COMPLETE INDIAN SPECIFIC A TION
SUDAKAR PATENT 1
IDENTIFICATION AND PURIFICATION OF A NOVEL, POTENT GALACTOSEAMINE/GALACTOSESPECIFIC,Ca2+ DEPENDENT, HEMOLYTICLECTIN FROM FRESHWATER GASTROPOD VILLORITACYPRENOIDES

Introduction

The molluscs are a diverse phylum of coelomates that includes the familiar snails, slugs, clams, mussels, and octopus. Bivalvia (clams, oysters, mussels, etc.) are filter-feeding habits and accumulate large numbers of bacteria, which are both a source of nourishment and an immune challenge. As in other invertebrates, bivalves possess a suite of adhesion molecules that aid in self/nonself recognition and bind reversibly to carbohydrate-containing molecules of foreign cells (Mydlarz et al., 2006). Among the many weapons in the chemical arsenal of bivalves are humoral defense factors such as agglutinins (lectins), ROS, antimicrobial peptides, and lysozymal enzymes (Canesi et al. 2002). The properties of lectins and their recognition roles in bivalve host defense are well documented including mussels and oysters (Bulgakov et al. 2004, Fisher 1992). Among these lectins is the recently isolated manila clam lectin, which binds to the surface of hypnospores from Perkinsus sp., a protozoan parasite of manila clams and other bivalves (Bulgakov et al. 2004). In bivalves, multiple lysosymes are involved in self-defense against pathogenic bacteria (Olsen et al. 2003).The bivalve Chlamys islandica, an antibacterial lysosyme-like protein (chlamysn) isolated from the viscera inhibits all growth of Gram-positive and negative bacteria. Interestingly, this protein is active at cold temperatures but remains stable and active when heated (Nilsen et al. 1999). In addition, bivalve hemocytes secrete antimicrobial polypeptides; hemocytes from the clam Tapes decussates secrete a polypeptide that is toxic to Perkinsus sp. (Villalba et al. 2004). The antiviral defenses of bivalves; protease-inhibiting peptide isolated from the Pacific oyster Crassostrea gigas was effective against HIV-1 (Lee & Maruyama 1998), and oyster hemolymph was found to have broad antiviral activity against HSV, IPNV, and other viruses (Olicard et al. 2005). Responses to large-scale infections are similar throughout the animal kingdom. There exist the plasma and immunocyte-released factors lectins/agglutinins in sponges, insects, molluscs, and mammals that cause foreign particles to aggregate (Gold et al, 1974; Fisher and DiNuzzo, 1991; Koizumi et al, 1999; Ma and Kanost, 2000; Loimaranta et al, 2005; Watanabe et al, 2006; Yu et al, 2006; Gandhe et al, 2007). The immune cells from higher metazoan orders interact with and remove agglutinated bacteria (Loimaranta et al, 2005)

Materials and Methods

Collection of Animals

The clam, Villorita cyprinoides (Common Indian marsh clam) were collected by divers at approximately 3 - 7 m depth at Anchalikadavu in the tributary of Thamirabharany river near Athencode, Vilavancode taluk, Kanyakumari district, Tamil Nadu. Animal were kept in plastic tanks at the center for marine science and technology lab with a continuous supply of fresh water

Preparation of sample

The shell of the clam was cleaned with water and dried with an absorbent paper. The shell was gently cut open and dissected out the muscle and cleaned with distilled water. The dissected Clam Villorita cyprinoides muscle was homogenized in a blender and extracted with the presence of TBS buffer pH7.6 .The extract was then centrifuged at 25000 g for 1h at 4°C.The clear supernatant was dialysed against TBS buffer. After estimating the protein and testing the hemagglutinating activity, the dialysed extract was stored at -20'^C for further study

Determination of hemolytic lectin

Hemagglutination assays

Hemagglutination assay was performed using different human and animal erythrocytes. 25 aliquots of samples in TBS containing 10 mm CaCb were mixed with same volume of 1.5 % (v/v) suspension of erythrocytes in TBS. After incubation of 1h at room temperature, the extent of agglutination or hemolysis was examined visually. The hemagglutination activity was expressed as a titer, i.e., the reciprocal of the highest dilution giving detectable agglutination.

