Construction, expression and characterization of a chimaeric mammalian-plant aspartic proteinase

Abstract

Aspartic proteinases are a well-characterized class of proteinases. In plants, all nascent aspartic proteinases possess a 100-amino-acid, plant-specific sequence (PSS) within their C-terminal lobe, presumed to possess a targeting role in vivo. In this study, the PSS domain from the Arabidopsis thaliana aspartic proteinase was inserted into porcine pepsinogen at the identical location found in nascent plant aspartic proteinases, to create a chimaeric mammalian-plant enzyme. Based on enzymic activity, this chimaeric enzyme demonstrated increases in pH stability above 6 and temperature stability above 60 degrees C compared with commercial pepsin. Differential scanning calorimetry of the chimaeric enzyme illustrated an approx. 2 degrees C increase in denaturation temperature ( T (m)), with increases in co-operativity and similar enthalpy values. Kinetic analysis indicated an increase in K (m) and decreased k (cat) compared with pepsin, but with a catalytic efficiency similar to the monomeric plant aspartic proteinase from wheat. Using oxidized insulin B-chain, the chimaeric enzyme demonstrated more restricted substrate specificity in comparison with commercial pepsin. This study highlights the use of a chimaeric enzyme strategy in order to characterize unique protein domains within enzyme families, and, for the first time, a putative structure-function role for the PSS as it pertains to plant aspartic proteinases.

A cut above the rest: the regulatory function of plant proteases

“…lysosomal sphingolipid-activating enzymes in mammalian cells) and, therefore, more adequately described as the saposin-like domain (SLD). The SLD may contribute to the substrate specificity of APs (Payie et al 2003) and, because of its membrane-permeabilizing activity, a role in plant defense has been proposed (Egas et al 2000). Most importantly, the SLD was shown to be required for the exit of the proenzyme from the ER and targeting to the vacuole (Ramalho-Santos et al 1998;Kervinen et al 1999;To¨rma¨kangas et al 2001).…”

Molecular cloning of a potato leaf cDNA encoding an aspartic protease (StAsp) and its expression after P. infestans infection

“…Heterodimeric formation is a result of post-translational proteolytic modifications resulting in the removal of PSI prior to activation [14,31]. In contrast, monomeric aspartic proteinases retain their PSI structure [29]. The antimicrobial activity found in StAP1 and StAP3 [17,18] can be explained by the presence of PSI in the StAsp clone and the sequence homology of StAsp-PSI to NK-lysin, a protein with antibacterial activity and capacity to lyses tumor cells but not red blood cells [36]; amoebapores, the pore forming peptides of Entamoeba histolytica in bacteria and eukaryotic cells [25], and granulysin, a SAPLIPs with antimicrobial activity [40].…”

Structure–function characterization of the recombinant aspartic proteinase A1 from Arabidopsis thaliana

“…Nephentesin I, cardosin B and wheat G1AP have the broadest specificity (Athauda et al, 2004;Bleukx and Delcour, 2000;Bleukx et al, 1998). It has been hypothesized that the PSI has a functional role in defining substrate specificity since the insertion of the PSI in pepsin resulted in a modified cleavage profile with a greater specificity compared to wild-type pepsin (Payie et al, 2003).…”

“…Oxidized insulin b-chain has been used as a substrate to determine the specificity of various aspartic proteinases due to its high content of hydrophobic amino acids and the preference of APs for peptide bonds formed by hydrophobic amino acids (Athauda et al, 2004;Bleukx and Delcour, 2000;Bleukx et al, 1998;Guevara et al, 2004;Kervinen et al, 1993;Park et al, 2000;Payie et al, 2003;Simoes et al, 2007;Verissimo et al, 1995). Fig.…”

“…The physiological, structural and functional relevance of this domain is unknown (Guevara et al, 2002). It is thought that the PSI is not critical for enzymatic 0031 (Asakura et al, 2000), however, roles such as protein targeting to the vacuole, proper folding, vesicle leakage, and structure-function have been identified (Tormakangas et al, 2001;Egas et al, 2000;Payie et al, 2003;Simoes and Faro, 2004;Terauchi et al, 2006). The PSI was found to undergo proteolytic cleavage resulting in its partial or total removal during activation of the enzyme to produce a single-chain or a two-chain mature form or a complex mixture of products Simoes and Faro, 2004).…”