Purification, characterization and cloning of an aspartic proteinase inhibitor from squash phloem exudate

Abstract

Phloem exudate from squash fruit contains heat-inactivated material which inhibits pepsin activity. This inhibitory activity was purified by mild acid treatment, chromatography on trypsin-agarose, Sephadex G-75 and reverse-phase HPLC, resulting in the elution of three peaks with pepsin-inhibitory activity. N-terminal sequencing indicated a common sequence of MGPGPAIGEVIG and the presence of minor species with seven-or two-amino-acid N-terminal extensions beyond this point. Microheterogeneity in this end sequence was exhibited within and between two preparations. Internal sequencing of a major peak after a trypsin digestion gave the sequence FYNVVVLEK. The common N-terminal sequence was used to design a degenerate primer for 3′ rapid amplification of cDNA ends and cDNA clones encoding two isoforms of the inhibitor were obtained. The open reading frames of both cDNAs encoded proteins (96% identical) which contained the experimentally determined internal sequence. The amino acid content calculated from the predicted amino acid sequence was very similar to that measured by amino acid analysis of the purified inhibitor. The two predicted amino acid sequences (96 residues) had neither similarity to any other aspartic proteinase inhibitor nor similarity to any other protein. The inhibitors have a molecular mass of 10 552 Da, measured by matrix-assisted laser-desorption ionisation time-of-flight mass spectrometry and approximately 10000 Da by SDS/PAGE, and behave as dimers of approximately 21 000 Da during chromatography on Superdex G-75 gel-filtration medium. The calculated molecular masses from the predicted amino acid sequences were 10551 Da and 10527 Da. The inhibitor was capable of inhibiting pepsin (K i ϭ 2 nM) and a secreted aspartic proteinase from the fungus Glomerella cingulata (K i ϭ 20 nM). The inhibitor, which is stable over acid and neutral pH, has been named squash aspartic proteinase inhibitor (SQAPI).Keywords : aspartic-protease inhibitor ; pepsin; phloem; squash.Aspartic proteinases are found in a wide variety of organ-range of chemical approaches have been employed in designing new inhibitors, invariably synthetic approaches have mimicked isms, including viruses, fungi, plants and animals, where they carry out a diverse array of functions involving specific proteol-pepstatin in the provision of a gem diol (or analogue) of the key reaction intermediate [2Ϫ5]. ysis or more general degradation [1]. Well known examples include HIV protease, pepsin, chymosin, renin, and cathepsins D Aspartic proteinase inhibitors, that are themselves proteins, are scarce in nature. Only four sources of proteinaceous aspartic and E. These enzymes are characterized by their catalytic activity being dependent on a pair of aspartic acid residues in the proteinase inhibitor have been reported ; from potato [6], wheat [7], yeast [8] and the nematode (Ascaris spp.) [9]. The best charactive site and by generally operating at low pH. Most are inhibited by pepstatin, a non-proteinaceous microbial hexapeptide acterized of these, the...

Inhibition of cysteine and aspartyl proteinases in the alfalfa weevil midgut with biochemical and plant-derived proteinase inhibitors

“…That this inhibitor preparation was equal in effectiveness to that of pepstatin A, which inhibits pepsin as well as cathepsin D, suggests that cathepsin D is the major aspartyl proteinase in the extract. This conclusion is strengthened by the fact that a preparation of squash aspartic proteinase inhibitor (SQAPI), which inhibits pepsin but not cathepsin D (Christeller et al, 1998), had no effect on the extract (data not shown). Whether PDI will be useful in transgenic applications will hinge in large part on the effectiveness of the individual isoforms, as expressed from their cDNAs, on proteolytic activity.…”

“…The ability of PIs to interfere with insect growth and development has been attributed to their capacity to bind to, and thereby inhibit the action of insect digestive proteinases (reviewed by Jongsma and Bolter, 1997). Since there exists significant variation among the types and properties of endo-and exo-proteinases utilized by insects for dietary purposes (reviewed by Terra and Ferreira, 1994), and because numerous PIs of widely-ranging specificities (Keilova and Tomasek, 1976a;Abe et al, 1994;Brzin et al, 1998;Christeller et al, 1998;Pernas et al, 1998) towards these proteinases are available, it is necessary to characterize the gut proteolytic activities of each individual species in order to devise a rational insect control strategy utilizing PIs.…”

A Novel Function for the Cathepsin D Inhibitor in Tomato

“…In contrast to this broad distribution of the Ser PIN family, proteinaceous inhibitors of aspartic proteinases are less numerous and have been described in only a few plant species: potato Tomasek, 1976a, 1976b), tomato (Solanum lycopersicum; Werner et al, 1993), wheat (Triticum aestivum; Galleschi et al, 1993), Vicia sativa (Roszkowska-Jakimiec and Bankowska, 1998), Anchusa strigosa (Abuereish, 1998), and squash (Cucurbita pepo; Christeller et al, 1998). The biochemical characterization of the potato and V. sativa members showed that they were both cathepsin D inhibitors (CDIs), whereas the proteins from A. strigosa and squash inhibit pepsin, a digestive aspartic proteinase.…”

Characterization of Cucurbita maxima Phloem Serpin-1 (CmPS-1)

“…The lished that phloem PIs (3-10 kDa) are able to inhibit a wide range of proteinases (18,21,22), including a number of such enzymes extracted from the midgut of lepidopteran (chewing) larvae (23,24).…”