Hemolytic assay:

Hemolytic activity was determined either by visual examination of lysis of the erythrocytes under the same condition as for the Hemagglutination assay or by measurement of the absorbance of 550nm due to hemoglobin released from the erythrocytes.

Erythrocyte Agarose Diffusion test:

Haemolytic assay carried out as described by mohrig et ah, (1996). The hemolytic activity was detected using a 1mm thick agarose layer containing 1.5% erythrocytes in 0.9% NaCl wells of 4mm diameter were cutout from the agarose and filled with 5μ l of sample. After incubating the agarose gel 16 hours of room temperature clear zones of hemolysis were visible around the well. The diameter of plaques were measured and used as a degree of hemolytic assay.

Hemolytic Assay- Scanning experiments.

Photometric scans were performed with completely lysed rabbit erythrocytes (1.5% cells/ml"'; Preparation as for controls in the purified samples (Villorita cyprinoides). Absorption was scanned from 200 to 700nm with Elco UV/Visible spectrophotometer in a standard cuvette with an effective light path of 10mm

Cross Adsorption Tests

Muscle samples (300 μ l) were mixed with an equal volume of washed and packed native cow, hen, pig, goat, mouse, rabbit and human (A, B, O) RBC and incubated for 1h with frequent shaking at R.T. The suspension was centrifuged (400g, 5min, R.T.), the supernatant was collected and adsorbed for a second and third time under the same conditions. The supernatant sample was adsorbed finally and tested for hemolytic activity against all the nine RBC types.

Effect of pH and thermal stability

Clam muscle samples (500 μ,l) were dialysed against the buffers at pH ranging from 3.5 to 10, using acetate buffer, tris-HC1 and glycine NaOH. After dialysis, all the samples were finally equilibrated by dialysis against TBS-Ca. The dialysates were centrifuged and the supernatant was tested for hemolytic activity using rabbit RBC. The thermal stability of muscle lectin was examined by holding 100^1 of muscle samples for 30 min at temperature ranging from 10-80°C. All samples were centrifuged and the clear supernatant was used to determine hemolytic activity using rabbit erythrocytes.

Divalent cation Dependency and EDTA Sensitivity

The initial hemolytic activity of muscle samples (imtreated) was determined in TBS containing 10 mM CaCl2 TBS. The muscle samples (each 500 μ l) were dialyzed extensively against TBS (to test divalent cation dependency) or in TBS-EDTA (to examine EDTA sensitivity) at 15°C .The samples were dialysed against TBS-EDTA and were subsequent re- equilibrated by dialysis in TBS. After centrifugation (400g,5min,room Temperature.), the supernatant was used to determine the hemolytic activity using human and animal RBC in the presence of TBS that did or did not contain different concentration of CaCb, Mgcl2, MnCl2, HgCl2, BrCl2, MnSo4 and MgSo4 (pH7.6).

Hemolytic Inhibition Assay

Purified lectin (512 HU) was allowed to react with equal volume of several carbohydrates and glycoprotein (Sigma) solutions and incubation at room temperature for 1 hr. After incubation, 25 μ l of preprepared 1.5% rabbit erythrocyte suspension was then added to the mixture and after 1 hr incubation the hemolytic activity was examined. The results were expressed as the minimum concentration of the inhibitor required to completely terminate hemolytic activity.

