LECTIN AND METHOD FOR THE EXTRACTION THEREOF FROM SCALLOP

Abstract: A method for manufacturing scallop lectin, which comprises: a) extracting a lectin-containing product from the digestive glands of a scallop selected from the family Pectinidae and the genus Haliotis, and b) purifying the resulting lectin-containing extract; and a lectin-haemagglutinin extracted from the digestive gland of a scallop selected from the family Pectinidae and the genus Haliotis, such as Placopecten magellanicus and possessing activity against Gram-negative bacteria, characterized by: a) a molecular weight of from about 10.000 to 20.000 under denaturing conditions; b) an ability to agglutinate sheep erythrocytes; c) an ability to agglutinate picoplankton of the species Spinobacter and Synechococcus; d) marked agglutination ability with respect to Gram-negative bacteria of the species Agrobacterium tumefaciens, Escherichia coli and Serratia marcescens; e) an isoelectric point in the range of about pH 4-5; and f) resistance to the proteolytic enzymes proteinase K, pronase and trypsin.

Description: LECTIN AND METHOD FOR THE EXTRACTION THEREOF FROM SCALLOP This invention relates to a method of extracting scal¬ lop lectin from the digestive glands of scallops of the fami¬ ly Pectmidae . in particular the sea scallop, Placopecten maqellanicus . and to the use of the product in agglutina ion and separation procedures. Lectins are carbohydrate-binding substances which do not exhibit enzymic activity nor are they produced as a re¬ sult of specific lmmunological response. Lectins can aggluti¬ nate a variety of cell types and this agglutination may be inhibited by specific carbohydrates. A variety of microorga- msms, plants, invertebrates and vertebrates possess lectins, indicating wide occurrence. Among invertebrates, lectins have been found in the haemolymph of Octopus . Limulus . Carcmosco- pius . Tridacna . Aplvsia . Tachypleus , Chelicerata and Dolabel- la . Other tissues may contain lectins, for example, gonads of Aplvsia . the albumen gland of Helix , eggs of Anthocidaris and tentacles of Cerianthus . Various functions have been ascribed to lectins. Haemo¬ lymph lectins may function in a defence mechanism against pathogens, the albumen gland lectin of Helix may protect eggs, the sea urchin lectin may be involved in fertilization and the Aplysla lectin may bind fertilized eggs to the cap¬ sule. Lectin-binding specificity varies. For example the agg- lutinms of Limulus and Carc noscorpius bind to 2-keto-3- -deoxy-octonic acid, a specific component of Gram negative bacteria. The haemolymph lectin of A 1vsI binds to material containing N-acetyl ammo sugars . In particular, at the present time horseshoe crab (Li¬ mulus ) lectin is employed for example m determining Gram negative bacterial presence in clinical samples and for the measurement of the bacterial endotoxm content of a variety of materla1s . However, use of this l.ectin is subject to several draw¬ backs and limitations, including the heat lability of the product and its susceptibility to degradation by proteolytic enzymes, and furthermore, the limited availability and high current cost of extracting Limulus lectin. It is an object of the present invention to provide a method of extracting a useful lectin product which avoids the above-mentioned disadvantages. Accordingly, one aspect of the invention provides a method of extracting scallop lectin, which comprises: a) extracting a lectin-containing product from the diges¬ tive glands of a scallop selected from the family Pectinidae and the genus Haliotis . for example the sea scallop, Placo¬ pecten ma ellanicus . for example by treatment with Tris buf- fer, and b) purifying the resulting lectin-containing extract. The purification can be effected by a variety of me¬ thods, including a) heat treatment followed by ammonium sulfate precipita- tion, gel chromatography, chromatofocussing and chromato¬ graphy on hydroxylapatite; b) heat treatment, ammonium sulfate precipitation, gel chromatography and isoelectric focussing; and c) heat treatment, ammonium sulfate precipitation and af- finity chromatography. Other combinations of these techniques are also feasible. Another aspect of the invention provides a lectin-hae- magglutinin extracted from the digestive gland of a scallop selected from the family Pectinidae and the genus Haliotis . such as Placopecten magellanicus , and possessing activity against Gram—negative bacteria, characterized by: a) a molecular weight under denaturing conditions of from about 10.000 to 20.000, particularly about 14.000; b) an ability to agglutinate sheep erythrocytes; c) an ability to agglutinate picoplankton of the species Spinobac er and Synechococcus : d) marked ability to agglutinate Gram negative bacteria particularly those having accessible surface endotoxm, of the species Agrobacterium tumefaciens . Escherichia col and Serratla marceseens : e) an isoelectric point in the range of about pH 4-5, in particular about 4.52; f) an ammo acid composition resembling that given in the following Table A; and g) resistance to the proteolytic enzymes proteinase K, pronase and