Insulysin hydrolyzes amyloid beta peptides to products that are neither neurotoxic nor deposit on amyloid plaques

Abstract

Insulysin (EC. 3.4.22.11) has been implicated in the clearance of beta amyloid peptides through hydrolytic cleavage. To further study the action of insulysin on Abeta peptides recombinant rat insulysin was used. Cleavage of both Abeta(1-40) and Abeta(1-42) by the recombinant enzyme was shown to initially occur at the His(13)-His(14), His(14)-Gln(15), and Phe(19)-Phe(20) bonds. This was followed by a slower cleavage at the Lys(28)-Gly(29), Val(18)-Phe(19), and Phe(20)-Ala(21) positions. None of the products appeared to be further metabolized by insulysin. Using a rat cortical cell system, the action of insulysin on Abeta(1-40) and Abeta(1-42) was shown to eliminate the neurotoxic effects of these peptides. Insulysin was further shown to prevent the deposition of Abeta(1-40) onto a synthetic amyloid. Taken together these results suggest that the use of insulysin to hydrolyze Abeta peptides represents an alternative gene therapeutic approach to the treatment of Alzheimer's disease.

Fig. 1.

Purification of recombinant rat insulysin. Insulysin was purified as described in Materials and Methods, and 15 μg aliquots from various stages of purification were analyzed by SDS-PAGE on a 7.5% gel stained with Coomassie Blue. A,Sf9 cell extract. B, Nonbound proteins from the Ni-NTA-agarose column. C, Protein eluted from the Ni-NTA-agarose column with 20 mm imidazole.D, Protein eluted from the Ni-NTA-agarose column with 100 mm imidazole. E, Protein eluted from the Mono-Q column. The position of molecular weight markers (myosin, 200 kDa; β-galactosidase, 116 kDa; phosphorylase B, 97.4 kDa; bovine serum albumin, 66 kDa; and ovalbumin, 45 kDa) is shown on theleft.

Fig. 2.

HPLC profile of products generated from the cleavage of Aβ_1–40 by insulysin. Varying amounts of recombinant rat insulysin were incubated with 25 μmAβ_1–40 for 30 min at 37°C. Cleavage products were separated by a 5–75% gradient of acetonitrile on a C₄reverse-phase HPLC column. Product peaks are numbered according to their order of elution. The peaks designated Ca andCb refer to contaminants in the Aβ_1–40solution. These are not reacted on by insulysin, as is seen by their invariant peak areas in all the traces. A,Aβ_1–40 alone. B, Aβ_1–40incubated with 50 ng of insulysin. C,Aβ_1–40 incubated with 250 ng of insulysin.D, Aβ_1–40 incubated with 500 ng of insulysin. The HPLC scans are skewed ∼2 min to the left to permit overlapping peaks to be viewed. The time scale refers to traceA.

Fig. 3.

Positions of cleavage within the Aβ_1–40 and Aβ_1–42 sequences. Primary cleavage sites are noted with the thick arrows.

Fig. 4.

Effect of insulysin on the neurotoxic effects of Aβ peptides. Purified insulysin was added with Aβ_1–40(30 μm) or Aβ_1–42 (25 μm) to primary cortical neurons, and incubation continued for an additional 48 hr. The neurotoxic effect of the Aβ peptides was determined as described in Materials and Methods. The insulysin and heat-inactivated insulysin controls used 5000 ng of enzyme. A, Effect of incubation with insulysin on the neurotoxic effects of Aβ_1–40. B, Effect of incubation with insulysin on the neurotoxic effects of Aβ_1–42. *p < 0.01 relative to the Aβ-treated sample as determined by ANOVA.

Fig. 5.

Insulysin protects against Aβ_1–40mediated neurotoxicity. Rat cortical neurons were treated as described in Figure 4 in the presence or absence of 50 ng of insulysin. Cells were stained with Hoechst 33258 (A–D) or with the Aβ antibody 10D5 (E–H). Hoffman modulation contrast micrographs are shown in I–L.A, E, and I show untreated neurons. B, F, and J show neurons with 50 ng of insulysin added. C,G, and K show neurons treated with 30 μm Aβ_1–40. D,H, and L show neurons treated with 50 ng of insulysin and 30 μm Aβ_1–40.

Fig. 6.

Insulysin inhibits the deposition of Aβ_1–40 onto synthetic amyloid plaques. A,Effect of incubation with insulysin on the deposition of Aβ_1–40. Aβ_1–40 (0.1 nm) was mixed with the indicated amount of purified insulysin and then added to synthaloid in 96 well plates. Deposition was permitted to occur over a 4 hr time period. B, Effect of preincubation with insulysin on the deposition of Aβ_1–40. Aβ_1–40 (1 nm) was preincubated for 60 min with the indicated amount of purified insulysin. The incubation mixtures were then added to synthaloid in 96 well plates, and deposition was permitted to occur over a 4 hr time period. *p < 0.01 as determined by ANOVA.