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The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity.

ARTICLE INFO

Keywords: Antisnake venom glycoprotein from Mucuna pruriens seeds Infrared spectroscopy Protein structure Thermal stability Glycoprotein

ABSTRACT

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein.

All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.

[C] 2011 Elsevier GmbH. All rights reserved.

Introduction

Mucuna pruriens (family: Fabaceae; subfamily: Papilionoideae; genus: Mucuna; species: pruriens) is a tropical legume having nutritional qualities and exhibiting a reasonable tolerance to a number of environmental stresses. Mucuna pruriens is mainly a medicinal plant used by folk medicine to treat a variety of disorders (lauk et al. 1993) but in particular it is known for its anti-snake venom properties (Houghton and Skari 1994). In Nigeria, where the seeds are locally prescribed as an oral prophylactic for snake bite, it is claimed that when two seeds are swallowed, they protect the individual for a year against snake bites.

To clarify this unclear phenomenon in our previous experiments we set up an animal model injecting intraperitoneally the mice with the acqueous Mucuna pruriens seeds extract (MPE). These studies (Aguiyi et al. 1996; Guerranti et al. 1999, 2008) demonstrated that MPE is able to protect mice against the toxic and lethal effects provoked by the venom of Echis carinatus (family: Viperidae) through short and long term protection mechanisms.

The long term protection is due to an immunological mechanism based on anti-MPE antibodies cross-reacting with venom proteins and neutralizing them (Guerranti et al. 2002). On the other hand, since antibodies anti-venom are also able to bind MPE protein, we demonstrated that plant and venom proteins present some common epitopes which are located on the glycanic component of a MPE glycoprotein, named gpMuc (Guerranti et al. 2004).

Concerning the short term protection, which does not depend on an immunological mechanism, we hypothesized that it is caused by a direct action of plant extract components on the venom toxins. Echis carinatus venom (EV) toxicity is mainly due to some proteases which affect blood coagulation. Two metalloproteinases, ecarin (Nishida et al. 1995) and carinactivase (Yamada et al. 1996), active on prothrombin are most studied, but viper venoms also contain thrombin-like enzymes, showing a proteolytic effect on fibrinogen (Castro et al. 2004). The presence of all these enzymes determines the well-known phenomenon of consumptive coagulopathy or disseminated intravascular coagulation (DIC) which leads to the formation of small blood clots inside the blood vessels throughout the body. As the small clots consume coagulation proteins and platelets, normal coagulation is disrupted and abnormal bleeding occurs (Tanos et al. 2008).

Abbreviations: MPE, acqueous extract of Mucuna pruriens seeds; gpMuc, glycoprotein from Mucuna pruriens seeds (ConA fraction); gpMuca, Ettan LC fraction; gpMucB, EttanLC/gel filtration fraction; FTIR, Fourier transform infrared; Amide I', amide I band in (2) [H.sub.2]O medium; K-PI, Kunitz-type tryspin inhibitor.

In light of the foregoing it is clear that protease inhibitors play an important role for the development of a new anti-snake drug. Plant seeds contain a large number of different types of serine protease inhibitors that are known to block trypsin and chymotrypsin from animal, fungal and bacterial origins (Powers and Harper 1986; Kennedy 1998). In particular the Kunitz-type trypsin inhibitors (K-Pl) from a variety of leguminous (Fabaceae family) have been characterized (Bode and Huber 2000; Tanaka et al. 1997; Oliva et al. 2000). Machuka (2000) demonstrated that the total protein extract from MPE inhibited trypsin and chymotrypsin activities. In our previous paper (Guerranti et al. 2004) we confirmed the data of Machuka demonstrating that the N-terminal aminoacidic sequence of gpMuc contains the consensus sequence DDREPV-DT characteristic of soybean K-PI and homologous to other K-PI (Mello et al. 2001; Haq and Khan 2003).

The presence of protease inhibitors in Mucuna pruriens suggest its role as a potential source of anti-venom drug.

In order to better understand the Mucuna pruriens short term anti-snake properties, it is fundamental to characterize structurally its protein components responsible of anti-snake activity with particular regard to gpMuc. The structural data will be an important contribution to elucidate the MPE protein components function.

