Insulysin Hydrolyzes Amyloid Β Peptides to Products That Are Neither Neurotoxic Nor Deposit on Amyloid Plaques

The Journal of Neuroscience, December 1, 2000, 20(23):8745–8749 Insulysin Hydrolyzes Amyloid ␤ Peptides to Products That Are Neither Neurotoxic Nor Deposit on Amyloid Plaques Atish Mukherjee,1 Eun-suk Song,1 Muthoni Kihiko-Ehmann,2 Jack P. Goodman Jr,3 Jan St. Pyrek,3 Steven Estus,2 and Louis B. Hersh1 1Department of Biochemistry, 2Department of Physiology and Sanders-Brown Center on Aging, and 3Mass Spectrometry Facility, University of Kentucky, Lexington, Kentucky 40536-0298 ␤ ␤ Insulysin (EC. 3.4.22.11) has been implicated in the clearance of action of insulysin on A 1–40 and A 1–42 was shown to eliminate ␤ amyloid peptides through hydrolytic cleavage. To further study the neurotoxic effects of these peptides. Insulysin was further ␤ ␤ the action of insulysin on A peptides recombinant rat insulysin shown to prevent the deposition of A 1–40 onto a synthetic ␤ ␤ was used. Cleavage of both A 1–40 and A 1–42 by the recombi- amyloid. Taken together these results suggest that the use of nant enzyme was shown to initially occur at the His 13-His 14, insulysin to hydrolyze A␤ peptides represents an alternative gene His 14-Gln 15, and Phe 19-Phe 20 bonds. This was followed by a therapeutic approach to the treatment of Alzheimer’s disease. slower cleavage at the Lys 28-Gly 29,Val18-Phe 19, and Phe 20- Ala 21 positions. None of the products appeared to be further Key words: amyloid peptide metabolism; metallopeptidase; metabolized by insulysin. Using a rat cortical cell system, the insulysin; A␤ neurotoxicity; A␤ deposition; A␤ cleavage The major pathological feature of Alzheimer’s disease is the pres- Kurochkin and Goto (1994) showed that another zinc metal- ence of senile plaques in the brain of affected individuals. Although lopeptidase insulysin (insulin degrading enzyme, insulinase, EC ␤ controversy exists whether the formation of amyloid deposits is the 3.4.22.11) also cleaved A 1–40, although the products of the reac- primary cause of Alzheimer’s disease, they contribute to its etiol- tion were not identified. In a subsequent study McDermott and ␤ ogy and progression (Selkoe, 1994). These insoluble amyloid de- Gibson (1997) confirmed the degradation of A 1–40 by insulysin, posits contain as a major constituent amyloid ␤ peptides (A␤ identified a number of putative reaction products, and showed that ␤ peptides) derived by processing of the amyloid precursor protein A 1–40 displayed an IC50 in the low micromolar range. Because this (Goldgaber et al., 1987) by ␤ and ␥ secretases (Sinha et al., 1999; study used partially purified enzyme, the contribution of contami- Vassar et al., 1999). Considerable effort has been expended in nating peptidases cannot be ruled out. Qiu et al. (1998) showed that identifying these secretases, the goal being the development of a secreted form of insulysin was produced from microglial cells specific inhibitors that would block the formation of amyloid (BV-2) and provided evidence that primary rat brain cultures and plaques. The recent report of an aspartyl protease that appears to differentiated rat adrenal pheochromocytoma (PC12) cells ex- be a true ␤ secretase (Vassar et al., 1999) provides optimism that pressed a cell surface form of insulysin (Vekrellis et al., 2000). this approach can soon be tested. Recently Perez et al. (2000) showed that insulysin represents an Tseng et al. (1999) showed that amyloid formation involves the abundant A␤ degrading activity in human brain soluble extracts. deposition of monomeric A␤. Thus, inhibition of monomeric A␤ In this study we have used homogeneous recombinant rat insu- ␤ aggregation or deposition represents an alternative strategy for the lysin to study the reaction of this peptidase with A 1–40 and ␤ ␤ treatment of Alzheimer’s disease. Compounds that prevent A A 1–42. We have identified cleavage sites and studied the cleavage aggregation have been reported (Soto et al., 1996; Tjernberg et al., reaction and its effect on the neurotoxic properties of the A␤ ␤ 1996; Tomiyama et al., 1996; Wood et al., 1996), and a high peptides and the ability of A 1–40 to deposit onto preformed throughput screen has been developed (Esler et al., 1997). Another synthetic amyloid fibrils. The results of this study suggest that approach is to hydrolyze A␤ peptides before they deposit onto insulysin may represent an alternative therapeutic approach for the amyloid plaques. Howell et al. (1995) showed that the zinc metal- treatment of Alzheimer’s disease. lopeptidase neprilysin (neutral endopeptidase, enkephalinase; EC ␤ 3.4.24.11) degraded A 1–40, whereas Iwata et al. (2000) showed MATERIALS AND METHODS that inhibition of neprilysin in rat brain produces an increase in Materials. A␤ and A␤ were obtained from Bachem (Torrance, CA). ␤ 1–40 1–42 A 1–42 concentration and the formation of diffuse amyloid