Identification and proteolytic processing of procardosin A

Abstract

Plant aspartic proteinases contain a plant-specific insert (PSI) of about 100 amino acids of unknown function with no similarity with the other aspartic proteinases but with significant similarity with saposins, animal sphingolipid activator proteins. PSI has remained elusive at the protein level, suggesting that it may be removed during processing. To understand the molecular relevance of PSI, the proteolytic processing of cardosin A, the major aspartic proteinase from the flowers of cardoon (Cynara cardunculus L.) was studied. Procardosin A, a 64-kDa cardosin A precursor containing PSI and the prosegment was identified by immunoblotting using monospecific antibodies against PSI and the prosegment. Procardosin A undergoes proteolytic processing as the flower matures. PSI was found to be removed before the prosegment, indicating that during processing the enzyme acquires a structure typical of mammalian or microbial aspartic proteinase proforms. In vitro studies showed that processing of PSI occurs at pH 3.0 and is inhibited by pepstatin A and at pH 7.0. Sequence analysis allowed the identification of the cleavage sites, revealing that PSI is removed entirely, probably by an aspartic proteinase. Cleavage of the PSI scissile bonds requires, however, a conformation specific to the precursor since isolated cardosins and pistil extracts were unable to hydrolyse synthetic peptides corresponding to the cleavage sites. In view of these results, a model for the proteolytic processing of cardosin A is proposed and the molecular and physiological relevance of PSI in plant aspartic proteinase is discussed.Keywords : aspartic protease ; milk-clotting enzyme; cardosin ; proteolytic processing; saposin.Aspartic proteinases (AP) are a widely distributed class of kingdom [5]. In the majority of plants AP are located in seeds, endoproteases that share significant similarities at the amino-whether quiescent or germinating. They are believed to particiacid-sequence level and at the structural level [1, 2]. The typical pate in storage-protein cleavage, which is necessary for germinacharacteristics of the family are an acidic pH optimum, inhibi-tion [5,10]. AP have also been found in leaves of some plants. tion by pepstatin A, preference for bonds between hydrophobic In leaves they have been implicated in mechanisms of defense amino acids, and sequence similarities, in particular the conser-against pathogens [11,12]. Species of the genus Cynara contain vation of the catalytic triads Asp-Thr-Gly or Asp-Ser-Gly [1Ϫ considerable amounts of AP in flowers. Recent data indicate that 4]. AP have been implicated in diverse physiological processes, the major AP from cardoon (Cynara cardunculus L.), cardosin such as digestion or blood-pressure regulation, and in some A, may be involved in the sexual reproduction of the plant [6] pathological conditions, including infection by fungi and retro-and may have a defensive role as well [13]. viruses or cancer [5, 6].A few primary structures deduced from the cDNAs of AP Like many other proteases, A...

Cardosins in postembryonic development of cardoon: towards an elucidation of the biological function of plant aspartic proteinases

“…4). A band at about 45 kDa was also detected at almost all time points studied and corresponds to an intermediate form of the protein, which has already been described (Ramalho-Santos et al 1998b). The processed mature form (31 kDa) could be very faintly detected in the first hours after imbibition until stage 6 (60 h), reappearing before the seedling stage.…”

“…A systematic study of cardosin A and B expression in various developmental stages and organs has been undertaken, particularly in reproductive structures (Ramalho-Santos et al 1996, 1997, 1998bVerissimo et al 1996;Faro et al 1998;Vieira et al 2001;Duarte 2005;Figueiredo et al 2006). In the course of these studies, the temporal and spatial accumulation patterns in cardoon postembryonic seed development were determined.…”

Structure and Mechanism of the Saposin-like Domain of a Plant Aspartic Protease

“…In some plant APs, the release of PSI occurs via proteolytic cleavage upon acid-induced autoactivation of the precursor protein and subsequent processing, albeit via self-cleavage (6,8,75). Interestingly, influenza A hemagglutinin fusion peptide is inaccessible to membranes at neutral pH; however, a drop of the pH inside the endosome below a critical threshold induces a large conformational change in the parent protein and is subsequently activated by a protease (plasmin) that cleaves the precursor polypeptide (76) into two disulfide-linked polypeptides and a fusion peptide (77).…”

“…This structural oddity among APs is called the plantspecific insert (PSI), also known as the plant-specific sequence, which belongs to the saposin-like protein (SAPLIP) family (12, 13). Plant APs are found in either monomeric or heterodimeric forms (9, 14); the latter result from post-translational proteolysis, which includes the removal of part or all of the PSI, whereas the PSI is retained in monomeric plant APs (6,8).…”

“…This structural oddity among APs is called the plantspecific insert (PSI), also known as the plant-specific sequence, which belongs to the saposin-like protein (SAPLIP) family (12, 13). Plant APs are found in either monomeric or heterodimeric forms (9, 14); the latter result from post-translational proteolysis, which includes the removal of part or all of the PSI, whereas the PSI is retained in monomeric plant APs (6,8).In general, members of the SAPLIP family have various physiological functions, all of which entail membrane interaction (14 -16) manifested in three principal ways: membrane binding, membrane perturbation without permeabilization, and membrane permeabilization (15). Examples of SAPLIP functions include roles in exohydrolase degradation of sphingolipids in the lysosome (saposins) (17), antimicrobial activity (granulysin and NK-lysin) (18), tumor lysis (NK-lysin) (19), pulmonary surfactant surface tension regulation (surfactant protein B) (20), and bacterial/eukaryotic cell lysis (amoebapores) (21).…”