In order to gain insights into the structural properties of gpMuc, this fraction was further purified to homogeneity (gpMucB) and structural experiments were performed by using FTIR and mass spectroscopy. The data show that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein with no a-helix content. All data suggest that gpMucB may belong to the K-PI family.

Materials and methods

Materials

Natural products: Mucuna pruriens seeds were collected in the Rukuba area in Jos, Nigeria with the aid of a traditional healer by the Department of Pharmacology and Clinical Pharmacy, University of Jos.

Chemicals: Ammonium sulfate, NaCl and acetonitrile were obtained from J.T. Baker; Concanavalin A was from Vector Laboratories; Bradford protein assay reagent was obtained from BIO-RAD; [MnCl.sub.2] was from Sigma; [CaCl.sub.2] was obtained from Reidel; deuterium oxide (99.9% (2) [H.sub.2]O) and (2)HC1 were purchased from Aldrich; Tris, citric acid trisodium dehydrate, methyl [alpha]-D-manno-pyranoside, [KH.sub.2][PO.sub.4], bovine serum albumin (66 kDa), carbonic anhydrase (29 kDa), cytochrome C (12.5 kDa) and trypsin were obtained from Sigma.

All other chemicals were commercially available and of the purest quality.

Columns: Superdex 75 HR 10/30, Mono [Q[TM]PC and Mono S[TM]PC 1.6/5 were purchased by GE Healthcare.

Methods

Preparation of Mucuna pruriens seed extract Sun dried seeds of Mucuna pruriens were ground to a paste of uniform consistency, 50 g of which was soaked in 100 ml [H.sub.2]0, extracted for 24 h at 4 [degrees]C, centrifuged at 10,000 X g for 20 min, and the supernatant lyophilized to a powder known as MPE which was stored at 4 [degrees]C and prepared freshly for use.

Concanavalin A affinity chromatography of MPE proteins

MPE proteins precipitated with 80% ammonium sulfate were loaded on a Concanavalin A packed column (31 ml column volume, CV) and attached to FPLC AKTA Purifier (GE Healthcare). The column was pre-equilibrated with binding buffer (10 mM Tris/HCl, pH 8, containing 0.15 M NaCl, 1 mM [MnCl.sub.2],1 mM [CaCl.sub.2] and 0.02% [NaN.sub.3]) at a constant flow rate of 0.1 ml/min. 1.5 ml of a 3.5 [micro].g/[mu].1 (5.25 mg total) solution of MPE proteins was diluted to 15 ml with binding buffer and was injected. Unbound fractions (ConA-) were washed out with 3 CVof binding buffer at a flow rate of 0.1 ml/min and collected in a single aliquot. gpMuc bound to the Concanavalin column was eluted with 3 CV of elution buffer (binding buffer containing 0.1 M[alpha]-methyl-D-manno pyranoside). Eluted fractions were automatically collected in 2 ml aliquots. During the run, [E.sub.280nm] was monitored. Both unbound and gpMuc eluted from the column were assayed for protein concentration and purity and then lyophilized.

Purification by tandem anionic-cationic exchange chromatography

gpMuc was separated using the ETTAN LC (GE Healthcare) system. This permits a tandem anionic-cationic exchange protein separation by the use of two different, connected columns. Mono Q[TM]PC 1.6/5 (anionic exchange) and Mono S[TM]PC 1.6/5 (cationic exchange), both previously equilibrated with buffer A (50mM Tris-HCl, pH 7.6). 20 [micro].g of gpMuc diluted with the same buffer were injected and eluted at a flow rate of 0.1 ml/min in both columns. Elution was achieved by the use of buffer B (50 mM Tris-HCl, 1 M NaCl, pH 7.6). During the whole run, [E.sub.280nm] was monitored. The peak of the eluted protein called gpMucA was assayed and stocked at -20 [degrees]C until use.

