M16 peptidases. The four domains of the enzyme fold to enclose a chamber, just large Introduction enough to accommodate peptides of up to about 50 amino acids. Additional selectivity is provided by favor- Insulin-degrading enzyme (IDE) is an evolutionary well able interactions between the enzyme and the N-termi- conserved zinc-dependent metalloendopeptidase of the nus as well as the cleavage sites within the substrate pitrilysin family and belongs to the M16A family of pep- peptides. Moreover, the charge distribution in the cata- tidases, according to the MEROPS classification (Raw- lytic cavity explains why peptides with positive charges lings et al., 2004). M16 peptidases are characterized by in proximity of their C-termini are usually poor substrates the conserved HXXEH motif for zinc binding, which is a (Shen et al., 2006). functional inversion of the more common HEXXH motif IDE, as the name suggests, has first been implicated of other metalloproteases, and they have thus been in the regulation of insulin signaling, and experimental evidence confirming its role in the intracellular degrada- a Present address: Plant Biology and Pest Control, Jules Verne tion of this hormone continues to cumulate since its University of Picardie, F-80000 Amiens, France. activity was first described 50 years ago (Duckworth 2008/236 Article in press - uncorrected proof 92 Y. Huet et al. et al., 1988, and references therein). More recently, atten- present in the purified fraction and likely represent deg- tion has focused on the capacity of IDE to degrade b- radation or arrested translation products as they immu- amyloid peptides involved in the neurodegenerative noreact with an antibody directed against the N-terminus Alzheimer disease (Kurochkin, 2001; and references of the fusion protein, including the GST moiety and the therein). Indeed, IDE-deficient rodents suffer not only first 142 N-terminal residues of SlIDE (not shown). The from hyperinsulinemia and glucose intolerance, but also presence of such low-molecular weight fragments during accumulate high levels of amyloid peptides in the brain bacterial expression appears to be a recurring problem, (Farris et al., 2003; Miller et al., 2003). A broader role for as it was also reported for the recombinant human IDE IDE is also supported by the fact that IDE cleaves many (Chesneau and Rosner, 2000). biologically important peptides other than insulin, by its GST-SlIDE was confirmed as a bona fide IDE using localization in cell types that do not bind or internalize MALDI-TOF mass spectrometry to characterize its activ- insulin (Duckworth, 1988). Moreover, IDE homologs are ity in vitro. The oxidized insulin B chain was rapidly pro- found in organisms lacking insulin-related peptides and cessed at the Glu13-Ala14, Ala14-Leu15 and Tyr16-Leu17 pep- insulin signaling, including plants. We report here the bio- tide bonds (complete processing in less than 5 min under chemical characterization of IDE from tomato which is the chosen reaction conditions), while cleavage at His10- the first report on a M16A peptidase from any plant Leu11, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 and Pro28- source. Lys29 occurred at a slower rate (complete processing required more than 30 min; Figure 3A). Six of these cleavage sites are identical with those generated by human Results IDE (Duckworth et al., 1989; Figure 3A). In contrast, E. coli pitrilysin, also an inverzincin from the M16A family, 16 17 A partial cDNA showing similarity to human IDE was cleaves the insulin B chain exclusively at the Tyr -Leu cloned from a tomato shoot cDNA library in a functional bond (Anastasi and Barrett, 1995; Figure 3A). To rule out screen for proteases cleaving the peptide AVQSKPPSKR the possibility that the observed activity is due not to DPPKMQTD (systemin), and its 59end was completed by SlIDE but rather to a contaminating E. coli protease, a RACE-PCR. The full-length cDNA of 3358 bp (accession mock purification was performed using a bacterial culture number AJ308542) encompassed an open reading frame expressing the anti-sense SlIDE cDNA, which resulted in of 2913 bp coding for a protein of 971 amino acids with a protein preparation devoid of any proteolytic activity a calculated molecular mass of 112 kDa. The tomato (not shown). (Solanum lycopersicum) sequence, called SlIDE, shared Further characterization of SlIDE activity using syn- the highest degree of amino acid identity with plant thetic peptide substrates revealed a certain preference orthologs, including two Arabidopsis sequences (68 and for proline one or two residues amino-terminal of the w 62% identity with At2g41790 and At3g57470, respect- hydrolyzed bond i.e., the P1 and P2 sites, according to x ively), and is 39% identical to human IDE (Figure 1). The the terminology of Schechter and Berger (1967) . How- predicted domain organization of SlIDE resembles that of ever, this is not an absolute requirement as glucagon, a human IDE. In both enzymes, the catalytic M16 domain 29-amino acid peptide lacking proline residues, was (Pfam domain PF00675; http://www.sanger.ac.uk/Soft- nevertheless cleaved at two unrelated sites (Figure 3B). ware/Pfam/) is located close to the amino-terminus. The Consistent with the identification of SlIDE in a screen for M16 domain (amino acids 34–172 in SlIDE) includes the proteases active against systemin, this peptide was conserved HXXEH motif, which is characteristic for the processed efficiently at the Lys14-Met15 bond (Figure 3B). inverzincin family of metalloproteases (Figure 1). It is fol- There are no common sequence elements at the cleav- lowed by two M16-C domains (PF05193; amino acids age sites in different synthetic peptide substrates (Figure 197–378 and 665–853, respectively), which have been 3B), and therefore SlIDE does not seem to require any implicated not in catalysis but rather in substrate binding specific amino acid motif for substrate recognition.

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