ISOLATION, PURIFICATION AND FUNCTIONAL STUDY OF ARABINOGALACTAN PROTEIN FROM GROUNDNUT

Abstract

Cell surface macromolecules play a significant role in the growth and development of almost all organisms from microbes to higher animals and plants. Besides, they were also found to be of great therapeutic importance. Among them, glycoconjugates (glycoproteins and proteoglycans) have drawn more attention due to their diverse biochemical and medical potential (Ooi and Liu, 2000). The search for a novel glycoconjugate originates from the main fact that most of the commonly used antitumor drugs, chemicals and radiotherapy which are found to be cytotoxic to the cancerous cells are also toxic to normal cells (Kim et al., 1996; Borchers et al., 1999). Thus the discovery and identification of new safer drugs, without severe side effects, has become an important area of research in biomedical sciences (Han et al., 2001). The glycoconjugates, mainly the polysaccharide-protein complexes are found to be more suitable alternative because of its high water solubility, immunostimulatory and antitumor activities. During past two decades, a number of polysaccharides and proteoglycan/glycoproteins with immunostimulatory and antitumor activities have been isolated from diverse sources such as mushrooms (Han et al, 1999; Kim et al, 2003), lichen (Zhang et al, 2002) and plants (Hauer and Anderer, 1993; Classen et al, 2000; Classen et al, 2006; Duan et al, 2003). One such group of molecules, which play important role in cell growth and development processes are natural plant derived glycoconjugates known as Arabinogalactan proteins (AGPs) and like molecules. AGPs are a group of proteins which contain both carbohydrate and protein as their constituents and are collectively known as Hydroxyproline rich glycoproteins. The protein backbone of AGP is typically hydroxyproline rich and the carbohydrate chain is arabinogalactan (AG) type which primarily consists of galactose and arabinose which are covalently attached to the protein backbone. The AG chain is usually a branched polysaccharide consisting of (l,3)-P-D-galactan backbone, having (l,6)-p-galactan side chains, which are terminally modified by arabinose with some exceptions where other less-abundant sugar may be present (Gasper et al., 2001; Showalter, 2001). On the basis of protein backbone, AGPs can be classified into two broad groups designated as "classical" and "non-classical". The classical AGP protein back bone has a predicted domain structure and typically rich in hydroxyproline, alanine, serine, threonine and glycine. The classical AGP's core is unique in their organization, comprising of an N-terminal secretion signal, hydroxyproline rich glycosylation domain and a C-terminal hydrophobic sequence with a site for GPI-anchorage (Du et al, 1996b; Schultz et al, 1998). They are attached to the membrane via a GPI anchor and during GPI anchor addition the rest of the C- terminal amino acids are removed. On the other hand nonclassical AGPs do not have GPI anchor and their protein backbone differ from classical protein core in many ways as they are relatively Hyp poor, rich in Asn and Cys (Schultz et al, 2000, 2002; Johnson et al, 2003; Mashiguchi et al, 2004). AGPs are widely distributed throughout the plant kingdom from bryophytes to angiosperms (Showalter, 1993; Nothnagel, 1997; Tischer et al, 2002; Letarte et al, 2006; Tang et al, 2006; Pereira-Netto et al, 2007). AGPs as a group are widely distributed in various organs and tissues. At organ level they are found in stems, leaves, roots, floral parts, vascular tissues and seeds (Nothengel, 1997; Coimbra and Duarte, 2003; Motose et al, 2004; Huang et al, 2007; Nguema-Ona et al, 2007). AGPs are found in many tissues, and are especially abundant and well documented in xylem, stylar transmitting tissues and cell suspension culture. Inside cell they have been found in extracellular space, plasma membrane, and the cytoplasmic organelles (Fincher et al, 1983; Komalavlas et al, 1991; van Hengel et al, 2004; Coimbra et al, 2007). AGPs are found to be highly polymorphic molecules and implicated to play roles in plant growth and development including cell fate, cell proliferation, cell expansion, sexual reproduction, programmed cell death (Gao and Showalter, 1999; Majewska and Nothngel, 2000; Chaves et al, 2002; Lee et al, 2005; Yang and Showalter 2007), cell signaling and somatic embryogenesis (Knox, 1995; Thomson and Knox 1998; Samaj et al, 1999; van