Applications To Health Promotion And Disease Prevention

Biotechnology applications of the protein seed content of

Macrotyloma axillare seed lectin (MaL) 901

The availability of a great number of lectins displaying distinct glycidic specificities has resulted in the utilization of such molecules as useful tools in medical and biological research. Lectins

FIGURE 106.2

1D gel separation of two bio-products isolated from M. axillare. BBI, Bowman-Birk inhibitors at approximately 8 kDa after reduction and alkylation of cysteine residues; MaL, M. axillare lectin at approximately 28 kDa (monomer form). BBI and MaL are stained in Coomassie. MaL*, M. axillare lectin stained using the PAS method to reveal its carbohydrate content.

FIGURE 106.2

1D gel separation of two bio-products isolated from M. axillare. BBI, Bowman-Birk inhibitors at approximately 8 kDa after reduction and alkylation of cysteine residues; MaL, M. axillare lectin at approximately 28 kDa (monomer form). BBI and MaL are stained in Coomassie. MaL*, M. axillare lectin stained using the PAS method to reveal its carbohydrate content.

can be used to explore the cell surface through its ability to recognize and bind the glycidic moieties of glycoproteins and glycolipids of the cell glycocalix. Alternatively, immobilization of lectins onto inert matrices offers great potential for affinity chromatography and isolation of glycoproteins, an important primary step towards the characterization of the glycoproteome. Due to their inherent functions, these versatile molecules are also used for blood-typing, are employed as mitotic agents (e.g, lectins such as concanavalin A stimulate mitosis in certain types of lymphocytes), and can act as probes to detect the alterations associated with cell development and transformation. Nowadays, lectin derivatives are also commercially available. These include the covalent attachment of fluorescein isothyocianide, biotin, or radioactive isotopes to lectins to facilitate their detection whilst preserving their biological activity (Kennedy etal., 1995).

Lectins from leguminous plants are used as model systems to study the molecular events associated with the interaction between a protein and a specific carbohydrate in biological research. This choice is based on the relative ease in purifying and isolating significant amounts of lectins from seed extracts. Once isolated, the lectin specificity can be evaluated by biophysical techniques such as X-ray crystallography, nuclear magnetic resonance, and microcalorimetry. Although a great variety of glycidic specificities have been observed for the isolates, a remarkable conservation of amino acid sequence is reported among leguminous lectins (Loris et al., 1998).

The lectin isolated from M. axillare (MaL) seeds, and its counterpart, purified from Dolichos biflorus (DBL) seeds, are specific to the carbohydrate N-acethyl-a-D-galactosamine (GalNAc). This finding offers biotechnological relevance, given that this sugar is an antigenic determinant of blood type A from the ABO system. Routinely, DBL is used to differentiate subgroups A1 from A2, the difference residing in the number of antigens on the erythrocyte surface (greater in A1 compared to A2) (Santana, 2008). Subgroup A1 is then identified by positive hemagglutination, and the test is negative for subgroup A2 under the use of a standard specific activity (Figure 106.3). Considering that DBL and MaL are closely related to each other and share the same sugar specificity (Haylett & Swart, 1982), methodologies to obtain the lectin isolated from M. axillare can be useful, as this leguminous plant can be cultivated and obtained in vast amounts in tropical countries.

| Native erythrocytes J Erythrocytes trypsin treated

Erythrocytes of the ABO system

FIGURE 106.3

Macrotyloma axillare seed Lectin (MaL) specificity on the ABO system. MaL specificity study on native and trypsin-treated erythrocytes for the human ABO antigen system. Reproduced from Santana et al. (2008), Intl J. Biol. Macromol., 43, 352—358, with permission.

CHAPTER 106

M. Axillare Seeds — Biotechnology Applications

MaL was first isolated by Haylett and Swart (1982) by affinity chromatography, essentially as proposed for the isolation of DBL. The protocol revealed a good protein yield; however, the method used for elution involved the utilization of a low pH buffer as an alternative to the use of GalNAc, as initially described by Etzler and Kabat (1970) during isolation of DBL. Further studies from our research group, at the Federal University of Ouro Preto, have revealed that a low pH can interfere with MaL activity as a tretamer form, which led us to propose a new protocol for the isolation of MaL. The procedure involved the utilization of fewer and simple steps resulting in high lectin yields and maximum retention of biological activity, and therefore suitable for scaling up for industrial production (Santana et al., 2008). The detailed technique was patented, and is now deposited under the accession BR200401517-A (Guerra de Andrade et al., 2004).

The Bowman-Birk inhibitors from M. axillare

Bowman-Birk type inhibitors, commonly called BBI, are cytoplasmic protein inhibitors of serine proteases. BBI were first isolated from soybean seeds by Bowman in 1946, and then characterized by Birk in 1961. Since then, BBI from other leguminous plants have been isolated and characterized (Norioka & Ikenaka, 1983). In general, BBI are low molecular mass proteins, of approximately 8 kDa, consisting of a single amino acid chain exhibiting a significant content of cysteine residues which confer a rigid tridimensional folding highly conserved among the different isolates of the Plantae Kingdom, to which they are exclusive. The main enzymes inhibited by BBI are trypsin and chymotrypsin. BBI are classified as bivalent inhibitors, as their tridimensional folding defines two inhibition loops or "heads," one being specific for the binding of trypsin and the other for the binding of chymotrypsin (Prakash et al., 1996). Other enzymes reported to be inhibited by BBI are cathepsin G, elastase, and chymase. Crystallographic analyses have shown that the tridimensional structure of this protein is mainly stabilized by seven disulfide bridges, followed by a number of intramolecular hydrogen bondings and a contribution of the hydrophobic core of the molecule (Chen et al., 1992). As a result, BBI are highly resistant molecules supporting extremes of temperature, ionic strength, and pH, being soluble in the pH range