trypsm. Thus, in accordance with the method of the invention, scallop lectin is extracted from the digestive gland of e.g. Placopecten maαellamcus . The yield of lectin is generally approximately 20 mg from thirty large scallop digestive glands having a wet weight of about one pound. Although di¬ gestive glands are the prime source of the lectin, other tis¬ sues may also be .extracted and yield lectin. Digestive glands are potentially available from the scallop fishery although at present they are discarded into the sea. Approximately 5.000 tons of scallop muscles are har¬ vested annually in Eastern Canada and a similar quantity of digestive glands would be available for extraction. The po¬ tential availability of scallop lectin is 200 kg per annum. The current price of horseshoe crab (Limulus) lectin which has similar specificity is about $100 per mg . In a preferred embodiment of the invention, the lectin can be extracted from the scallop digestive glands by homo¬ genizing the glands in Tris buffer containing sodium chloride or Tris buffer supplemented with n-ammocaproic acid, EDTA, benzamidme, dithiothreitol , phenylmethylsulfony1 fluoride, sodium azide and sodium chloride. n-Aminocaproic acid, EDTA, benzamid e, and phenyl ethylsulfony1 fluoride are added to lessen degradation by digestive gland enzymes while sodium azide is added to inhibit microbial growth. The digestive glands were homogenized to effect the desired extraction. The lectin can be purified from the extract by various procedures, including classical protein techniques and affi- nity chromatography. Using standard protein purification procedures, namely, heat treatment, ammonium sulfate precipitation, gel chroma¬ tography, chromatofocussing (or isoelectric focussing) and chromatography on hydroxylapatite, a lectin preparation can be obtained which gave a single band following SDS-PAGE elec- trophoresis. Comparison with markers of known molecular weight indicated the lectin had a molecular weight of about 14.000. PAGE electrophoresis under non-denaturing conditions as well as gel chromatography suggest that the lectin is com¬ posed of subunits which may assemble into aggregates of higher molecular weight. The purified lectin can be labelled 14.- by reductive methyla ion using radioactive formaldehyde and sodium cyanoborohydride as the reductant (Jentoft and Dearborn, 1979) . Electrophoresis (SDS-PAGE) gives a single band of radioactivity with a molecular weight of 14.400. Alternatively, the lectin can be purified from the ex¬ tract by affinity purification. This procedure is more suited for commercial production as the procedure permits rapid and specific purification of the lectin. A suitable ligand con¬ tains 2-keto-3-deoxy-octonate (KDO) or sialic acid in poly¬ meric form. The ligand used for affinity chromatography is obtained by trifluoroacetolysis of lipopolysaccharide (LPS) from a heptoseless mutant of E .coli . The LPS can be extracted by the method of Darveau and Hancock (1983) or Galanos, Lude- ritz and Westphal, European J. Biochem. 9.: 245-249 (1959) . The latter procedure is simpler and for this reason is pre¬ ferred. Electrophoresis of LPS preparations indicated the LPS preparation contained a single band of high mobility as de- tected by silver staining. Tri luoroacetolysis of the LPS preparation yielded a pentasaccharide consisting of 3 mole¬ cules of KDO (2—k.eto-3-deoxy-octonate) and 2 molecules of glucosamine. The pentasaccharide is purified by extraction with ether, deacetylation (ammonium hydroxide in methanol, IK, 1 hour, 20°C), chromatography on Sephadex G-25 and HPLC using a column of porasil and ethyl acetate: acetic acid: water (3:1:1) as solvent. The free amino group of glucosam e is the reactive group used to attach the ligand (KDO) to af- figel 15 (Biorad Laboratories Ltd.) . The extract of the di¬ gestive glands was heated (100°C for 1 hour), cooled to 2°C and centrifuged OOOOxg, 20 min) . Ammonium sulfate was added to the supernatant fluid and proteins precipitated between 40-60% saturation collected by centrifugation OOOOxg, 20 mm) . The protein fraction was dissolved in distilled water, dialyzed and freeze dried. An aliquot (lg) was dissolved in Tris buffer (0.02 M, pH 7.6, 10 ml) containing sodium chlo- ride (0.9%) and sodium azide (0.02%) . The solution was passed through a KDO ligand-af igel column (1.5 x 15 cm, 1.6 mg KDO) at a flow rate of 10 ml/h. The column was washed with Tris buffer (50 ml) and the bound lectin was eluted with Tris buf¬ fer (5 ml) containing the KDO-glucosam e pentasaccharide (4mg) followed by washing with Tris buffer (20 ml) . Protein- -contammg fractions were pooled and placed in an Amicon Ultraflltration cell equipped with a UM 10 filter. The penta¬ saccharide was recovered by elut g the ultraflltration cell with distilled water and chromatographmg the KDO-containing sugars in the filtrate on Sephadex G-25. The lectin was reco¬ vered from the ultrafiltration cell by freeze drying. Ammo acid analysis carried out on samples of the scal¬ lop lectin gave the following results: TABLE 1 Ammo acid composition of P .Maσellanicus lectin. Kolar ratio Amino acid Analysis 12 Analysis 11^ Alanine 9.5 9.5 Arginine 2.4 2.4 Aspartic acid 7.4 8.6 Cysteic acid — 2.2 G Glluuttaammiicc aacciidd 7 7..44 7.3 Glycine 8.7 13.5 Histidine 2.0 2.0 Isoleucine 6.0 5.0 Leucine 6.0 6.0 L Lyyssiinnee 3 3..22 3.3 Methionine 1.0 0.4 Phfenylanine 2.9 2. ' 7 Proline 4.6 3.9 Serine 10.7 9.4 T Thhrreeoonniinnee 2 200..55 17.4 Tyrosine 3.0 i.l Valine 4.5 5.4 * ■ Several unidentified peaks were detected n each analysis. 