STUDIES ON TRYPSIN INHIBITORS FROM PLANTS OF RUTACEAE FAMILY

Abstract

Serine proteinase inhibitors are present in leaves, flowers, seeds and tubers of many plants as their defensive agent against insect pests. Many plant serine proteinase inhibitors have been purified and characterized particularly from the seeds of Leguminosae, Cucurbitaceae, Solanaceae and Gramineae family. Among them, the Kunitz trypsin inhibitor super-family has gained particular importance for its specific activity against trypsin-like serine proteinases. These proteinase inhibitors have been shown to inhibit the proteolytic activity of several lepidopteran pests which largely depends on presence of serine proteinases for protein digestion. Their role in retarding growth and development of insect pests fed on diets containing inhibitor has been demonstrated in many studies. However, insect pests overcome the effect of plant proteinase inhibitors by expressing new proteinases which are either insensitive or can degrade them. Due to adaptive strategy adopted by insect pest against host proteinase inhibitors, it is important to identify effective and potent inhibitors of insect proteinases from unrelated nonhost plants. One of the major focuses of plant biotechnology is to develop crops resistant to particular insect pests. One of the strategies could be to express effective proteinase inhibitors against insect gut proteinases from non-host plants in affected crops. A Kunitz-type trypsin inhibitor from the seeds of Murraya koenigii belonging to Rutaceae family has been purified earlier in our lab. Biochemical characterization of Murraya koenigii trypsin inhibitor (MKTI) has shown that it is highly resistant to proteolytic degradation by selected proteinases and is a major seed storage protein. In the present work, in vitro and in vivo effects of MKTI on two lepidopteran insect pests namely Helicoverpa armigera and Spodoptera litura were studied. Both are polyphagous lepidopteran insect pests and affects many important crops causing severe economic losses. This work demonstrated the inhibitory potential of MKTI against gut proteinases and their effect on growth and development of the two pests. Also, the cloning, sequencing, expression and homology modeling of MKTI were performed. The MKTI gene was cloned form both genomic and cDNA. This work also includes the cloning, sequencing and homology modeling of another Kunitz-type trysin inhibitor from the plant Murraya paniculata belonging to Rutaceae family. The thesis is divided into six chapters. Chapter 1 reviews the literature in the area of plant proteinase inhibitors particularly serine proteinase inhibitors. Chapter 2 assesses the in vitro and in vivo effects of Murraya koenigii trypsin inhibitor on Helicoverpa armigera, a polyphagous lepidopteran insect pest. MKTI inhibited the trypsinlike and total proteinases activity of H. armigera gut proteinases (HGP) by 78.5% and 40% respectively. In feeding experiments, MKTI was found to adversely affect the growth and development of larvae, pupae and adult in dose dependent manner. A reduction of 69% in larval weight after 10 days of feeding and 40% larval mortality was observed in larvae fed with diets containing 200 uM inhibitor. Likewise, pupation, pupal weight, adult survival, adult emergence, fertility and fecundity were adversely affected. Also, malformed pupae and adults were observed because of abnormal development. The effect of MKTI feeding on endogenous levels of proteinases showed a substantial decrease in trypsin-like activity and similar increase in chymotrypsin-like activity after being fed with inhibitor supplemented diet. The MKTI was found to be completely stable against proteolysis by HGP up to 72 h with inhibitory activity intact. The results clearly demonstrated the efficacy of MKTI as a plant defense agent against H. armigera. Chapter 3 evaluates the effect ofMurraya koenigii trypsin inhibitor (MKTI) on digestive proteinases and growth of Spodoptera litura. MKTI inhibited the trypsin-like and total proteinases activity of S. litura gut proteinases (SGP) by over 81% and 48% respectively. The effect of MKTI feeding on endogenous levels of proteinases showed an 8-fold decrease in trypsin-like activity and approximately 4-fold increase in chymotrypsin-like activity in fifth instar larvae after being fed with inhibitor supplemented diet. MKTI was found to be remarkably stable against proteolysis by SGP even after 72 h of incubation with total gut proteinases. Incorporation of MKTI in artificial diet adversely affected the growth and development of larvae, pupae and adult in dose dependent manner. Larval feeding studies showed a reduction of 44.8% in larval weight after 10 days of feeding and larval mortality increased to 43.3% when fed with diets containing 200 uM inhibitor. These results showed that MKTI can be an effective plant defense agent against S. litura. Chapter 4 describes cloning, sequencing, expression and homology modeling of Murraya koenigii trypsin inhibitor. A 648 base pair open reading frame was obtained after sequencing of the MKTI PCR product. DNA sequence analysis of both cDNA and genomic DNA showed same nucleotide sequence in the coding region, implying thatgenomic clone lacks intervening sequences. The deduced amino acid sequence ofMKTI comprises a polypeptide of 215 amino acid residues of which first 25 N-terminal residues form a signal peptide and remaining 190 residues form mature protein. The open reading frame of mature protein was overexpressed as the glutathione S-transferase (GST)-fusion protein in E. coli TGI cells after subcloning it in-frame in pGEX-5X-3. The recombinant MKTI was obtained from GST-MKTI fusion protein after treatment with factor Xa. Both, the fusion protein and isolated reMKTI showed identical trypsin inhibitory activity. Chapter 5 describes amino acid sequence analysis which showed that MKTI forms a distinct cluster with miraculin-like proteins, a Kunitz family member, in phylogenetic analyses. in It showed maximum homology (57% identity & 74% similarity) with miraculin-like proteins. The amino acid sequence of reactive loop of MKTI showed major differences from other Kunitz-type inhibitors. The MKTI sequence showed existence of possible glycosylation, phosphorylation and myristoylation sites. The structure prediction by homology modeling showed significant differences between MKTI and other soyabean Kunitz family inhibitor structures despite similar overall fold. The major differences were observed in intermolecular contacts between MKTI reactive loop and trypsin when compared to soyabean Kunitz inhibitor. The present study explores the unique features of MKTI structure based on the amino acid sequence and homology modeling. Chapter 6 describes the cloning, sequencing and homology modeling of a trypsin inhibitor from Murraya paniculata. In comparative protein profiling, a corresponding band similar to MKTI was observed in Murraya paniculata seed extract. But weak trypsin inhibitory activity has been observed in the M. paniculata seeds extract compared to MKTI crude extract. A PCR fragment of 570 bp was obtained from genomic DNA amplification. Sequence analysis has shown that it belongs to kunitz family of trypsin inhibitor. Murraya paniculata trysin inhibitor (MPTI) shares 96% homology matching with the amino acid sequence of MKTI. MPTI also forms a distinct cluster with miraculin like protein and distantly related with kunitz type trypsin inhibitor. Structure prediction by homology modeling revealed that it has overall three fold internal symmetry with (3- trefoil fold. MPTI is a weak trypsin inhibitor as compared to MKTI. This study analyses its differences with MKTI based on the amino acid sequence and homology modeling.