functional alteration of a dimeric insecticidal lectin to a monomeric antifungal protein correlated to its oligomeric status功能改变的二聚的杀虫植物血凝素单体的抗真菌蛋白相关的低聚物的状态.pdfVIP

Functional Alteration of a Dimeric Insecticidal Lectin to a Monomeric Antifungal Protein Correlated to Its Oligomeric Status 1 1.¤ 1. 1 2 1 Nilanjana Banerjee , Subhadipa Sengupta , Amit Roy , Prithwi Ghosh , Kalipada Das , Sampa Das * 1 Division of Plant Biology, Bose Institute, Kolkata, India, 2 Department of Chemistry, Bose Institute, Kolkata, India Abstract Background: Allium sativum leaf agglutinin (ASAL) is a 25-kDa homodimeric, insecticidal, mannose binding lectin whose subunits are assembled by the C-terminal exchange process. An attempt was made to convert dimeric ASAL into a monomeric form to correlate the relevance of quaternary association of subunits and their functional specificity. Using SWISS-MODEL program a stable monomer was designed by altering five amino acid residues near the C-terminus of ASAL. Methodology/Principal Findings: By introduction of 5 site-specific mutations (-DNSNN-), a b turn was incorporated between the 11th and 12th b strands of subunits of ASAL, resulting in a stable monomeric mutant ASAL (mASAL). mASAL was cloned and subsequently purified from a pMAL-c2X system. CD spectroscopic analysis confirmed the conservation of secondary structure in mASAL. Mannose binding assay confirmed that molecular mannose binds efficiently to both mASAL and ASAL. In contrast to ASAL, the hemagglutination activity of purified mASAL against rabbit erythrocytes was lost. An artificial diet bioassay of Lipaphis erysimi with mASAL displayed an insignificant level of insecticidal activity compared to ASAL. Fascinatingly, mASAL exhibited strong antifungal activity against the pathogenic fun