Purification and MW determination by gel filtration chromatography

Using Superdex 75 HR 10/30 (CV = 25ml), on a AKTA Purifier (GE Healthcare) and previously equilibrated with running buffer (10 mM [KH.sub.2][PO.sub.4]. 0.150M NaCl. pH 7), 20[micro].g of gpMucA were separated by gel filtration at a flow rate of 0.5 ml/min. For MW determination, a protein mix composed of Bovine Serum Albumin (66 kDa), carbonic anhydrase (29 kDa) and cytochrome C (12.5 kDa), was injected as standard. During the whole run, [E.sub.280nm]. [E.sub.230nm]. and [E.sub.210nm] were monitored. The peak of the eluted protein called gpMucB was assayed and stocked at -20[degrees]C until use.

SDS-PAGE

Proteins were assayed according to Bradford (1976) and separated by SDS-PAGE according to Laemmli (1970) using a 12% or 14% gel (w/v) in a Bio-Rad Mini protein III system. The sample for the non reducing SDS-PAGE was pretreated with the Laemmli buffer in the absence of [beta]-mercaptoethanol. LMW or prestained protein molecular weight markers were used for estimation of MW. Total protein detection was achieved by the silver staining protocol.

Native PAGE. For native PAGE the protein was pretreated with sample buffer 2X (20% glycerol and 0.125 M Tris-HCl, pH 8.9) in the absence of SDS and [beta]-mercaptoethanol. Total protein detection was achieved by the silver staining protocol.

Tryptic digestion MALDl-TOF MS. The gpMucB fraction and the SDS-PAGE separated bands were analysed by MALDl-TOF in the native state and after peptide mass fingerprinting as described (Hellman etal. 1995). Briefly, after detection by the mass spectrometry Coomassie staining protocol, the electrophoretic bands were excised, destained and dehydrated with acetonitrile for subsequent rehydration with trypsin solution. Tryptic digestion was carried out overnight at 37 [degrees]C. Each protein band digest (0.75 [micro].1) was spotted onto the MALDI instrument target and allowed to dry. Then 0.75 ([micro] of the instrument matrix solution [saturated solution of alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.5% (v/v) trifluoroacetic acid] was applied to the dried sample and dried again. Mass spectra were obtained using an ETTAN MALDl-TOF mass spectrometer from Amersham Biosciences (Upsala, Sweden).

The same protocol, except for the bands destaining and dehy-dratation, was followed also for the MALDl-TOF peptide mass fingerprinting analysis of a solution of gpMucB.

Preparation of samples for infrared measurements

gpMucB was studied at [p.sup.2]H 8.0 and [p.sup.2]H 4.0 using 50mM Tris/(2)HC1 (buffer A) and 25 mM Citric acid trisodiumdihydrate/(2)HCl (buffer B) buffers, respectively. Typically. 1.5 mg of lyophilized protein were dissolved in 200 [micro].1 of buffer (A) or buffer (B), and the solution was centrifuged in a 10 K Centricon micro-concentrator at 5000 X g and at 4 [degrees]C to a final volume of approximately 35 [micro].1. Then, 200 [micro].1 of buffer (A or B) were added and the solution was concentrated again. This procedure was repeated several times in order to completely hydrate the protein with the chosen buffer. During the preparation of the protein sample at [p.sup.2]H 4.0 the formation of a small pellet was observed. The infrared analysis was performed on the supernatant (35 [micro],l).

Infrared spectra

The concentrated gpMuc solution was injected into a ther-mostated Graseby Specac 20500 cell (Graseby-Specac Ltd., Orpington, Kent, UK) fitted with [CaF.sub.2] windows and a 25[micro].m Teflon spacer. FTIR spectra were recorded on a Perkin-Elmer 1760-X Fourier transform infrared spectrometer using a deuterated triglycine sulfate detector and a normal Beer-Norton apodization function.

At least 24 h before, and during data acquisition the spectrometer was continuously purged with dry air at a dew point of -70 [degrees]C. Spectra of buffer and samples were acquired at [2 cm.sup.-1] resolution under the same scanning and temperature conditions. FTIR spectra were recorded at temperatures ranging from 20 [degrees]C to 95 [degrees]C, with typical 5 [degrees]C increments. The cell was thermostated using an external bath circulator (HAAKE F3), and the actual temperature in the cell was controlled by a thermocouple placed directly onto the [CaF.sub.2] window. Spectra were processed using the SPECTRUM software from Perkin-Elmer. Correct subtraction of (2)[H.sub.2]0 was adjusted for the removal of the (2)[H.sub.2]0 bending absorption close to 1220 [cm.sup.-1] (Tanfani et al. 1997). Second derivative spectra were calculated over a 9-data point range (9 [cm.sup.-1]). The deconvoluted parameters for the amide I band were set with a gamma value of 2.5 and a smoothing length of 65.