2 Lectin was hydrolyzed by 6N HC1 at 100°C for 24 hours. 3 Lectin was oxidized with performic acid, then hydrolyzed by 6N HC1 at 100°C for 48 hours. The scallop lectin will agglutinate most Gram negative bacteria indicating that the lectin recognizes a cell surface feature common to most Gram negative bacteria as illustrated by the following Table 2. TABLE 2 Cell type Specific activity % lectin (un11s/mg) bound 2 I Erythrocytes - sheep 512 2 II Bacteria A. Picoplankton Spinobacter sp, 32 Synechococcus bacillaris 64 Synechococcus sp.(WH 7803) Synechococcus sp.(WH 7805) 32 B.Gram positive Bacillus cereus Bacillus megaterium Bacillus subtilis " 16 Brevibacteπum linens Staphylococcus aureus C.Gram negative Aeromonas hydrophila Aeromonas punctata 2 Agrobacterium tumefaciens 32 Chromobacterium violacem 4 Citrobacter freundn 2 15 Enterobacter aerogenes 8 9 Erwinia amulovora 4 21 Escherichia coli B 128 Escherichia coli(heptoseless )64 57 Klebsiella pneumomae 4 Proteus vulgaris 8 Providentia alcalifaciens 4 Pseudomonas aerogenosa 8 Pseudomonas fluorescens 2 6 Serratia marcescens 64 cont . Table 2 Shigella flexneri 4 Vibrio anguillaru 4 Yersinia enterocolitica 4 Zymomonas mobills - III Phytoplankton Isochrysis glabana ND 41 Chaetoceros calagrans ND 67 Chaetoceros gracilis ND 18 - = no agglutination ND = not determined 2 Phytoplankton have a pronounced tendency to self aggluti¬ nate, therefore ^C-labelled lectin was used to demonstrate binding of lectin to phytoplankton. Such binding could be attributable to epiphytic bacteria. Gram negative bacteria which were agglutinated by the least amount of lectin (highest specific activity) were either rough strains, e.g. heptoseless E.coli . or had compro¬ mised surfaces, e.g. E. coli B, indicating the recognition site of the lectin lies close to the cell surface and may be masted by cell surface components. This assists in explaining the variation in specific activity obtained with different Gram negative bacteria. Two Gram negative bacteria not agglu¬ tinated by scallop lectin were Aeromonas hydrophila and Zymo¬ monas mobilis . These bacteria have LPS which does not contain KDO, suggesting the feature of LPS recognized by scallop lec- tin contains KDO. This was confirmed by affinity chromato¬ graphy as more than 70% of the labelled (^ 4 C) lectin bound to an affinity column which had a KDO-containing pentasaccharide as the ligand. A portion of the bound lectin (29%) could be eluted from the affinity column with pentasaccharide from LPS of heptoseless E. coli . Monomeric KDO was unable to elute any bound lectin under the same conditions that KDO-containing pentasaccharide was able to elute bound lectin. This suggests the lectin binding site may consist of two or more KDO sugar residues. Bacterial LPS contains three KDO residues. Scallop lectin agglutinates sheep erythrocytes as well as human erythrocytes from Groups 0, A, B and AB. Fetuin, thyroglobulm and human acid glycoprotein all contain sialic acid (N-acetylneurammic acid) and were able to block agglu¬ tination of sheep erythrocytes by scallop lectin. Proteins which do not contain sialic acid were unable to block agglu¬ tination of sheep erythrocytes. Sialic acid is similar in structure to KDO. Sialic acid-contammg material could elute bound lectin from the affinity column described above, howe¬ ver, the small quantity of lectin eluted suggests the lectin has a much higher affinity for the KDO-containing penta¬ saccharide than for sialic ac d. The scallop lectin of the present invention possesses several unique features of advantage, including firstly heat stability. For example, agglu ination titer was not dimmish- ed by heating a solution of the lectin for 1 hour at 100°C. Limulus lectin, which is presently used for some of the pur- poses anticipated for scallop lectin, is heat labile and once hydrated its activity decreases rapidly. Another feature of the scallop lectin of the invention is its resistance to proteolytic enzymes. For example, pro- te ase K, pronase and trypsin were unable to dimmish the titer of a lectin preparation during overnight incubation thereof with each of the above proteolytic enzymes. This att¬ ribute probably arises from the anatomical location of the lectin, namely, the digestive gland of the scallop, which presumably would contain proteolytic enzymes. Resistance to proteolytic enzymes increases the long term stability of the lectin and may permit unique applications. The specificity of scallop lectin appears to be unique in requiring at least two adjacent KDO residues for binding. Limulus lectin also binds to KDO or sialic-contaimng ate- rials and the degree to which Lim lus lectin and scallop lec¬ tin have similar binding sites is not at present full-y known. A number of other lectins which recognize sialic acid-contai- ning materials are known, although Limulus lectin appears to be the only lectin commercially available. The scallop lectin of the invention is useful in agglu¬ tinating Gram negative bacteria for affinity purification of lipopolysaccharide from Gram negative bacteria, removal of bacterial endotoxin from biological material, for example injectable pharmaceuticals, and for chromatography of sialic acid-containing glycoproteins and polysaccharides . The following are potential specific applications of the scallop lectin of the invention. 