Preparation of the DEAE cellulose

Commercially available DEAE cellulose was used the as anion exchange chromatography. 5g DEAE cellulose powder was taken and mix TBS buffer allowed to settle. Discard the supernatant. The slurry of the matrix washed with distilled water 5 min under agitation for two times and allowed to settle, discard supernatant. The distilled water washed matrix was allowed to wash with IN NaOH solution. Slowly add DEAE-cellulose to IN sodium hydroxide 300 ml with gentle stirrer for 30 min (pH reached to 13). Discard the sodium hydroxide solution and wash the resin with double distilled water until pH reached to pH 8.0. Remove the fine particles after some minutes settling. Then replace solution with 1 N hydrochloric acid with gentle stirrer for 30 min (pH reached to 1.0). Wash the resin with double distilled water until pH reached to 3.0. Discard the distilled water and replace it with 10x buffer (500mM tris HC1 pH 7.6) and gentle stirrer for 30 min. Discard the 10x buffer and then equilibrate the resin with 50 mM tris HC1 pH 7.6, degassed and the fines removed before the suspension of DEAE-cellulose resin was transferred into a glass column in sudden use or the equilibrated DEAE-Cellulose slurry was resuspending in buffer containing sodium azide and stores it in 2-8®C.

DEAE-cellulose column chromatography

DEAE-cellulose powder (Himedia) was swollen and pretreated in appropriate buffer. The resin was packed into a glass column (1x30 cm) to reach approximately 15 cm high, then equilibrated with 3 volumes of 50 mM tris-HC1, pH 7.6 at 6-8 °C with flow rate of 0.7-0.8 ml/min using peristaltic pump (Manipul 3, Gilson). The dialyzed crude extract was applied to a column of DEAE-cellulose column (HiMedia). Proteins were eluted with 100 ml of 50 mM tris-HC1, pH 7.6 and followed with 100 ml a stepwise gradient of 0.1-3M NaCl in 50 mM tris-HC1, pH 7.6. The column effluent was collected with fractionation, 2 ml per fraction. All fractions were monitored at 280 nm using spectrophotometer (Shimadzu, UV-1201). From elution pattern, the absorption peaks were collected and concentrated to a small volume by lyophilization. The samples were then dialyzed against 25 mM tris-HC1, pH 7.6 and again concentrated and freezed (-70°C) for storage until used.

Affinity chromatography

Sample preparation
The DEAE-Cellulose purified active fraction was used for affinity chromatography purification. The sample was dialyzed and neutralized by using cold TBS, pH7.6, overnight at 4®C. The neutralized sample was centrifuged at 10,000 rpm at 10 min at and collects supernatant and stored at -80°C.For the affinity purification, the store sample taken from the freezer and thawed and used.

Solutions in CNBr-activated fetuin Sepharose 4 Fast Flow affinity chromatography

1. CNBr activated sepharose (1 g makes ~3.5 ml) from Pharmacia ,

2. Galactose (needs to be very pure and between 1 and 4 mg/ml), dialyzed against coupling buffer

3. Coupling buffer, pH 9.0 0.1MNaHC03
0.5 M NaCl

4. Blocking buffer, pH 8.0 (make fresh just before use)
1 M ethanolamine in coupling buffer (sterile filtered) or 1 M Tris-Base, pH 9.0 in coupling buffer (sterile filtered)
1mM HC1, sterile-filtered

5. Low pH Wash
0.1 M acetic acid
0.5 M NaCl

Preparation of CNBr-activated fetuin Sepharose 4 Fast Flow
Commercially available CNBr-activated sepharose 4 Fast Flow was used for preparation of affinity matrix. 2.5g freeze dried CNBr-activated sepharose 4 Fast Flow powder was Swell CNBr Sepharose beads in 1 mM HC1 for -15 min. at room temperature. Transfer gel to sintered glass funnel and wash with 200 ml of 1 mM HC1. Stir slurry with glass rod for about 20 minutes until matrix is swollen, then apply vacuum to remove liquid. Wash matrix with 3 x 100ml 1mM HC1. Wash to dryness each time.