Results

Purification and SDS-PAGE analysis of MPE protein

A complete purification scheme for MPE is reported in Fig. 1. MPE proteins precipitated by 80% ([NH.sub.4].[sub.2])[SO.sub.4] were loaded on an ConA affinity chromatography column and two peaks were obtained as shown in Fig. 1A. The first peak contains the unbound proteins whilst the second one contains the glycoproteic fraction called (gpMuc). This fraction was further purified by tandem anionic-cationic exchange (Fig. IB) and by gel filtration chromatographies (Fig. 1C); after both purificative steps a single symmetric peak was obtained called gpMucA and gpMucB, respectively, implying that the purity of these fractions is similar to that of gpMuc as confirmed by SDS-PAGE using a 12% gel (w/v) (Fig. 2A).

[FIGURE 1 OMITTED]

The gpMucB fraction was further analysed by SDS-PAGE using a 14% gel (w/v) both in reducing and not reducing conditions (Fig. 2B). These analytical conditions allowed to see that gpMucB fraction presents the same electrophoretic pathway in both conditions, and that it is characterized by three bands with apparent MW ranging from 20 to 24 kDa. Fig. 2B also shows a different profile of native gpMucB: a single large band was observed but in these conditions it is only possible to show the mobility of the protein. To calculate the MW of native gpMucB the retention time of the peak eluted from the gel filtration column was used. The analysis of gpMucB (Fig. 1C) compared with a protein standard mixture and a relative calibration curve (y = -7.168 +180.93. [r.sup.2] =0.97). indicates that this peak has a MW of about 70 kDa. This data is apparently in contrast with the MW obtained by SDS-PAGE of the same fraction and with our previous results which demonstrated that gpMuc was a glycoprotein with a MW ranging from 20 to 26 kDa (Di Patrizi et al. 2006). This observation suggests that in its native form, gpMucB is most likely a trimer.

[FIGURE 2 OMITTED]

MALDl-TOF peptide mass fingerprinting

Mass spectroscopy was used to calculate the mass of native protein eluted from gel filtration (gpMucB). Moreover, MALDl-TOF peptide-mass fingerprinting of each band obtained by 14% gel (w/v) SDS-PAGE was analysed (bands 1-3 in Fig. 2B).

The MS-spectra analysis of the fraction eluted by gel filtration shows that gpMucB is a multiform protein, as shown by two main peaks with a molecular mass of about 21 and 23 kDa (Fig. 3) and a small broad peak of about 19 kDa. Each gpMucB SDS-PAGE band (bands 1-3 in Fig. 2B) shows a similar peptide mass fingerprint demonstrating that they belong to the same protein (Table 1).

[FIGURE 3 OMITTED]

gpMucB secondary structure

The structural properties of gpMuc were analysed by FTIR spectroscopy. The infrared absorbance spectrum of a protein is characterised by several bands, but the one most used in structural studies of proteins is the amide I' band. The amide I' band is quite broad, covering a 1700-1600[cm.sup.-1] range. It consists of some component bands due to the secondary structural elements present in proteins. These elements may be highlighted by resolution enhanced (second derivative and/or deconvoluted) spectra (Byler and Susi 1986; Arrondo et al. 1993).

