Purification, characterization and cDNA cloning of a trypsin inhibitor from Pentaclethra macroloba.

Abstract

The larval growth of the European corn borer, Ostrinia nubilalis , was reduced when grown on artificial diet containing either purified PmTI or PmSTI fusion protein expressed in E. coli. Likewise, high mortalities were observed with the free-living nematode Caenohabditis elegans cultured on E. coli expressing PmSTI

Two types of trypsin inhibitors, designated PmLTI and PmSTI, were isolated and purified from seeds of Pentaclethra macroloba. The two predominant forms of PmLTI have estimated molecular weights of 43 and 39 kDa, whereas the major forms of PmSTI have molecular weights around 7 to 8 kDa. SDS-PAGE analysis of samples indicated that PmLTI exists as a dimer, and PmSTI appears to be a monomer. Inhibitory activity of PmLTIs was about 2.78 and 3.93 IU/mgTI against bovine trypsin and Heliocoverpa zea midgut trypsin, respectively. Inhibitory activity of PmSTI was 50.94 and 14.23 IU/mgTI against bovine and H. zea midgut trypsin, respectively. PmSTI was still active after boiling for 30 minutes but lost activity immediately when boiled with reducing agent. PmSTI is the first low molecular weight TI isolated from seeds of the legume subfamily Mimosaceae.

A PmSTI cDNA clone was successfully expressed in tobacco plants after Agrobacterium-mediated plant transformation. Transformation and expression of the PmSTI coding sequence were confirmed by Southern blot analysis, trypsin inhibitor assay and Western blot. When the effects of the expression of the PmSTI transgene in tobacco plants was evaluated, results indicated that the transgenic plants exhibited some degree of protection to the tobacco hornworm (Manduca sexta).

Amino acid sequencing revealed that two PmSTI variants, one 60 and the other 61 amino acid residues in length, were present in seeds. Analysis of PmSTI sequence indicates that it belongs to the Bowman-Birk inhibitor family. Although PmSTI has an estimated pI of 8.15 based on experimental data, PmSTI did not bind to either an anion exchange column at pH 9.0 or a cation exchange column at pH 5.0. PmSTI is a tight binding inhibitor of trypsin with an inhibition constant of 0.1 nM against bovine trypsin, but did not inhibit chymotrypsin. A full-length 558-bp cDNA sequence encoding the precursor of PmSTI has been isolated and characterized. The cDNA sequence possesses typical sequence motifs observed in plants. These include a Kozak ribosome binding site (GAAG ATGG), a cis-acting signal for the 3' -end mRNA formation which consists of a TATGTA domain upstream of the transcription stop signal AATAAA and a GT or T rich downstream region. The deduced PmSTI precursor has 120 aa residues which comprises a 52 residue N-terminal peptide, a mature PmSTI sequence and an 8-residue carboxyl-terminal extension peptide.