Coupling of Fetuin with CNBr-activated sepharose 4 Fast Flow

Coupling is done after washing of activated matrix. Then activated matrix wash with 50ml coupling buffer (Coupling buffer, pH 9.0, 0.1 M NaHC03, 0.5 M NaCl). Dilute 5 mg ligand (Fetuin) in 15ml coupling buffer. Quickly transfer activated Sepharose to a flask containing the ligand solution. Shake gently overnight at 4°C. The degree of coupling is checked by reduction of fetuin in the coupling medium and to remove 100 μ l aliquot for later analysis. Filter matrix on sintered glass funnel and collect flow through. Remove 10 μ l for gel analysis (compare to 10 μ l of starting solution to estimate binding efficiency). Keep remainder until you know whether the coupling reaction has worked. Wash matrix with 100ml coupling buffer on sintered glass filter. Transfer matrix back to flask and incubate with 50 ml blocking buffer at room temperature for two hours or 4°C overnight (Blocking buffer, pH 8.0 (make fresh just before use) 1 M ethanolamine in coupling buffer (sterile filtered).The adsorbent is washed thoroughly on a sintered glass funnel with 0.2 M NaCl and finally with distilled water. The processed CNBr- activated fetuin sepharose 4 Fast Flow is stored in cold TBS, pH 7.6, containing 0.02% sodium azide at 4®C until use.

CNBr-activated fetuin Sepharose 4 Fast Flow Affinity chromatography

2 ml of DEAE-Cellulose purified active fraction was used for affinity chromatography purification and applied to 2.5g of CNBr-activated fetuin sepharose 4 Fast Flow glass column ,previously equilibrated with TBS (pH7.6) at 4°C. After column packing, washed with TBS until attain 0.002 OD at 280 nm. This is to remove unwanted or unbinding proteins from the column, so as to obtain homogenous lectin protein. The elution was done with elution buffer containing O.IM galactose and the fractions were collected in 2ml polypropylene tubes at the rate of 0.4 ml/min. Fractions were immediately collected and stored at 4®C .the fraction were taken OD at spectrophotometer of each fraction. The high protein contented fractions were identified using hemolytic assay. After identification of active fraction, dialyzed against distilled water for overnight at 4'^C.The dialysates was the aliquot and stored at -20®C.The protein content was estimated using Lowry method.

Purification of Clam hemolytic lectin by Gel filtration chromatography using

Sephadex G-75 Preparation of Gel filtration matrix SephadexG-75

In gel filtration and gel permeation chromatography - also known as size exclusion chromatography - separation is based on differences in the size and/or shape of the analyte molecules, which governs the analytes' access to the pore volume inside the column packing particles. The exclusion limit of a size exclusion packing indicates the molecular weight, for a particular polymer type, above which analytes are fully excluded from entering the pores and thus will not be separated. 2g of Sephadex G-75 powder swell using dissolved in distilled water and stir it well, allowed to settle. Remove fines from the supernatant by suction. Then this slurry equilibrated with 50mM TBS buffer pH7.6 and stir it well, allowed to settle and decanted the supernatant by suction. The elution buffer (TBS with 50mM Tris 140mM NaCl pH7.6) is then added to the swelled gel at 4 or 5 times the settled gel volume. The Sephadex is gently stirred and allowed to settle; and fines removed by suction. Buffer addition, gel settling, and decantation are repeated three times. The equilibrated gel is kept at 2"C-4°C overnight before packing the columns.

Purification of clam lectin

A fore equilibrated, stored, swelled Sephadex G-75 gel taken from the freezer and attain the room temperature. Degassed the swelled gel and packed in the glass column (0.6 X 100cm). Sephadex gel was uniformly packed with out form air bubbles. Then the gel was equilibrated with elution buffer. After equilibration, the dialyzed affinity purified active sample was applied to the column. Elution was done with help of elution buffer and the fractions were collected 2ml propylene tubes observe the OD value of each fractions .Active fractions were identified through the hemolytic assay and protein content was estimated using lowry method.