1) Affinity purification of LPS from most Gram negative bacteria is possible using scallop lectin to yield highly purified LPS preparations. In many cases, the antigenicity or serotype of a Gram negative bacterium is determined by the nature of its LPS. Affinity purification of LPS could provide very pure antigens for screening monoclonal antibodies for diagnostic purposes or to act as an€igens for ligands for antibody production/purification of known LPS specificity. For example, scallop lectin purified by affinity chro- matography is capable of recognizing LPS extracted from the inner core of E. coli (heptoseless rough mutant, 5162) . The inner core region of this Gram negative bacterium contains 2-keto-3-deoxy-octonate (KDO) which is recognized by the scallop lectin if the KDO is in polymeric form. 2) Scallop lectin recognizes sialic acid-containing glycoproteins and, consequently, scallop lectin could be used for affinity purification of sialic acid containing glycopro¬ teins . 3) Scallop lectin may be used to remove LPS from vacci- ne preparations or other injectable pharmaceuticals. The li- pid A moiety of LPS elicits many biological activities in mammals. Consequently in recognition of these toxic proper¬ ties LPS is also called endotoxin. The prefix "endo" indica¬ tes it is attached to the bacterial surface. Many vaccine preparations contain endotoxin and it is desirable to remove endotoxin from any injectable pharmaceutical. One commercial ® product available for this purpose is Detoxi-Gel . Scallop lectin attached to a suitable supporting gel would serve the same purpose but would have the advantage of increased speci¬ ficity. Non-specific binding of other components besides en- CD dotoxin may sometimes be a problem with Detoxi-Gel , particu- larly when protein is present. 4) The scallop lectin may also find utility in measure¬ ment of endotoxin levels in injectable pharmaceuticals and foods, which is presently accomplished using Limulus amebo- cyte lysate (LAD assay. While the above disclosure is primarily concerned with extraction of lectin from the sea scallop, P.. mage1lameus , useful lectms can also be extracted and employed from other molluscs of the family Pectinidae . for example Chlamvs slan- dica . Chlamys rubida and Arctica islandic . The following Table 3 shows the activity of several other molluscs of inte¬ rest . TABLE 3 Lectin activity of representative molluscs in addition to Placopecten magellamcus . NAME Group Scientific Common Lectin Heat Inhibi- activi resis- ted by ty 1 (u- tant 2 LPS 3 Scallops Argooeeten Bay seal- 1 Yes Yes rradians lop Chl mys Iceland 4 Yes Yes lslandica scallop Chlamvs - 1 Yes Yes hostata cont. Table 3 Ch1amvs Yes Yes rubida Crassadoma Big seal- Yes Yes gigantea lop Clams , Mvtilus Califor- 1 Yes Yes musse1s Californi- nia and cus mussel oysters Spisula Stimpson's 2 Yes Yes polvurvma surf clam Cvstodria Porpellor Yes Yes syllogua clam Arc ica Ocean Yes Yes islandica quahaug Crassostrea Japanese Yes Yes iqas oyster Limpets Lucapina so . Key hole Yes Yes limpet Snails Moon snail Yes Yes In contrast to the results given in Example 1 for ex¬ traction of the digestive gland of P .magellanicus . the lectin activity is based on extraction of the whole animal by the method used to extract P .magellanicus . Lectin activity was measured after heating the extract to 100°C for 30 minutes. Lectin activity was tested in the presence of E . coli LPS (serotype 055) . Of the molluscs extracted other than P.. magellanicus the lectin from Chlamvs slandica was studied most intensive¬ ly. C . slandica lectin was similar to lectin from P .magella- mcus with respect to heat resistance, resistance to proteo¬ lytic enzymes, agglutination blockage by bacterial LPS and sialic acid-contammg glycoproteins. Methods used to purify P .magellanicus lectin gave similar results when applied to C . islandica lectin. The following Examples illustrate the invention. EXAMPLE 1 Approximately thirty sea scallops having shell diame¬ ters in the range of 10-20 cm were obtained from Mushaboom, Nova Scotia ( 44°51 ' ; 62°34 ' ) by divers. The live scallops were kept m sea water at 2-6°C. Within 24 hours their digestive , glands were removed and homogenized .in Tris buffer (0.02 M, pH7.6 , 500 ml) supplemented with n-aminocaproic acid (0.01 M), EDTA (0.005 M) , benzamidme (0.005 M) , dithiothreitol (0.001 M) , phenylmethylsulfonyl fluoride (0.001 M) , and so- dium azide (0.02%) and sodium chloride (0.9%) using a Poly- tron ultrasonic-mechanical homogenizer for 2 