Fig. 4A shows the absorbance (a), deconvoluted (b) and second derivative (c) spectra of gpMucB at [p.sup.2]H 8.0 and at 20 [degrees]C. In the amide I' region, the resolution-enhanced spectra show seven bands. The most intense band is located at 1635.4[cm.sup-1], typical position for [beta]-sheet structure (Arrondo et al. 1993). The asymmetry of the 1635.4 [cm.sup.-1] band in the second derivative spectrum (c) suggests the presence of another component revealed by the shoulder located at 1642 [cm.sup.-1]. a position that may be assigned to unordered structures (Arrondo et al. 1993). The 1660 [cm.sup.-1] band is typical of turns absorption (Krimm and Bandekar 1986; Arrondo et al. 1993), whilst both turns and [beta]-sheets may absorb in the 1670-1690 [cm.sup.-1] region (Krimm and Bandekar 1986). Hence, the spectra indicate that gpMucB is an all-beta protein without a-helices content. Bands below 1620 [cm.sup.-1] may be assigned to amino acid side-chain absorption (Barth 2000). In particular, the 1582.4 and 1565.2 [cm.sup.-1] bands are due to the ionized carboxyl group of aspartic and glutamic acid, respectively whilst the 1515.1 and 1612.4 [cm.sup.-1] bands are attributed to tyrosine absorption. It must be pointed out that these two bands may be due in part to contamination of gpMucB by L-DOPA since the structure of L-DOPA is similar to the tyrosine structure (inset in Fig. 4A). Indeed, the presence of L-DOPA was always observed in all and different gpMucB preparations which appeared with a different grey intensity, depending on the "age" of seeds used for the protein extraction. The grey colour could be due to oxidation products of L-DOPA, which is bound to the protein apparently with high affinity.

[FIGURE 4 OMITTED]
Table 1 gpMucB SDS-PAGE bands (bands 1-3 in Fig. 2B) peptide mass
finger printing.

 m/z      Band number

          1  2  3

930.515   x
966.255   X  x  x
1017.367  X  x
1024.311  X
1147.592     X  X
1280.513     X  X
1307.472  X  X  x
1377.699  X  x  X
1504.724  X  x  X
1533.867  X
1591.742     X  X
1595.735  X
1630.885  X  X
1639.621  X  X  X
1717.796     x  x
1794.796        X
2158.085  X  X
2168.034        X
2199.049     X  X
2214.097     X
2215.084        X
2314.187     X  X
2380.006        X
2381.179     x
2395.205  X  X  X
2411.131     X  X
2418.057        X
2427.118     X  X
2496.264  X  X  X
2504.157  X     X
2574.247        X
2587.114     X  X
3053.186        X
3053.712  X  X


The spectrum of gpMucB at [p.sup.2]H 4.0 is similar to the spectrum obtained at [p.sup.2]H 8.0 (Fig. 4B and C). In particular, at [p.sup.2]H 4.0 the position of [beta]-sheet bands (1635.4, 1687.4 [cm.sup.-1] in second derivative spectrum at [p.sup.2]H 8.0) are shifted to higher wavenumbers (1637 and 1689.9 [cm.sup.-1], respectively). The band-shift to higher or lower wavenumbers is caused by lesser or greater (1)H/(2)H exchange of amide hydrogens, respectively (Pedone et al. 2003). Hence, this finding indicates that at acidic [p.sup.2]H the protein underwent less (1)H/(2)H exchange due to a lower accessibility of the solvent ((2)[H.sub.2]O) to the protein, suggesting in turn a more compact structure at [p.sup.2]H 4.0 than at [p.sup.2] H 8.0 (Pedone et al. 2003). The confirmation of the smaller extent of (1)H/(2)H exchange at [p.sup.2]H 4.0 comes from the higher absorption close to 1550 [cm.sup.-1] (spectral region of amide II band) at [p.sup.2]H 4.0 than at [p.sup.2]H 8.0 (Fig. 4B and C). Indeed, the amide II band intensity is a measure of the accessibility of the solvent ((2)[H.sub.2]O) to the protein since it is particularly sensitive to (1)H/(2)H exchange. In(1)H20 the amide II band maximum is located close to 1550 [cm.sup.-1] whilst in (2)[H.sub.2]O it shifts to about 1450 [cm.sup.-1]. As a consequence, proteins studied in (2)[H.sub.2]O medium display a low absorption close to 1550 [cm.sup.-1] (residual amide II band) (Pedone et al. 2005) whose value depends on the extent of (1)H/(2)H exchange. The lower the absorption close to 1550 [cm.sup.-1] the higher the (1)H/(2)H exchange. Hence, in our case, the higher intensity of the residual amide II band at [p.sub.2]H 4.0 indicates that at acidic [p.sup.2]H the protein structure is less accessible to the solvent than at [p.sup.2]H 8.0.