Preparation of Villorita cyprinoides hemolytic lectin - streptokinase conjugate
Villorita cyprinoides hemolytic lectin - streptokinase conjugate was prepared by using glutaraldehyde as bifunctional cross linking reagent .Solution of VCL (18mg/ml) and streptokinase (5mg/ml) were prepared in sodium phosphate buffer(0.1M pH 7.8) containing IM NaCl and 0.003M MgC12.The VCL(500 μ l) and Streptokinase (1000 μ l) were mixed and cooled at 4°C. A cold 25% aqueous glutaraldehyde solution (20 μ l) was slowly added with constant mixing. The mixture was allowed to stand for 30min at 4''C , after which it was directly loaded on a Sephadex G-75 column (0.6X 100) of 98 ml bed volume equilibrated with sodium phosphate buffer 0. 1M(pH 605). The elution was carried out with same buffer. Flow rate was maintained at lml/8min.Fractions of 1 ml was collected. The clot lysis assay was carried out in all active fractions.

SDS-PAGE

The purity and approximate molecular mass of purified lectin was estimated by SDS- PAGE in reduced condition with a 10% running gel and in the buffer system of Laemmli (1970) using mini vertical gel electrophoresis system (GeNei™).Visualization of protein band was performed by silver staining.

Results

Hemolytic activity was observed in the muscle extract of u.v using different types of erythrocytes such as, human ABO, rabbit, goat, dog, pig, hen, turkey, squirrel and mouse table(l). The muscle extract gave the maximum hemolytic activity titre with rabbit erythrocytes. In mouse erythrocytes gave better activity as compared with other animal erythrocytes. Among human ABO erythrocytes, the human B group erythrocytes gave strong hemolytic titre and the low activity was recorded in the A and O group erythrocytes. But the sub group of human Al erythrocytes was exceptionally high lytic activity as compared with human A group erythrocytes. The turkey erythrocytes gave very low activity was detected. Based on hemolytic activity titre obtained the erythrocytes can be graded as rabbit>mouse>pig>human B>squirrel=goat=hen>dog>human 0=turkey=cow>human A as respectively.

Table 1: Identification of natural lectin from clam muscle

Table 1

Hemolytic assay (EAPT)

The hemolytic activity was assessed by using diffusion hemolytic assay the erythrocyte agarose plate assay(EAPT).In this assay the clear hemolysis was shown the agar plate. The clear hemolytic plaques (6mm in diameter) were shown for crude as well as purified lectin. These experiments clearly proved the sample was hemolytic property.

Table II. Determination of hemolytic activity

Table II
The hemolytic activity was detected using spectrophotometer assay the measurement of absorbance at 550imi due to hemoglobin released from the erythrocytes. In this assay the rabbit erythrocytes showed highest OD value in compared with other human erythrocytes. In human O group erythrocytes was vary to OD value, and in B group also high OD value as compared with human A and C er)^ocytes. The hemoglobin content was rabbit>human B>human 0>human A respectively. This shows the muscle extract contained protein with hemolytic activity.

Table III. Enzyme treated erythrocytes on hemolytic activity

Table III

Enzyme treated erythrocytes are also tested the hemolytic activity as shown in the (table 2). From this results trypsin treated rabbit and human ABO erythrocytes gave very much or significantly argumented the hemolytic titers where as neuraminidase and papain treated erythrocytes were reduced the hemolytic titer as low as half of the original activity.

Scanning experiments
The scanning of completely lysed rabbit erythrocytes from 200 - 700 revealed peaks of absorption at 238, 264, 364, 444, 542, 578 respectively (Fig ).Absorption at 264 was higher Additionally, a scan of a purified lytic lectin was showed .

