Abstract
Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.
Highlights
Legumain belongs to the C13 subfamily of cysteine proteases represented by asparaginyl endopeptidase (AEP), and was first found in legume seeds [1,2]
To determine the corresponding legumain sequences, we obtained a transcriptome assembly of M. cochinchinensis using total RNA isolated from fresh seeds that was sequenced by the Beijing Genomics Institute (BGI)
This study reports the discovery and characterization of a splicing legumain designated McPAL1 that was isolated from the squash plant M. cochinchinensis and Journal Pre-proof is involved in the production of trypsin inhibitors MCoTI-I and MCoTI-II
Summary
Legumain belongs to the C13 subfamily of cysteine proteases represented by asparaginyl endopeptidase (AEP), and was first found in legume seeds [1,2]. Legumain-mediated protein splicing, recognized as early as 1985 to play a role in concanavalin A (ConA) maturation, combines both hydrolytic and ligase activity in tandem during protein processing and involves excision of intervening sequences and religation of the remaining sequences to induce a different circular-permutated structure [13] This post-translational splicing process mediated by enzyme jack bean AEP was described in 1994 by Min and Jones [12]. Using a panel of Asx-containing peptides including the MCoTI-II linear precursor and other bioactive peptides, we examined the unusual trimodal catalytic mechanism of McPAL1 that involves pH- and P1-residue-dependent activities that confer its multi-functionality as a splicing enzyme, hydrolase, and ligase. We characterized the exceptionally high tolerance of McPAL1 to heat and basic pH
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