minutes. The homogenized extract was centrifuged (lO.OOOxg, 60 minutes) and the supernatant fluid was dialyzed against distilled water at 4°C for 24 hours. The distilled water was changed twice during the 24 hour period. The dialyzed extract was freeze dried and assayed for agglutination activity. In order to obtain a purified lectin product, the ex¬ tract was dissolved in saline then heated to 90-100°C using a boiling water bath and kept at that temperature for 1 hour. Insoluble material was removed by centrifugation (lO.OOOxg, 60 minutes) and the supernatant fluid was dialyzed against distilled water at 4°C for 24 hours then freeze dried. The dried extract was dissolved in 200 ml of distilled water and any insoluble material removed by centrifugation (lO.OOOxg, 30 minutes). Ammonium sulfate was added to the supernatant fluid m increments of 20% from 0 to 100% saturation. The precipitates which formed at each 20% increment were removed by centrifugation (lO.OOOxg, 60 minutes) . The five precipi¬ tates were dialyzed against distilled water at 4°C for 24 hours with frequent changes of distilled water, freeze dried and assayed for lectin activity. The most active fraction (40-60% ammonium sulfate satu¬ ration) was chromatographed on Fractogel 65F (2.5x42 cm) de¬ veloped with the same buffer used for lectin extraction at a flow rate of 50 ml/h. 10 ml fractions were collected and tes¬ ted for lectin activity. Active fractions were pooled, dia- lyzed against distilled water at 4°C for 24 hours and freeze dried. The dried material was chromato ocussed on an ion ex¬ change PBE 96 column (1.6x20 cm) equilibrated with imidazole buffer (0.025M, pH7.2) supplemented with n-aminocaproic acid (0.01M), EDTA (0.005 M) and sodium azide (0.02%). The lectin was eluted with ^ a mixture of imidazole buffer and polybuffer 74 (Pharmacia Ltd.) in a ratio of 8:1 at pH 3.9 and 5 ml fractions were collected and tested 'for activity. The pH and absorbance at 280 nm of selected fractions was also determi- ned. Active fractions were pooled, dialyzed and freeze dried. Analysis of the freeze dried material by gel electro¬ phoresis indicated the presence of low molecular weight ma¬ terial suggesting contamination by polybuffer 74. To further purify the lectin, the material was dissolved in potassium phosphate buffer (0.5 M, pH 7,5) supplemented with sodium azide (0.02%) and chromatographed on a hydroxylapatite column (1.5 x 16 cm) developed with a step gradient of phosphate buffer from 0.01 M to 0.5 M. Active fractions were pooled, dialyzed against distilled water and freeze dried. Purification by chromatofocussing followed by chroma¬ tography on hydroxylapatite may be replaced by isoelectric focussing. For isoelectric focussing, the density gradient was formed with sucrose and ampholyte (pH 4-6.5, Pharmacia Ltd) was used at a concentration of 1.5% in a total volume of 440 ml. Triton X-IOCT was added to the light and dense solu¬ tions to give a final concentration of 20% CV/"V) . The freeze dried lectin-containing material (600 mg) from gel chromato- graphy was dissolved in the light solution (215 ml) . After filling the column, the voltage was adjusted to keep the pow¬ er input at 6 Watts. After 36 hours at 5°C, focussing was terminated and the column emptied in 10 ml fractions. Frac- tions containing precipitated protein were centrifuged and the pellet washed four times with buffer at the same pH as the fraction. Each protein fraction was tested for agglutina¬ tion activity and active fractions were pooled. The increase in specific activity for the lectin sample at each stage of purification is evident from the results given in the following Table 4. TABLE 4 Purification Weight Speci- Total Recovery Fold step (mg) fie ac- acti- (%) puri- tivity vity . fica- (u ts/ (units, tion mg) xlO 3 ) Crude extract 1470 512 752 - 1 Heat treatment 1308 512 669 89 1 (NH 4 ) 2 S04 pre¬ 550 1,024 563 75 2 cipitation Fractogel 65F 310 2,048 634 84 4 Chromatofo- 18 18, 125 326 41 35 cussing hyd- roxylapat te Polyacrylamide gel electrophoreses (PAGE) was carried out using a gradient gel (PAA 4/30, Pharmacia Ltd.) equil - brated with buffer containing Tris (0.09 M) , boric acid (0.08 M), Na 2 EDTA (0.93g/L) at pH 8.4. An electrical potential of 300 volts was applied for 10 minutes then the voltage was reduced to 150 volts for 16 hours. Gels were stained with Coomassie blue (0.02%) in acetic acid (7%, 2 hours) or PAS stain for carbohydrate. In the latter procedure, parallel gels were treated with aqueous periodic acid, Schiff's rea¬ gent then desta ed with 1 M HC1 containing potassium meta- bisulphite C5%) . SDS-PAGE electrophoresis of proteins was carried out using a gradient gel CPAA 4/30, Pharmacia Ltd.) equilibrated with buffer containing Tris (0.04 M) , sodium acetate (0.02 M) EDTA C2mM) and SDS (0.2%) at pH 7.4. Samples were heated C100°C, 10 minutes) in Tris buffer (lOmM, pH 8.0), EDTA (1 mM) with SDS (2.5%) and mercaptoethanol (5%) and applied to the gel. An electrical potential of 300 volts was applied for 10 minutes then the voltage was reduced to 150 volts. The mobility of bromo phenol blue tracker dye was observed and the