Hengel et al, 2001; Letarte et al, 2006; Lamprot et al, 2006). Expression level of AGPs have been found to be developmentally regulated and also depend on biotic and abiotic stress, wounding, pathogen invasion, heat shock and phytohormones (Li and Showalter, 1996; Gilson et al, 2001; Park et al, 2003; Sun et al, 2004). Because of its significant role in cell growth and development, AGPs from various important crops have been isolated, structure determined and a number of genes have been cloned and characterized (Sommer-Knudsen et al, 1997; Serpe and Nothnagel, 1999; Schultz et al, 2000; Gaspar et al, 2004). In addition, they have been found to be of commercial value due to its immunomodulatory and antitumor activities (Duan et al, 2003; Thude et al, 2006; Classen et al, 2006), as antimicrobial agents (Singha et al, 2003, Kottakis et al, 2007), as emulsifier and as adhesive (Whistler, 1993). Medicinal plants having immune enhancing property such as Baptisia tinctoria (Egert and Beuscher, 1992), Thuja occidentalis, Angelica acutiloba (Kiyohara et al, 1987), Curcuma longa (Gonda et al, 1993) also contain AGPs. It has been reported that active constituent of important medicinal plants preparation has been found to be AGP (Classen et al, 2000). The active constituent of the most potent, commonly used immunomodulator from Echinacea purpurea has also been found to be AGP (Alban et al, 2002). There are a number of reviews highlighting structure and functions of AGPs in plant growth and development (Majewska and Nothnagel, 2000; Gaspar et al, 2001; Showalter, 2001; van Hengel et al, 2004). Due to its polymorphic nature variations have been found in structure, functions and therapeutic potential of AGPs depending upon it source. Thus to get further information about their structure, functions and to get new potential AGPs of therapeutic importance, there is always a need to explore the AGPs from new sources. In present work efforts are being made to isolate AGP from groundnut and to explore its possible functions and applications. Groundnut (Arachis hypogaea L.) is one of the major oilseed crops and is a member of genus Arachis and family Leguminoceae. A. hypogaea is an alleotetrapoloid (2n = 4x = 40) species native to South America (Sharma and Bhatnagar-Mathur, 2006). Two major producers in Asia are India with 8.2 million hectare and China with 4.6 million hectare which constitutes 55.9% and 31.6% . area of Asia, respectively (Swamy et al, 2003). The seeds of groundnut contain 44-56% oil and 22-30% protein on dry seed weight basis. Oleic and Linoleic are two major fatty acids together account for 75-80% of the total fatty acids in groundnut. Groundnut is an important commodity in many developing countries for both direct human food and oil production, particularly in India where the nitrogen (N)-rich crop residues are also used as fodder. In addition, it is a good source of minerals like P, Ca, Mg and K. Seeds also contains vitamins E, K and B group (Dwivedi et al, 1996). The production of groundnut in India needs to be increased from the current 8 million tones to about 14 million tones by 2020 to meet the increasing demand of the oil and confectionery industry (Girdhar, 2004). However, the production of groundnut is affected greatly due to various abiotic stresses such as salt, drought and diseases like tikka disease of groundnut, groundnut rosette disease, late leaf spot and rust disease. Thus to meet the high demand of groundnut there is need for development of abiotic stress tolerant and disease free varieties. One of the strategies to achieve this would be to identify the biomarkers which may be helpful in development of salt stress tolerance and disease free crops. AGP could be one of the potential molecules due to two main facts. Firstly, AGPs have found to induce somatic embryogenesis (Chapman et al, 2000; van Hengel et al, 2001) and secondly AGPs have also been reported to have association with salt stress (Lamport et al, 2006). Although AGPs have been identified as important molecules from several economically important plants, no study regarding these has been carried out in A. hypogaea which is one of the most important crops of India. As well variations have been reported in AGPs functions and its therapeutic applications depending upon it source. Therefore, in present study an attempt is being made to isolate the AGP from groundnut {A. hypogaea) seedling and to explore its possible cell functions and therapeutic potential