Fig.
1

Photometric scan of completely lysed rabbit erythrocytes 1.5% cells/ml ' and purified hemolytic lectin from Villorita cyprinoides

Wavelength [nm]

• lysed erythrocytes

o- purified hemolytic lectin

Table IV. Effect of temperature of clam lectin

Table IV

Effect of temperature:

When the sample was incubated at different temperatures, the muscle extract of Villorita cyprinoides (table .3) as well as purified lectin was its activity was stable between 25-30°C. After 30°C, the lectin activity was gradually reduced and when the sample was measured at 62°c the total activity was abolished. So this hemolytic activity was heat sensitive.

Table V. Effect of pH:

Table V
The muscle extract of Villorita cyprinoides, the hemolytic activity was over wide range of pH (6-9.5) and the activity was high at 7.5-8.5 Table (4). The haemolytic activity was gradually reduced after pH8.5 and completely loses its activity after pH 9.5.

Table VI. Cross adsorption test:

Table VI.

Cross adsorption studies were carried out to assess whether the hemagglutination activity is due to single agglutinin or due to multiple agglutinin in the hemolymph investigated. Absorption of the samples with any one of the erythrocytes used was sufficient to remove the hemagglutination activity for other erythrocytes used. Human ABO, rabbit, cow, hen, pig and goat were used for Cross adsorption test. As shown in (table 7) adsorption of sample thrice with any one of the RBC types resulted in a complete removal of the lytic activity against that RBC type. . In this cross adsorption tests each RBC types was found to completely or partly adsorb hemolytic activity for other RBC types and the efficiency of adsorption differ among the eight RBC types tested. When the sample was adsorbed to rabbit erythrocytes, it lost only small amount of hemolytic activity against human B and 'O' and hen erythrocytes but rabbit erythrocytes lost its activity. In pig erythrocytes adsorbed with sample and cross adsorption test with other RBC tests. This is interesting to that rabbit completely lost its activity.. This shows the hemolytic lectin was highly specific to the pig erythrocytes surface carbohydrates. In other RBC types adsorbed with samples, and cross adsorption with other RBC types mostly reduced their activity.

Table VII. Metal ions:

Table VII

In this occasion, the hemolytic activity of the sample was tested with different concentration of divalent cations such as CaCl2, MgCl2, ZnCl2, MnCl2, FeCl2, HgCl2, BaCl2, MnSo4,and MgSo4 (table 5). In this experiment MgCl2 and BaCl2 gave higher activity at 50 mm and 25 mmol. The FeCbl2 and HgCl2 have no activity this means these ions were not involved dining other hemolytic activity. MnSo4 and MnCl2 recorded very low activity. The requirement of divalent cations was very much involved in the hemolytic activity. Increasing concentration MgSo4 showed the highest activity but lower activity in lower concentration. In 25mM and 12.5mM concentration of CaCl2 gave higher activity and decreasing concentration showed lower activity.

Table VHI. Cation dependency and EDTA sensitivity:

Table VHI

When the hemolytic activity of the sample was tested in the presence of varying concentration of EDTA, there was great change in hemolytic titre was observed with very low concentration of EDTA gave very low activity, but without CaCl2, the hemolytic activity was greatly reduced as compared with the presence of CaCl2. This shows that this hemolytic lectin was highly calcium dependent activity.
Table IX. Inhibition of muscle hemolytic lectin of Villorita cyprynoides by various sugars.

Table IX

The sugar specificity hemolytic lectin in the muscle of Villorita cyprinoides were assessed by hemolytic inhibitor efficiency of various sugar and glycoproteins of the 27 carbohydrates (encompassivy mono-di-oligo and polysaccharide) and glycoprotein. The hemolytic activity of the sample tested against trypsinised rabbit erythrocytes (table 7). In this experiment low inhibitory activity was recorded by the sugar arabinose at 200mm concentration. The simple hexose galactose and their amino derivative such as galactosamine and glucosamine were moderately inhibit hemolytic activity at the concentration of 25mM, 25mM and 50mM respectively. By contrast their N-acetylated derivatives such as N-acetyl galactosamine (Ga1NAc) as well as N-acetyl glucosamine (G1cNAc) did not inhibit the hemolytic activity at concentration up to 200mM. Similarly the hemolytic activity was not inhibiting the N-acetylated simple sugars such as N-acetyl neuraminic acid (Neu Ac). Such that other carbohydrate except galactose, galactosamine, glucosamine, arabinose did not inhibit even at low or high concentrations. In the case of glycoprotein such as fetuin and asialofetuin shows the inhibitory activity. The fetuin was one of the potent inhibitor of the hemolytic activity at the concentration of 0.625mg. Furthermore desialylation of these glyco proteins either slightly reduced or completely abolished the inhibitory potency. Bovine sub maxillary mucin did not inhibit any hemolytic activity even at a concentration of 10mgml"'. This result shows this hemolytic lectin shows galactose specific lectin.