electrical field was applied until the dye was at the edge of the gel. Proteins were fixed and SDS removed electro- phoretically at 24 volts for 30 minutes in 25% isopropanol, containing 10% acetic acid. Gels were stained with Coomassie blue (0.02%) in acetic acid (7%) overnight. Agglutination .of two cell types,, sheep erythrocytes and S .bacillaris. was used to determine specific activity and to assess activity in fractions obtained during purification. Both cell types gave similar results but only those results obtained using sheep erythrocytes are reported. As indicated in Table 2, the crude extract of the digestive gland had a specific activity of 512 units/mg- which did not decrease on heating, indicating the lectin was heat stable. Ammonium sul¬ fate precipitation increased the specific activity two-fold and chromatography on Fractogel 65F indicated that the lectin eluted near the void volume indicating a high apparent mole¬ cular weight as illustrated in the accompanying Figure 1. Chromato ocussing indicated that the lectin was eluted at pH 3.8-4.2 as seen from Figure 2.. This step substantially in- creased specific activity, however, gel electrophoresis de¬ tected the presence of contaminating- material of high mobi¬ lity which probably originated from the polybuffer used for chromatofocussing. This was removed by chromatography on hyd¬ roxylapatite as shown by Figure 3. The final product had a specific activity of 18 x 10 3 units/mg which represented a 35-fold purification. Gel electrophoresis with and without SDS indicated the presence of one major diffuse band. The bands were also stai¬ ned by the PAS procedure indicating the presence of carbohyd¬ rate. Gel electrophoresis without SDS demonstrated the pre- sence of high molecular weight aggregates. The molecular weight of these aggregates decreased to about 14.000 the presence of SDS. Replacing the chromatofocussing purification step with isoelectric focussing resulted in a 64-fold purifi¬ cation of the final product and indicated the lectin had an isoelectric point of 4.52 using ampholytes in the range 4-6.5. EXAMPLE 2 The anatomical distribution of the purified lectin ob- tamed in Example 1 was assessed by two methods. Firstly, the tissues were exposed to fluorescem isothiocyajate labelled E. coli LPS (1 mg/ml, 10 minutes, serotype 055:5B, Sigma Che¬ mical Co.) and unbound LPS was removed by repeated washing with saline. The tissue was examined by fluorescent micro- scopy (Wild Leitz Dialux 20 EB microscope, excitation 450 490 nm, beam splitter 510 nm, and barrier filter 515 nm) . The control tissue was not exposed to fluorescent LPS but other¬ wise received identical treatment. Secondly, tissues were extracted as previously descri- bed and the extracts analyzed for lectin activity. Haemocytes were collected from haemolymph by centrifugation (5.000xg, 10 minutes) and washed with saline. The washed haemocytes were suspended in distilled water, cooled to 0°C in an ice bath and treated for 30 seconds with a Branson cell disruptor Mo- del 200 at 15 Watts. Cell debris was removed by centrifuga¬ tion (5,000xg, 10 minutes) and the agglutination activity of the supernatant fluid measured. The protein content of the supernatant fluid was determined by the method of Warburg and Christian (1941) . Fluorescent microscopy demonstrated that the digestive gland, lamella and mantle bound fluorescent LPS with similar intensity. Other tissues bound LPS less well and the muscle did not bind LPS at all. Tissue extraction verified the pre¬ sence of lectin in the digestive gland and mantle,. Extracts of other tissues had no lectin activity although, in some cases, the tissue could bind fluorescent LPS. This may indi- cate the presence of lectin in these tissues in a form not extractable by Tris buffer. Disruption of haemocytes released hae agglutination activity indicating that, in addition to the digestive gland and mantle, haemocytes also contained extractable lectin activity. The results are shown in the following Table 5. TABLE 5 Anatomical location of the lectin Tissue Method 1 Fluorescent LPS 2 Extraction 3 Digestive gland +++ 512 Labial palps + Gonads ++ Lamella +++ Mantle +++ 8 Haemocytes N.D. 210 Foot -t- Heart ++ Muscle - Crystalline style ++ Hind gut N.D. Plasma N.D. 1 N.D. = not determined; - = no fluorescence or no aggluti¬ nation. 2 Controls showed no fluorescence for all tissues. +++ = strong fluorescence, ++ = moderate fluorescence, + = weak fluorescence . 