plaques. Solutions were prepared by dissolving the peptide in dimethylsulfoxide However, it was observed that neprilysin inhibitors were less effec- (DMSO) to give a stock concentration of 200 ␮M. The peptide stock was ␤ ␤ lyophilized and stored at Ϫ80°C until use. The aggregation state of A␤ tive in altering the A 1–40 concentration, suggesting that A 1–40 peptide stock solutions was checked by electron microscopy (Ray et al., might be cleared by a different mechanism or peptidase (Iwata et 2000) and found to be predominantly, if not exclusively, monomeric. For ␮ ␤ al., 2000). the in vitro reactions with insulysin, a final concentration of 25 M A 1–40 was obtained after bringing the lyophilized peptide into solution with ␤ ␤ double-distilled water. For cytotoxicity studies A 1–40 and A 1–42 peptides Received June 28, 2000; revised Sept. 8, 2000; accepted Sept. 15, 2000. were dissolved in sterile N2 medium (Life Technologies, Rockville, MD). This research was supported in part by National Institute on Drug Abuse Grants DA ␤ Human -endorphin1–31, obtained from the National Institute on Drug 02243 and DA 07062 and National Institute on Aging Grant AG 05893. We thank Dr. Abuse drug supply system, was dissolved in water to give a stock solution Richard Roth of Stanford University for providing us with the cDNA clone to rat of 300 ␮M. Trifluoroacetic acid (Sigma, St. Louis, MO) was diluted into insulysin and for insulysin antisera, Dr. John Maggio and Jeffrey R. Marshall of the water to produce a 5% working solution. University of Cincinnati for helping us establish the A␤ deposition assay, and Drs. Expression and purification of recombinant insulysin. A rat insulysin Mark Lovell, Chengsong Xie, and William Markesbery of the Sanders-Brown Center cDNA, (pECE-IDE), kindly provided by Dr. Richard Roth of Stanford on Aging, University of Kentucky for helping us in preliminary neuronal toxicity University (Stanford, CA), was subcloned into the baculovirus-derived studies. vector pFASTBAC (Life Technologies) through BamHI and XhoI restric- Correspondence should be addressed to Dr. Louis B. Hersh, Department of tion sites such that a His6-affinity tag was attached to the N terminus of the Biochemistry, University of Kentucky, Chandler Medical Center, 800 Rose Street, protein. Generation of recombinant virus and expression of the recombi- Lexington, KY 40536-0298. E-mail: [email protected]. nant protein in Sf9 cells was performed according to the manufacturer’s Copyright © 2000 Society for Neuroscience 0270-6474/00/208745-05$15.00/0 directions. For the purification of recombinant insulysin, a 1:10 (w/v) 8746 J. Neurosci., December 1, 2000, 20(23):8745–8749 Mukherjee et al. • Insulysin Degradation of A␤ Peptides suspension derived from a 50 ml culture of viral infected Sf9 cells was prepared in 100 mM potassium phosphate buffer, pH 7.2, containing 1 mM dithiothreitol (K-PO4/DTE buffer). The suspension was sonicated 10 times, each burst for 1 sec, using a Branson sonifier (setting 3 at 30%) and then centrifuged at 75,000 ϫ g for 30 min to pellet cell debris and membranes. The supernatant containing recombinant rat insulysin was loaded onto a 0.5 ml nickel-nitrilotriacetic acid (Ni-NTA) column (Qiagen, Valencia, CA) that had been equilibrated with the K-PO4/DTE buffer. After extensive washing of the column with starting buffer, and then with 20 mM Imidazole-HCl, pH 7.2, the enzyme was eluted with 0.1 M Imidazole-HCl, pH 7.2. The enzyme was further purified over a 1 ml Mono-Q anion exchange column (Pharmacia Biotech, Piscataway, NJ) in 20 mM phosphate buffer, pH 7.2. A linear salt gradient of 0–0.6 M KCl, equivalent to 60 column volumes, was applied to the column with the enzyme eluted at 0.28 M KCl. SDS-PAGE of the insulysin was conducted on a 7.5% gel. Insulysin activity determination. Insulysin activity was assayed by measuring the disappearance of ␤-endorphin by isocratic reverse-phase HPLC (Safavi et al., 1996). A 100 ␮l reaction mixture containing 40 mM potassium phosphate buffer, pH 7.2, 30 ␮M ␤-endorphin, and enzyme was incubated for 15 min at 37°C. The reaction was stopped by the addition of 10 ␮lof5% trifluoroacetic acid to give a final concentration of 0.5%. The reaction mix was loaded onto a C4 reverse-phase HPLC column (Vydac, Hisperia, CA), Figure 1. Purification of recombinant rat insulysin. Insulysin was purified and products were resolved isocratically at 32% acetonitrile. The ␮ ␤-endorphin peak was detected by absorbance at 214 nm using a Waters as described in Materials and Methods, and 15 g aliquots from various 484 detector. The reaction was quantitated by measuring the decrease in stages of purification were analyzed by SDS-PAGE on a 7.5% gel stained the ␤-endorphin peak area. with Coomassie Blue. A, Sf9 cell extract. B, Nonbound proteins from the Determination of sites of cleavage of A␤ peptides.

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