STUDIES ON PROTEASE INHIBITOR, ITS COMPLEX WITH TRYPSIN AND CHILECTIN FROM Tamarindus indica

Abstract

Seeds of leguminous plants are known to contain proteins that are important for a number of biological processes. Proteinase inhibitors, chitinases, lectins, globulins etc. from these seeds are the most studied ones, since Leguminosae family members are recognized as an excellent source of these biologically significant proteins. The Kunitz type inhibitors of serine proteases form an important field of scientific investigation due to their multifunctional abilities. The Kuntz STI inhibitors are included as Kunitz-P inhibitors in the I3A family of the MEROPS database (http://merops.sanger.ac.uk). Specific inhibitors are reported as insecticidal, anti-inflammatory, anti-cancer, anticoagulant, antiviral, antiparasitic etc. These multifunctional plant Kuntz type inhibitors are crucial tools enabling us to gain knowledge of the basic principles of protein interactions and could be important for human beings in control of many diseases by using them to discover highly potential drugs. So, it is necessary to search for novel Kunitz inhibitors with important functions. Applications of these types of multifunctional inhibitors are very extensive which give an inspiration to perform their structure based study to correlate their-structure with their activities. The structural properties of inhibitors that control the activity of the proteinases could be targeted as potential pharmaceutical products. Plant lectins are heterogeneous and highly diverse class of non-immune origin (glyco) proteins because of their carbohydrate-binding specificity, differences in molecular structure and biochemical properties. They are classified into twelve diverse families of evolutionary and structurally related lectin domains. There is no precise description of the biological functions of plant lectins because of their diverse classes and carbohydrate specificities. Several functions of specific 'lectins are reported which include antifungal, insecticidal, antiviral, antiproliferative, apoptosis-inducing and symbiosis mediating between nitrogen fixing microorganisms and legume plants. Lectins show extensive structural diversity and considering the structural folds, plant lectins are grouped into seven folds. Previous reports showed that plant lectins are also members of chitinase family in fold while they might possess or lack chitinase activity. To gain further insight into structure-function relationship of lectins in general, it is necessary to carry out structural study of these types of proteins which will also lead to understand their evolution and how they acquire new functions using the same parental fold. My thesis encompasses the cDNA cloning, isolation, purification, characterization, crystallization and structural studies of a Kunitz type inhibitor, its complex with trypsin and purification, biochemical and structural studies . of chitinase..~ike lectin (chilectin) from Tamarindus indica. The first chapter gives a general introduction and a brief review of the work done nationally and internationally for Kunitz type inhibitors and lectins. The second chapter describes purification and biochemical characterization of a Kuntiz type inhibitor from seeds of a leguminous plant, Tamarindus indica named as TKI (Tamarind Kunitz type inhibitor). TKI was isolated using a series of columns, initially Affi-blue column followed by DEAE an anion exchange column. The approximate molecular mass of the TKI was determined to be 21 kDa from SDS-PAGE analysis. The protein yield was 0.6 mg per gram of seeds and consisted of a homogenous mixture. The trypsin inhibitory activity and N-terminal amino acid sequence analysis revealed the isolated protein to be a protease inhibitor belonging to Kunitz type inhibitor family. The N-terminal amino acid sequence of TKI showed 79% homology with factor Xa inhibitor (BuXI) from Bauhinia ungulata and 64% with Trypsin inhibitor (BvTI) from Bauhinia variegata. TKI was tested for its anticoagulant, factor Xa inhibitory and antiproliferative activities and was found to possess anticoagulant and FXa inhibitory activities. Furthermore it also showed dose dependent suppression of the cancer cell growth of MCF-7, HepG2 and PC-3 cells. The third chapter contains cDNA synthesis, cloning, expression and purification of TKI. Briefly, the total RNA was isolated using three month old seeds after flowering and cDNA was synthesized. It was further amplified and cloned into pGEM-T easy vector and sequenced. The cDNA fragment amplified with PCR was —700 bp in length consisting of 3' UTR region and poly (A+) tail. A 555 bp ORF obtained after sequencing of the TKI gene coded for a polypeptide of 185 amino acid residues with a calculated molecular mass of 20575 Da. Sequence analysis showed that TKI belongs to Kunitz type STI family, however, has distorted Kunitz signature signature sequence due to insertion of Asn15 in the signature motif. TKI has