3 Agglutination activity is reported as units/mg dry weight of the digestive gland and mantle extracts, units/mg protein for the haemocyte extract, and units/mg dry weight of plasma EXAMPLE 3 To assess the ability of scallops to filter picoplank¬ ton from sea water, a suspension of S .bacillaris (4L, appro- ' ximately 4x10' cells/ml) was divided equally into two 4L bea- kers . Three scallops were placed into one of the beakers and both beakers were aerated and mixed under similar coditions of air flow and agitation. The number of S . bacillaris in sus¬ pension was determined by direct count using a Petrof f-Hauser bacteria counter at the beginning and 18 and 24 hours later. The effect of exposure to S .bacillaris on the lectin content of the digestive gland was assessed by maintaining thirty scallops flowing sea water at 5°C for one month, then dividing the scallops into two groups. One group of 10 animals was maintained in a 200L tank with flowing sea water at a rate of 15L per hour to which was continuously added a suspension of S . bacillaris (3.7 x 10 7 cells/ml) at a flow rate of 238 ml/hour. The other group comprising 12 animals was maintained in a separate identical tank without the addi¬ tion of S . baci1laris . After 1 and 2 months, the digestive glands of five animals were pooled and the lectin content determined by extraction so that two measurements were made for each group. The results are as shown in the following Table 6. TABLE 6 Filtering of 5.bacillaris from sea water by scallops Treatment Time (h) Cell count 1 (number/ml) Control (without seal- 0 3.7 ± 0.4 x 10 7 lops) 18 4.4 ± 0.3 x 10 7 24 4.8 ± 0.8 x 10 7 With scallops 0 4.2 ± 0.4 x 10 7 18 1.8 ± 0.3 x 10 7 24 1.6 ± 0.2 x 10 7 Average of five determinations It can be seen from Table 6 that scallops removed approxima¬ tely two-thirds of S .bacillaris cells suspended in sea water within 24 hours. The lectin content of the digestive gland of scallops maintained in flowing sea water declined within one month from 512 units/mg extract to 8 units/mg. Exposing scal¬ lops to S .bacillaris partly restored the lectin titer so that after 1 month the titer was 32 units/mg and after 2 months the titer was 64 units/mg. Controls maintained without added S. bacillaris had titers of 4 and 1 unit/mg after 1 and 2 months respectively. Titers were determined using an extract of five digestive glands. EXAMPLE 4 To assess the ability of scallop lectin to remove bac- terial LPS from solution, an affinity gel was constructed as follows. Lectin from P.magellanicus was attached to Af igel 15 using the method recommended by the manu acturer. The re¬ sulting affinity gel contained 5 mg/ml of lectin. A column (0.9 x 6 cm) was constructed using this gel and LPS (45 mg containing 134 μ,g KDO) was placed on the column. The column was washed with Tris buffer (0.02 M, pH 7.6 containing 0.9% NaCl and 0.02% Na azide) . The eluant contained 94μg KDO indi¬ cating that approximately 30% of the LPS (40μg KDO) bound to the affinity gel. ' A column of similar size constructed of Affigel 15 which had been blocked with ethanolamine did not bind any LPS as indicated by the quantitative recovery of KDO in the eluant . EXAMPLE 5 Following the procedure of Example 1 digestive gland extracts were obtained from several abalone species. Lectin contents of said extracts were measured by agglutination of sheep erythrocytes (RBcs) and E. coli cells before and after heating to 100°C for 30 minutes. E. coli units were measured using E . coli B which had grown for 24 hours at 37°C on Tryp- tic Soy Broth (Difco) . Cells were washed three times with saline and then suspended in saline to give an optical densi ty of 0.5 at 690 nm. The results of the measurements are given in Table 7 below. TABLE 7 Scientific name Unheated Heated RBC units/ E.coli RBC units/ E.coli mg units mg mg units/mg Haliotis rufescens 800 0 200 800 Haliotis pulqens 683 0 171 1365 Haliotis cracne- 800 0 200 1600 rodii Haliotis corrugata 656 82 656 Haliotis kamt- - 34 2133- schatkama In this disclosure the lectin activity expressed as "RBC units/mg" was determined as follows: Measurement of lectin activity: Lectin activity was measured using sheep erythrocytes and the picoplankton, Synechococcus bacillaris . To each well of a Cook microtiter plate, Tris buffer (lOOμl, 0.02 M, pH 7.6) supplemented with sodium chloride (0.9%) and sodium azi¬ de (0.02%) was added. The material to be assayed was dissol¬ ved in saline (20 mg/ml), insoluble material removed by cen- trifugation (5,000 x g, 10 min) and the supernatant fluid (100 μl) was added to the first well, mixed and the mixture (100 μl) was transferred to the second well. This process was continued giving a serial dilution of the preparation being assayed. To each well in a series, a suspension of sheep ery¬ throcytes (15 μl, 0.35 absorbance at 620 nm or 1.1 x 10 6 cells/ml) or a suspension of E.coli cells (15μl, optical ab¬ sorbance of 0.5 at 690 nm) was added. The microtiter plate was gently shaken and incubated at 4°C for 24 hours. The ti- ter was the highest dilution capable of cell agglutination. Specific activity is a reciprocal of this titer divided by the dry weight of material added to the first well in the dilution series. Unless stated otherwise, all specific acti- vities were determined with sheep erythrocytes. To determine agglutination activity of fractions obtai¬ ned during purification of lectin, an aliquot (lOOμl) of each fraction was tested " for activity as outlined above. The E.coli agglutinating lectin of Haliot s kamtschat- kama was shown to have a molecular weight of between 10.000 and 20.000 by SDS-PAGE electrophoresis under denaturing con¬ ditions. Isoelectric focussing indicated that the agglutina¬ ting activity had an isoelectric point between 4.0 and 5.0, in particular at a value of 4.6.