In the amide I' region (1700-1600 [cm.sup.-1]) the most evident difference is the higher intensity of the main [beta]-sheet band (band close to 1635 [cm.sup.-1]) in the spectrum recorded at [p.sup.2]H 4.0. A minor difference is represented by the appearance of a small band at 1622.7 [cm.sup.-1] (Fig. 4C) probably due to [beta]-strands particularly exposed to the solvent (Ausili et al. 2004), and by the absence of the 1642 [cm.sup.-1] band due to unordered structures (Fig. 4C). All these differences suggest that lowering of the [p.sup.2]H induces some conformational changes in gpMucB involving the secondary and probably the tertiary structure of the protein. However, these conformational changes are not remarkable since at [p.sup.2]H 4.0 gpMucB maintains all the characteristics of an all-beta protein as in the case of [p.sup.2]H 8.0. Other small differences between the spectra of the protein at different [p.sup.2]Hs can be observed in the 1580-1560 cm '. The lower intensities of the bands close to 1582 and 1565 [cm.sup.-1] observed at [p.sup.2]H 4.0 may be ascribed to deuteration, at least partial, of ionized carboxyl group of aspartic and glutamic acid (Barth 2000).

gpMuc thermal stability

In order to characterize further the structural properties of gpMucB the thermal stability of the protein was analysed (Fig. 5). In particular, the set of spectra (A) and (C) (left panels of Fig. 5) show that in the 20-60 [degrees]C temperature range the spectra do not undergo remarkable changes either at [p.sup.2]H 8.0 (set of spectra A) and 4.0 (set of spectra C). On the other hand, in the 55-99.5 [degrees]C temperature range the spectra undergo evident changes with the increase in temperature (set of spectra (B) and (D)). In particular, with the increase in temperature the amide I' band maximum shifts to higher wavenumbers and the amide I' bandwidth and intensity increase and decrease, respectively. Temperature-dependent changes can be observed also in the amide II region (1600-1500 [cm.sup.-1]) and in particular the increase in temperature leads to the decrease in intensity close to 1550 [cm.sup.-1]. This phenomenon is due to further (1)H/(2)H exchange induced by high temperatures as a consequence of increased molecular dynamics and protein thermal denaturation (Ausili et al. 2004).

[FIGURE 5 OMITTED]

Panels (E) and (F) summarise the temperature-induced changes in the absorbance spectra. In particular. Fig. 5E shows that gpMucB at [p.sup.2]H 4.0 (Tm = 80.3) is less thermostable than at [p.sup.2]H 8.0 (Tm = 87.5), whilst Fig. 5F shows the decrease in intensity of the residual amide II band, reflecting the temperature-dependent extent of (1)H/(2)H exchange (Ausili et al. 2004).

More detailed information on temperature-dependent spectral changes may be obtained by second derivative spectra recorded at different temperatures (Fig. 6). At [p.sup.2]H 8.0 (Fig. 6A) the main [beta]-sheet band (1635.4 [cm.sup.-1]) decreases in intensity with the increase in temperature with the greatest decrease at 85 [degrees]C (thick line). A similar situation can be observed at [p.sup.2]H 4.0 (Fig. 6B), but in this case the greatest decrease in [beta]-sheet band intensity occurs at 80 [degrees]C (thick line); these temperatures are close to the Tms obtained from graph 5E. At temperatures higher than 85 [degrees]C(at [p.sup.2]H8.0) or 80 [degrees]C(at [p.sup.2]H 4.0) the spectra are characterised by a broad band whose maximum is comprised in the 1648-1640 [cm.sup.-1] range, characteristic of unordered structures. Moreover, the spectra show two bands close to 1615and 1685 [cm.sup.-1] due to intermolecular interactions (protein aggregation) brought about by protein denaturation (Ausili et al. 2004). The two bands are more intense at [p.sup.2]H 4.0 than at [p.sup.2]H 8.0, indicating an higher propensity of the protein to aggregate at acidic [p.sup.2]H.