Hemolytic inhibition potency of the sugars are graded as Fetuin >galactose>galactosamine > glucosamine= asialofectin> arabinose

DEAE-cellulose column chromatography purification

When the clam muscle extract of Villorita cyprinoides sample was pass through a DEAE- cellulose column. In these results, three major peaks were appeared. One major peak was in unabsorbed fraction and two were adsorbed fraction. In this chromatographic purification, the desired protein was not adsorbed m this purification matrix. In the unabsorbed fraction number 3'"'' was very high active one. In other fractions 2, 4, 5, 6,7th gave lytic property also. The remaining fractions did not showed any activity. The high active fraction was identified through hemolytic assay. The protein content was estimated and stored.

CNBr-activated fetuin Sepharose 4 Fast Flow Affinity chromatography

DEAE-cellulose isolated protein fractions were introduced into the CNBr-activated fetuin Sepharose 4 Fast Flow Affinity chromatography column. DEAE-cellulose yielded, upon affinity chromatography, gain three peaks .One major peak and two minor peak .The adsorbed fraction that is 59*'^ fraction had high hemolytic activity and 57, 58, 61, 62nd fraction also the hemolytic activity. Furthermore two minor peaks are also appeared but devoid of activity.

Purification of Clam hemolytic lectin by Gel filtration chromatography using Sephadex G-75

Affinity purified active fraction was chromatographer on gel filtration chromatography using Sephadex G-75. From this result, one major peak that was high active fraction .Fraction number 13th fraction gave hemolytic property .that fraction was analyzed in native PAGE and SDS PAGE.

Fig .3

Fig .4

Fig .5

Fig .6

Fig .7

S- Streptokinase , B-plant lectin; C- Clam lectin

Fig .8

Enzyme-lectin conjugate(STREPTOLECTIN) is found to have enhanced effect in clot lysis process, hi this experiment, the streptokinase is clot lytic enzyme and the lectin was hemolytic action. These two are well characterized and documented. Streptokinase was cross linked to lectin using glutaraldehyde and the purification was done by gel filtration chromatography using Sephadex G-75.the eluted fraction consists of the conjugate of lectin with the enzyme which is termed as (STREPTOLECTIN). In the clot lysis experiment, streptokinase activity as slow, that which require more time to lysed a clot. But hemolytic lectin, hemolytic process was very quick and it had required very short time. In the conjugated compound (STREPTOLECTIN)., that clot lytic process was very much enhanced activity as compared to the activity of streptokinase alone and it require very short time. The STREPTOLECTIN had an 309% increase (Fig. 9 and Table 10) in clot lysing activity in 10 mins as compared to normal pure commercial Streptokinase.

Fig .9 Clot lysis of STREPTOLECTIN as compared to normal Streptokinase

Table: 10. Comparision of Clot lysis by (STREPTOLECTIN, clam lectin and Streptokinase conjugate purified ).with other samples

Native PAGE

SDS-PAGE analysis

In the SDS-PAGE, the purified protein contain tow sub units,that are 42 KD a and 39 KD a

References
Mydlarz L.D., Jones E.L. and Harvell C.W. Innate Immunity, Environmental Drivers, and Disease Ecology of Marine and Freshwater Invertebrates Annu. Rev. Ecol. Evol. Syst. 2006. 37:251-88.