Claims: CLAI MS 1. A method for manufacturing scallop lectin, which comprises : a) extracting a lect -containing product from the diges- tive glands of a scallop selected from the family Pectinidae and the genus Haliotis . and b) purifying the resulting lectin-containing extract. 2. A method according to claim 1, wherein the scallop is selected from the family Pect dae . such as the sea scal- lop, Placopecten magellanicus . 3. A method according to claim 1, wherein extraction is effected by treatment with a suitable buffer. 4. A method according to claim 3, wherein the buffer is Tris buffer or phosphate buffer. 5. A method according to claim 1, 3 or 4, wherein puri¬ fication is effected by heat treating the lectm-conta mg extract, followed by ammonium sulfate precipitation, gel chromatography, chromatofocussing and chromatography on hyd¬ roxylapatite . 6. A method according to claim 1, 3 or 4, wherein puri¬ fication is effected by heat treating the lectm-contammg extract, followed by ammonium sulfate precipitation, gel chromatography and isoelectric focussing. 7. A method according to claim 1, 3 or 4, wherein puπ- fication is effected by heat treating the lectm-contammg extract, followed by ammonium sulfate precipitation and affi¬ nity chromatography. 8. A method according to claim 3 or 4, wherein the buf¬ fer is supplemented by a degradation inhibitor and/or an antimicrobial agent. 9. A method according to claim 3 or 4, wherein the buf¬ fer is supplemented v/ith one or more reagents selected from n-ammocaproic acid, EDTA, benzamidine, dithiothreitol , phe¬ nyImethylsulfony1 fluoride, sodium azide and sodium chloride. 10. A method of manufacturing scallop lectin, which comprises : a) extracting a lectin-containing product from the diges¬ tive glands of the sea scallop, Placopecten magellanicus using Tris buffer or phosphate buffer; and b) purifying the resulting lectin-containing extract by heat-treatment, followed by ammonium sulfate precipitation and chromatography. 11. A ' method according to claim 10, wherein the chroma- tographic treatment includes at least one treatment selected from gel chromatography, chromatofocussing, " chromatography on hydroxylapatite, and affinity chromatography. 12. A lectin-haemagglutinin extracted from the digesti¬ ve gland of a scallop selected from the family Pectinidae and the genus Haliotis. such as Placopecten magellanicus and possessing activity against Gram-negative bacteria, characte- rized by: a) a molecular weight of from about 10.000 to 20.000 under denaturing conditions; b) an ability to agglutinate sheep erythrocytes; c) an ability to agglutinate picoplankton of the species Spinobacter and Synechococcus : d) marked agglutination ability with respect to Gram nega¬ tive bacteria of the species Agrobacterium tume aciens . Es¬ cherichia coli and Serratia marcescens : e) an isoelectric point in the range of about pH 4-5; and f) resistance to the proteolytic enzymes proteinase K, pronase and trypsin. 13. A lectin product as defined in claim 12, having a molecular weight of about 14.000 when denatured and a speci¬ fic activity against Gram negative bacteria substantially as defined in Tables 2 and 3 hereinabove. 14. A lectin product as defined claim 12 or 13, ha¬ ving an amino acid composition resembling that given in Table 1 hereinabove.

Inventors: BROWN ROBERT GEORGE, GILL SIKANDER SINGH, BROWN, ROBERT, GEORGE, GILL, SIKANDER, SINGH
Applicants: MEDICARB AB
Application year: 1987
Main classification: C07K15
IPC codes: A61K38, C07K1, C07K14, G01N33