[FIGURE 6 OMITTED]

At [p.sup.2]H 4.0 a new band close to 1626 [cm.sup.-1] starts to appear at 90 [degrees]C. This phenomenon, not observed at [p.sup.2]H 8.0, is unusual and the position of this band is typical of [beta]-structures. Hence, the spectra suggest that at high temperature a part of unfolded polypeptide chain might assume a regular conformation. However, the 1626 [cm.sup.-1] band may represent another phenomenon. For instance, at acidic pH and high temperature the protein may undergo hydrolysis and thus the 1626 [cm.sup.-1] band could represent the absorption of a particular product of protein degradation.

The spectra indicated as (*20 [degrees]C) in panels (A) and (B) represent the spectra obtained by cooling the samples heated at 99.5 [degrees]C down to 20 [degrees]C. At [p.sup.2]H 8 (panel (A)) the spectrum shows a 1631.5 [cm.sup.-1] band due to [beta]-sheet and two bands at 1679 and 1612.8 [cm.sup.-1]. The former band may be due to [beta]-sheets and/or turns (Krimm and Bandekar 1986), whilst the latter band may be due to tyrosine (and L-DOPA) absorption (Barth 2000). However, the two bands could also be due to protein aggregation since a small absorption close to 1684 and 1614 [cm.sup.-1] was present also at 99.5 [degrees]C. In the case of the spectrum recorded at [p.sup.2]H 4.0 (panel (B)) the presence of protein aggregates is clearer since the 1683.4 and 1615 [cm.sup.-1]bands are more intense and can be clearly seen with a similar intensity and position at 99.5 [degrees]C. The spectrum ( * 20 [degrees]C) also shows the presence of the 1627 [cm.sup.-1] band that could represent absorption due to [beta]-structure and/or the absorption of a particular product of protein degradation. In synthesis, the spectra (*20 [degrees]C) indicate that the protein at [p.sup.2]H 8.0 refolds into [beta]-sheets and that it could be slightly aggregated. At [p.sup.2]H 4.0 part of gpMucB may refold into a [beta]-structure, and another part is represented by protein aggregates.

Discussion

Whilst the long-term anti-snake venom effect is based on a common epitope between gpMuc and venom [PLA.sub.2], the short-term anti-snake venom activity of MPE can be explained only with a direct action of some plant components on venom toxins since the immunological mechanism is excluded in 24 h. In a previous paper, the high heterogeneity of gpMuc was reported partially justified by a different kind of glycosilation (Guerranti et al. 2004; Di Patrizi et al. 2006). Our recent data suggest that gpMuc is more complex and that heterogeneity it is not only due to glycosilation. Here we suggest that the explanation for the short-term activity also resides in gpMuc structure.

In order to avoid protein contamination, in this work we applied different purification steps to MPE and performed detailed structural studies on the fraction purified to homogeneity (gpMucB).

Both tandem anionic-cationic exchange and gel filtration chromatograms, showed the presence of a homogeneous single symmetric peak (gpMucA, gpMucB). Moreover, these purifications do not improve or modify the electrophoretic pattern. The SDS-PAGE demonstrated that all purified fractions show more bands with apparent masses ranging from 20 to 24 kDa without any differences both in non-reducing and in reducing conditions so that the presence of disulfide bonds can be excluded. MALDl-TOF MS analysis of gpMucB shows two main protein forms having molecular weights of 21 and 23 kDa and a small component of 19 kDa, confirming previous data on gpMuc (Di Patrizi et al. 2006).

The MW analysis of native gpMucB, obtained by gel filtration, demonstrated that the homogeneous peak of gpMucB corresponds to a 70 kDa protein. This MW is three times higher than those obtained by SDS-PAGE and MALDl-TOF suggesting that the native protein is a trimer. The subunits are linked together with non-covalent bounds and separate only in the presence of SDS.

The peptide mass fingerprinting of each gpMucB SDS-PAGE band shows the presence of similar digested peptides, demonstrating that native gpMucB is a single multiform protein whose heterogeneity is due to different glycosilations, as previously hypothesized (Di Patrizi et al. 2006).

