Cloning and Functional Analysis of the Bifunctional Agglutinin/Trypsin Inhibitor from Helianthus tuberosus L.

In order to find new insect resistance genes, four homologous cDNAs, hta-a, hta-b, hta-c and hta-d with lengths of 775, 718, 784 and 752 bp, respectively （GenBank accession numbers AF477031-AF477034）, were isolated from a tuber cDNA expression library of Helianthus tuberosus L. Sequence analysis revealed that all four cDNAs contain an open reading frame of 444 bp, coding a polypeptide of 147 amino acid residues, and that the sequences of the cDNAs are very similar to those of the mannose-binding agglutinin genes of the jacalin-related family. In hemagglutination reactions and hapten inhibition assays, affinity-purified HTA （Helianthus tuberosus agglutinin） from induced Escherichia coli BL21（DE3） expressing GST-HTA shows hemagglutination ability and a higher carbohydrate-binding ability for mannose than other tested sugars. Trypsin inhibitory activity was detected in the crude extracts of induced E. coli BL21（DE3） expressing HTA, and was further verified by trypsin inhibitory activity staining on native polyacrylamide gel. The mechanism of interaction between HTA and trypsin was studied by molecular modeling. We found that plenty of hydrogen bonds and electrostatic interactions can be formed between the supposed binding sites of HTA-b and the active site of trypsin, and that a stable HTA/trypsin complex can be formed. The results above imply that HTA might be a bifunctional protein with carbohydrate-binding activity and trypsin inhibitory activity. Moreover, Northern blotting analysis demonstrated that hta is predominantly expressed in tubers of H. tuberosus, very weakly expressed in stems, but not expressed at all in other tissues. Southern blotting analysis indicated that hta is encoded by a multi-gene family. The insect resistance traits have been described in another paper.

Structural diversification of phenylspirodrimane lactams by employing a biosynthetic intermediate

Phenylspirodrimanes are a kind of meroterpenoids with structural diversity and complexity,exhibiting a wide of biological properties,especially for the lactam derivatives consisting a y-lactam moiety and N-linked side chains.These compounds were derived from multi-step combination of enzymatic and non-enzymatic conversions of intermediates in their biosynthetic pathways.Stachbotrydial(2)with an o-phthalaldehyde unit was supposed as the high-reactivity intermediate of phenylspirodrimane lactams via nonenzymatic reaction with amines.In the present work,an effective and non-enzymatic diversification strategy was developed for the structural diversification of phenylspirodrimane lactams including monomers and dimers from 2 by feeding structurally various mono-and diamines in the fungus Stachybotrys chartarum cultures.In total,24 phenylspirodrimane lactams(1,3-25)including 18 new compounds were synthesized.Among them,stachybocin A(1),a bioactive phenylspirodrimane lactam dimer,was produced with the yield of 18.7 mg/g of cell dry weight.The structures of these compounds were elucidated by extensive spectroscopic data,single-crystal X-ray diffraction(Cu Kα),and calculated electronic circular dichroism(ECD)analyses.Bioassay revealed that compounds 1,17,and 24 displayed significant inhibitory effect on the inactivated state of hNav 1.2 channels with IC_(50) values of 0.22,2.08,and 0.53μmol/L,respectively.In addition,1 showed potent protein tyrosine phosphatase 1B(PTP1B)inhibitory N-methyl-b-aspartate(NMDA)receptor antagonistic,and anti-inflammatory activities.