Substrate Specificity of Barley Cysteine Endoproteases EP-A and EP-B1

Abstract

The cysteine endoproteases (EP)-A and EP-B were purified from green barley (Hordeum vulgare L.) malt, and their identity was confirmed by N-terminal amino acid sequencing. EP-B cleavage sites in recombinant type-C hordein were determined by N-terminal amino acid sequencing of the cleavage products, and were used to design internally quenched, fluorogenic peptide substrates. Tetrapeptide substrates of the general formula 2-aminobenzoyl-P 2 -P 1 -P 1 -P 2 -tyrosine(NO 2 )-aspartic acid, in which cleavage occurs between P 1 and P 1 , showed that the cysteine EPs preferred phenylalanine, leucine, or valine at P 2 . Arginine was preferred to glutamine at P 1 , whereas proline at P 2 , P 1 , or P 1 greatly reduced substrate kinetic specificity. Enzyme cleavage of C hordein was mainly determined by the primary sequence at the cleavage site, because elongation of substrates, based on the C hordein sequence, did not make them more suitable substrates. Site-directed mutagenesis of C hordein, in which serine or proline replaced leucine, destroyed primary cleavage sites. EP-A and EP-B were both more active than papain, mostly because of their much lower K m values.Two EPs known to play a central role in the breakdown of barley (Hordeum vulgare L.) endosperm storage proteins (hordeins) are Cys EPs designated EP-A (Koehler and Ho, 1990a) and EP-B (Koehler and Ho, 1988). They are secreted by the scutellum and aleurone layer into the starchy endosperm during germination in response to GA 3 (Koehler and Ho, 1990b;Marttila et al., 1995). EP-B has an apparent molecular mass of 30 kD, is identical to MEP-1 (Phillips and Wallace, 1989), and has a possible homolog with an apparent molecular mass of 31 kD (Zhang and Jones, 1996). The substrate specificity of this EP-B has been determined from the cleavage patterns of small proteins such as hordothionin, levitide, and cholecystokinin (Poulle and Jones, 1988;Zhang and Jones, 1996). There is little information about the substrate specificity of EP-A, although both EP-A and EP-B have been shown to digest hordein (Phillips and Wallace, 1989; Ho 1990a, 1990b). Characterization of the substrate specificity of barley Cys EPs provides an essential basis for an understanding of the activation of ␤-amylase by MEP-1 (ϭ EP-B) (Guerin et al., 1992) and limit dextrinase (Sissons, 1996) during germination, in which these enzymes are released (and activated) from bound and latent forms by proteolytic cleavage.Hordeins are unusual proteins, the amino acid composition and water insolubility of which present special problems as protease substrates. Pro and Gln comprise 40% of the total amino acids, and Pro causes particular problems for EPs, especially when it is at the protease scissile bond P 1 -P 1 Ј. (The substrate positions are denoted P i , . . . , P 2 , P 1 , P 1 Ј, P 2 Ј, . . . , P j , in correspondence with the binding subsites S i , etc., according to Berger and Schechter 1970). Hordeins, which are stored as compact protein bodies within the vacuoles of endosperm cells, comprise four major clas...

Purification and Characterization of Barley Dipeptidyl Peptidase IV

“…Based on our knowledge of EP-B specificity and its hordein cleavage pattern, particularly within the repetitive domains, it is known that many of the peptides released by its action have a Pro residue in the second and/or penultimate position (Davy et al, 1998). This is clearly illustrated by the cleavage products of C hordein, where KPFPVQ, QPFPQ, and QPQ are formed.…”

“…Proteins were visualized by silver staining (Bloom et al, 1987). For N-terminal sequencing, proteins were blotted onto polyvinylidene fluoride (PVDF) membrane (Immobilon P, Millipore, Bedford, MA) using a semidry electroblotter (Aricos, Ølstykke, Denmark), as previously described (Davy et al, 1998).…”

“…Pro residues confer unique structural constraints on the peptide chain and significantly influence their susceptibility to proteolytic cleavage (Yaron and Naider, 1993). Cys endoproteases EP-A and EP-B and the Ser carboxypeptidases I, II, and III play a major role in hordein degradation (Koehler and Ho, 1990;Dal Degan et al, 1994;Davy et al, 1998). A study of EP-A and EP-B specificity has shown that many of the hordein peptides released by their action have a Pro residue in the second and/or penultimate position (Davy et al, 1998).…”

“…Cys endoproteases EP-A and EP-B and the Ser carboxypeptidases I, II, and III play a major role in hordein degradation (Koehler and Ho, 1990;Dal Degan et al, 1994;Davy et al, 1998). A study of EP-A and EP-B specificity has shown that many of the hordein peptides released by their action have a Pro residue in the second and/or penultimate position (Davy et al, 1998). However, peptides containing Pro in the penultimate position are very poor substrates for the barley Ser carboxypeptidases I, II, and III (Breddam and Sørensen, 1987).…”

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination

“…Zhang and Jones (1995) reported that 27 Cys-proteases are among the 42 proteases that are involved in the germination of barley (Hordeum vulgare) seed. Until now, few of these proteases have been characterized (Koehler and Ho, 1990;Davy et al, 1998), and there are currently only limited data on the effects of the inhibitors required for their regulation (Martinez et al, 2005b). In contrast, sets of genes encoding transcriptional factors synthesized in response to phytohormones have been implicated in controlling hydrolase activity during barley seed germination (Gubler et al, 1999;Sreenivasulu et al, 2008).…”