In our previous work we demonstrated that the N-terminal sequence of gpMuc protein isoforms contains the entire consensus sequence DDREPV-DT characteristic of the conserved signature of soybean K-PI also reported by Machuka (2000) and that they also share homology with other K-PI (Mello et al. 2001; Haq and Khan 2003). Literature data and the results of this work support the suggestion that gpMucB may belong to the K-PI. In particular, CATH classification of K-PI indicates these proteins as mainly beta, and in particular the analysis of their crystal structure reveals 0-3% of a-helices and 30-40% of [beta]-sheet (Dattagupta et al. 1990; De Meester et al. 1998; Song and Suh 1998; Krauchenco et al. 2003, 2004). Moreover, plants proteinases inhibitors present a great stability to heat, pH extremes, and to hydrolysis by proteases (Macedo et al. 2007). All this information is consistent with the FTIR data that indicate that gpMucB is an all-beta protein, stable at high temperature and at acidic pH (Tm = 80.3 and 87.5 at [p.sup.2]H 4.0 and 8.0, respectively).

Moreover, the papaya K-PI shows some of the structural features found in gpMucB, such as a great stability at acidic pH and at high temperature (up to 80 [degrees]C), and an extensive [beta]-sheet structure content. In addition, the FTIR analyses of gpMucB and of the papaya K-PI (Azarkan et al. 2006) show that there is a good match between the shapes of both spectra, indicating that the two proteins adopt a very similar secondary structure.

Legume storage organs such as seeds and tubers contain a number of proteinase inhibitors (Laskowski and Kato 1980) representing 1-10% of the total protein content (Ryan 1981; Pearce etal. 1982; Ussufetal. 2001). The percentage of gpMucB with respect to all MPE proteins is about 10%.

Legume proteinase inhibitors (PI) are classified into two main families: K-PI, characterized by a molecular mass of about 20 kDa and by a low cystine content and Bowman-Birk type proteinase inhibitors (BB-PI), having a molecular mass of about 8 kDa and a high cystine content. The MW of gpMucB, ranging from 20 to 24 kDa, is compatible with that of K-PI.

Moreover gpMuc glycosilation confirmed by previous studies (Di Patrizi et al. 2006) is compatible with glycosilation of plant K-PI as found in several reports (Kobayashi 2001; Kress et al. 2004; Azarkan et al. 2006; Sumikawa et al. 2006).

In conclusion, all the similarities between gpMucB and K-PI further support the idea that gpMucB contains a K-PI domain and considering that Echis carinatus venom is a complex mixture of toxins in which proteases strongly contribute to its toxicity, the K-PI activity can explain the direct anti-snake venom neutralization of MPE.

Assuming that MPE protection against venom is also exerted through a long term protection, based on anti-gpMuc antibodies cross-reacting with venom proteins, the induction of both mechanisms by the same protein is very likely related to its structural complexity. Due to oligomeric and glycosilated structure of the protein, it is supposed that the immunogenic epitope of gpMuc can coexist with the functional K-PI domain in the same or in different subunits.

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Andrea Scire (a), Fabio Tanfani (a), Enrico Bertoli (a), Emiliano Furlani (b), Hope-Onyekwere NNadozie (b), Helena Cerutti (b), Alessio Cortelazzo (b), Luca Bini (c), Roberto Guerranti (b)

(a) Dipartimento di Biochimica, Biologia, e Genetica, Universita Politecnica delte Marche, Via Ranieri, 60131 Ancona, Italy

(b) Dipartimento di Medicina Interna, Scienze Endocrino-Metaboliche e Biochimica, Universita di Siena, Via A. Mow, 53100 Siena, Italy

(c) Dipartimento di Biologia Molecolare, Universita di Siena, 53100 Siena, Italy

* Corresponding author at: Dipartimeento di Medicina Interna, Scienze Endocrino-Metaboliche e Biochimica, Universita di Siena, Via A. Moro, 53100 Siena, Italy. Tel.: +39 0577234290; fax: +39 0577234285. E-mail address: guerranti@unisi.it(R.Guerranti).

doi:10.1016/j.phymed.2011.02.004
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Author:Scire, Andrea; Tanfani, Fabio; Bertoli, Enrico; Furlani, Emiliano; NNadozie, Hope-Onyekwere; Cerutti
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUIT
Date:Jul 15, 2011
Words:7057
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