Canesi L, Gallo G, Gavioli M, Pruzzo C. 2002. Bacteria-hemocytes interactions and phagocytosis in marine bivalves. Micros. Res. Tech. 57:469-76.

Bulgakov AA, Park KI, Choi KS, Lim HK, Cho M. 2004. Purification and characterization of a lectin isolated from the Manila clam Rudi tapes philippinarum in Korea. Fish Shellfish Immunol. 16:487-99.

Fisher W S. 1992. Occurrence of agglutinin in the pallial cavity mucus of oysters. J. Exp. Mar. Biol 162:1-13. / / .

Olsen OM, Nilsen IW, Sletten K, Mymes B. 2003. Multiple invertebrate lysozymes in blue mussel (Mytilus edulis). Comp. Biochem. Physiol. B 136:107-15.

Nilsen IW, 0verb0 K, Sandsdalen E, Sandaker E, Sletten K, Mymes B. 1999. Protein purification and gene isolation of chlamysin, a cold-active lysozyme-like enzyme with antibacterial activity. FEBS Lett. 464:153-58

Villalba A, Reece KS, OrdasMC,Casas SM, Figueras A. 2004. Perkinsosis in molluscs A review. Aquat. Living Resour. 17:411-32

Lee TG, and Maruyama S. 1998. Isolation of HIV-1 protease inhibiting peptides from the thermolysin hydrolysate of oyster proteins. Biochem. Biophys. Res. Commun. 253:604-8.

Olicard C, Renauh T, Torhy C, Benmansour A, Bourgougnon N. 2005. Putative antiviral activity in hemolymph from adult Pacific oysters, Crassostrea gigas. Antivir.Res. 66:147-52.

Claims

I claim that:

1. A novel Haemolytic Lectin from Villorita Cyprynoides wherein purified lectin showed single band on Native PAGE and two subunits on the SDS-PAGE of 42 KD a and 39 KD a.

2. The method of claim 1 wherein Lectin from the hemolymph of Villorita Cyprynoides was purified by Ammonium sulphate precipitation, Affinity chromatography, CNBr- activated fetuin Sepharose 4 Fast Flow Affinity chromatography and gel filtration chromatography. The purity was confirmed with RP-HPLC.

3. The method of claim 1 wherein Hemolytic inhibition potency of the sugars are graded as Fetuin >galactose>galactosamine > glucosamine> asialofectin> arabinose
4. The method of claim 1 wherein Affinity purified active fraction was chromatographed on gel filtration chromatography using Sephadex G-75. From this result, one major peak that was high active fraction .Fraction number 13th fraction gave hemolytic property .that fraction was analyzed in native PAGE and SDS PAGE.

5. The method of claim 1 wherein A novel therapeutic drug for human clot lysis was invented by Enzyme-lectin conjugate(STREPTOLECTIN) and is found to have enhanced effect in clot lysis process.

6. The method of claim 1 wherein Streptokinase was cross linked to lectin using glutaraldehyde and the purification was done by gel filtration chromatography using Sephadex G-75.the eluted fraction consists of the conjugate of lectin with the enzyme which is termed as STREPTOLECTIN).

7. The method of claim 1 wherein In the conjugated compound (STREPTOLECTIN)., that clot lytic process was very much enhanced activity as compared to the activity of streptokinase alone and it require very short time.

8. The method of claim 1 wherein The STREPTOLECTIN had an 309% increase in clot lysing activity in 10 mins as compared to normal pure commercial Streptokinase.

9. The method of claim 1 wherein any lectin conjugated to Streptokinase or related enzyme for human clot lysis, treatment for stroke, stroke related diseases, heart attack treatment.

10. The method of claim 1 wherein the application of this lectin in medical diagnostics, medical therapy, clot lysis, stroke, medical drug, fertility control, pest control, biosensor, microbial control, biomedical tool etc