maintained two characteristics of Kunitz STI family such as having molecular weight —20-22 kDa and conserved two disulphide bonds. The full length TKI encoding gene was cloned into pET-28cTEV vector and expressed Ninto BL21- DE3 competent Escherichia coli cells which showed expression and solubility of TKI but with negligible trypsin inhibitory activity which might be due to improper folding and disulfide bonds formed. Formation of correct disulfide bonds and proper folding might be ii required for TKI to maintain its inhibitory activity, so we expressed TKI in chemically competent E. coil K12 cells shuffle T7 which is engineered to form disulfide bonded proteins in -- the cytoplasm. SDS-PAGE analysis showed that rTKI was expressed as a soluble protein which has potent trypsin inhibitory activity like native TKI. Recombinant TKI was purified using Ni-NTA column and pure rTKI exhibited trypsin inhibitory activity. This result shows that rTKI is a stable protein and able to preserve its functional property and can be used further for biochemical studies. The fourth chapter encloses the structure of TKI, its complex with trypsin and molecular docking studies with factor Xa. Crystal structure of free TKI was solved at 1.9A showing Ji-trefoil fold consisting of 12 anti-parallel (3-strands with two disulphide bridges and an exposed reactive site loop having Arg at P1. To understand the mode of interaction of TKI with trypsin, crystal structure of TKT:pancreatic porcine trypsin (PPT) was determined at 2.2A. The stable complex formed by TKI blocked the Sl pocket of PPT by its exposed reactive site and-stabilized the complex by making interaction with the sub sites of PPT. The electron density for Asnl 5 was clearly visible in both free as well as TKI:PPT complex structure. In order to analyze the possible binding of TKI with FXa, molecular docking was performed. TKI Pl and P3 residues showed the interactions with Si and S4 pocket of FXa, respectively in a classical L-shaped substrate like conformation. Arg64, which. interacts with cation hole with its side chain occupying the aryl binding site of S4, is the most important residue of TKI which may provide specificity towards FXa. The Asn15 is important residue insertion in TKI which is involved in recognition and stabilization of complexes. Asnl5 interacts with four residues of PPT and two residues-of FXa and participates in stabilization of complex. Furthermore, it interacts with those residues which are important for specificity of FXa which point towards its role in recognition of specific proteases. Comparison of the reactive site of TKI with Kunitz STI inhibitors and factor Xa inhibitors show that it has distinct reactive site residues which possibly inhibit other trypsin like serine proteases and could play multifunctional roles. The fifth chapter encompasses purification, characterization, crystallization and structure determination of the chitinase-dike lectin (TCLL) and its complex with G1cNAc. The TCLL was purified using Affi-blue and DEAE column chromatography was found to be monomeric 34 kDa protein which shows negligible chitinase activity. The structure was determined at 1.49A and shows ((3a)8 barrel topology. The sequence of TCLL was proposed from crystallographic data and by mass spectroscopy. TCLL showed significant sequence similarity to the reported class III chitinase of GH18 family members. Evaluation of active site and substrate binding subsites revealed that it has mutations of key residues which are required for chitinase activity. Remarkably, the complex structure of TCLL with G1cNAc reveals that the latter binds in a pocket formed by two loops (34a4 and (35a5 which have different conformation in structures of GH1 8 members. From biochemical and structural studies, we concluded that TCLL is a chilectin which explored novel carbohydrate binding site other than chitintbinding groove in GH1 8 family members. However, TCLL does not fit anywhere into this classification of plant lectins by Van Damme et al.,(008[ Consequently, TCLL structure signifies a new class of plant lectins and confers evidence for evolutionary link of lectins With chitinases. The structure of TCLL also depicts how plants utilize the existing structural scaffolds ingenuously to attain new functions. So we propose a new class of lectin which is evolutionarily related to class III chitinase. Moreover, the one of the existing `Class V chitinase homologs' class of lectin and our proposed class `Class III chitinase homologs' have same fold. To avoid number of classes in lectin classification, we propose the new class "TIM barrel domain" rather than existing one and these two classes can be grouped into TIM barrel domain as one subclass. Furthermore, one cannot exclude the possibility that the glycosyl hydrolase family members or other ,families having TIM barrel topology from plants will not emerge as lectin. So the TIM barrel domain will be the main class and the forthcoming classes will be subclasses.