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Studies on Trypsin-modified Bovine and Human Lens Acylpeptide Hydrolase

https://doi.org/10.1006/exer.2000.0955Get rights and content

Abstract

Acylpeptide hydrolase removes the N -acetylated amino acids from the peptide substrates but not from intact proteins. Cleavage between amino acid residues 203–204 of the native acylpeptide hydrolase results in the formation of a 55 kDa truncated active enzyme in the bovine lens, in vivo. In this study we explored the hydrolytic properties of the truncated enzyme using lens β- and γ-crystallins as substrates. SDS–PAGE analysis indicated that the βB2-crystallin was cleaved by truncated acylpeptide hydrolase into several protein fragments (10–26 kDa). No cleavage of the γ-crystallins was observed under similar conditions. Both the acylpeptide hydrolase activity and the protease activity of the 55 kDa enzyme were completely inhibited by diisopropylfluorophosphate, p -chloromercuribenzoate and ebelactone, and moderately inhibited by N -tosyl phenylalanine chloromethyl ketone. SDS–PAGE analysis followed by fluorography of (3H) diisopropylfluorophosphate labeled human lens acylpeptide hydrolase preparation showed the presence of the 55 kDa truncated form of the enzyme, as observed in the bovine lens. The peptide (d)-AIKGDQFL-NH2—the amino acid sequence 200–207 of the native bovine acylpeptide hydrolase with an in vivo cleavage site of native protein—was hydrolysed by the lens protease(s) suggesting that the in vivo generation of the 55 kDa acylpeptide hydrolase may be mediated through a proteolytic processing. The protease(s) responsible for the cleavage of this peptide was inhibited by diisopropylfluorophosphate and p -chloromercuribenzoate.

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      For example, it has been reported that in bovine and human lens, ACPH catalytic subunit of 75 kDa removes N-acetylated amino acid residues from the αA-crystallin, and a truncated form of 55 kDa has an endoprotease activity that could play a role in the age-related cleavage of βB2-crystallins (Senthilkumar et al., 2001). Interestingly, the total ACPH activity was found to be decreased in human cataract lenses, (Senthilkumar et al., 2001) and this may contribute to the accumulation of N-terminally blocked peptides in the lens nucleus (Sharma and Kester, 1996). More relevant to our results is the finding reported by Yamin et al. (2007) indicating that ACPH degrades Aβ1–40 in vitro and that Alzheimer's disease brains express lower levels of ACPH mRNA than brains of age-matched controls (Yamin et al., 2007).

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    Author for correspondence: K. Krishna Sharma, Mason Eye Institute, University of Missouri, One Hospital Drive, Columbia, MO 65212, U.S.A. E-mail: [email protected]

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