Serine protease inhibitors react with the protease active site at the serine residue, forming derivatives of inactivated enzymes. This class of proteases is inhibited by Kunitz type inhibitors, BBI, the potato inhibitors type I and II, and the kazal type inhibitors (Losso, 2008). In this context, BBI isolated from M. axillare merit attention due to their highly specific inhibitory activity, especially considering BBI isolated from germinated seeds (Cesar, 2009). The interaction of BBI with either trypsin or chymotrypsin is able to strongly inhibit the activity of these proteases; in addition, simultaneous binding of these two proteases with a single BBI monomer has been reported. The conformational loop of the BBI reactive site is fully complementary to the active site of the enzyme to be inhibited, allowing for a strong protein—protein interaction to take place (Chen et al., 1992). Although this interaction is reversible, it occurs with an affinity compared to that observed for the formation of the complex resulting from the interaction between the protease and its protein substrate. The perfect docking of the inhibitor at the enzyme active site prevents necessary conformational changes to occur, resulting in an unfavorable energetic barrier to hydrolysis.

BBI have been the subject of scientific research involving protease inhibition, mainly due to its antichymotrypsin activity. The rationale relies on the observation that the inhibition of a cell's chymotrypsin activity is linked to an anticarcinogenic effect. BBI have also been shown to possess anti-inflammatory and radioprotective properties, also being able to inhibit the production of free radicals and some carcinogen-induced transformations. Although a wide spectrum of anticancer activities has been reported, the exact mechanisms these inhibitors

utilize to promote anticarcinogenic effects remain to be elucidated (Billings et al., 1990; Kennedy, 1994; Losso, 2008).

More recently, the activity of BBI has been shown to extend beyond their classical chymo-trypsin/trypsin targets. In this regard, Chen et al. (2005), using BBI isolated from soybeans, demonstrated that these molecules also inhibited the chymotrypsin-like acitivity of the 26S proteasome, a major nanomachine involved in intracellular proteolysis. In vitro and in vivo experiments confirmed the specific inhibition of the proteasome's chymotrypsin-like activity, using MCF7 cells isolated from breast cancer tissue. It was also verified that such inhibition is linked to an accumulation of ubiquitylated proteins, the natural proteasome substrates, and a reduction in the levels of regulatory cyclins involved in cell division. Altogether, the authors suggested that inhibition of the 26S proteasome activity by BBI could greatly contribute to their preventive effect on cancer.

Further evidence of the interaction of BBI and the proteasome has been reported by Saito et al. (2007), who also showed inhibition of the chymotryspin-like activity in osteosarcoma cells. More specifically, BBI inhibited the degradation of connexin 43 by the ubiquitin-proteasome system in these tumor cells. It was suggested that the antiproliferative effect observed was due to maintenance of connexin function through homeostatic balance promoted by GAP junctions.

M. axillare seed germination and BBI

The practice of seed germination and consumption as a health-promoting activity has been observed in different cultures. In this context, it was verified that germinated M. axillare seeds exhibit a significant increase in the activity of BBI, reaching up to a four-fold increase when compared to the activity of the inhibitors isolated from non-germinated seeds (Cesar, 2009). The activation process requires hydrolysis and reduction of the molecular mass of BBI, with the monomer found on 5-day germinated seeds being approximately 6 kDa, as opposed to the 8-kDa monomers found on dormant seeds (Figure 106.4). This reduction in size produces a notable effect on the pharmacokinetic properties of these inhibitors when they are administered to mice. The most evident effect observed was a significant increase in their distribution volume, allowing for a better diffusion of BBI throughout the body (Table 106.1).

The higher distribution volume and the increased activity of BBI present in the cotyledon strengthen the possibility of a better efficacy of these inhibitors on cancer prevention. The pharmacokinetic data of tissue distribution revealed a marked accumulation of BBI isolated

Seeds Germinated seeds

Macrotyloma axillare

FIGURE 106.4

Inhibitory activity of BBI from M. axillare. Relative inhibitory activity of BBI isolated from M. axillare dormant seeds compared to those isolated from 5-day germinated seeds. Up to a four-fold increase is observed for antichymotrypsin activity.

Seeds Germinated seeds

Macrotyloma axillare

FIGURE 106.4

Inhibitory activity of BBI from M. axillare. Relative inhibitory activity of BBI isolated from M. axillare dormant seeds compared to those isolated from 5-day germinated seeds. Up to a four-fold increase is observed for antichymotrypsin activity.

TABLE 106.1 Pharmacokinetic Parameters of BBI Isolated from M. axillare Seeds and Labeled with Radioactive Isotope (I125) Determined after Oral Administration to Swiss Mice3.

Pharmacokinetic parameter

Seeds

Cotyledons

a (distribution constant)

0.0405/min

0.0938/min

b (elimination constant)

0.0008/min

0.0016/min

VD (distribution volume)

4.76 ml

5.41 ml

t1/2 (plasma half life)

14.4 hours

7.2 hours

Andrade, M.H.G. and Santos, A.G. (2007), Patent number: BRPI0601377 (A), available at http://v3.espacenet.com/publication Details/originalDocument?CC=BR&NR=PI0601377A&KC=A&FT=D&date=20071127&DB=EPODOC&locale=en_EP.

Andrade, M.H.G. and Santos, A.G. (2007), Patent number: BRPI0601377 (A), available at http://v3.espacenet.com/publication Details/originalDocument?CC=BR&NR=PI0601377A&KC=A&FT=D&date=20071127&DB=EPODOC&locale=en_EP.

from M. axillare in the gastric tissue when compared to other organs when BBI are administered orally (Figure 106.5). This constitutes a striking feature of M. axillare BBI when compared with those isolated from soybeans (Glycine max).

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