AU603411B2 - Lectin and method for the extraction thereof from scallop - Google Patents

Description

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INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 88/ 04303 C07K 15/08, 15/14, 3/02 Al (43) International Publication Date: 16 June 1988 (16.06.88) (21) International Application Number: PCT/SE87/00577 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (Euro- (22) International Filing Date: 3 December 1987 (03.12.87) pean patent), DK, FI, FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), NO, SE (31) Priority Application Number: 525,121 (European patent).

(32) Priority Date: 11 December 1986 (11.12.86) Published (33) Priority Country: CA With international search report.

(71) Applicant: MEDICARB AB [SE/SE]; Bj6rnnisvagen A. O.J.P. 4 AUG 1988 21, S-113 47 Stockholm (SE).

(72) Inventors: BROWN, Robert, George 18 Swanton Drive, Dartmouth, Nova Scotia B2W 2C4 (CA).

GILL, Sikander, Singh Punjabi University, Dept. of Zoology, Patiala-147002

AUSTRALIAN

(74) Agents: HENNINGSSON, Gunnar et al.; Bergling JUN Sundbergh AB, Box 7645, S-103 94 Stockholm 0 JUN 1988 PATENT OfRFICE a (54) Title: LECTIN AND METHOD FOR THE EXTRACTION THEREOF FROM SCALLOP Optical Density 00 Un Agglutination -600 1,0 500 coo 0 S -300 S2-100 0, 100 103 100 200 300 400 500 600 700 (57) Abstract Volume (ml) A method for manufacturing scallop lectin, which comprises: a) extracting a lectin-containing product from the digestive glands of a callop selected from the family Pectinidae and the genus Haliotis, and b) purifying the resulting lectincontaining 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 resistance to the proteolytic enzymes proteinase K, pronase and trypsin.

Signature(s declarant(s Note: No lega other witness wo of Hlizatin nr WIU required To: The Commissioner of Patents Olle Larm (Managing director) 88/04303 PCT/SE87/00577 LECTIN AND METHOD FOR THE EXTRACTION THEREOF FROM SCALLOP This invention relates to a method of extracting scallop lectin from the digestive glands of scallops of the family Pectinidae, in particular the sea scallop, Placopecten magellanicus, and to the use of the product in agglutination and separation procedures.

Lectins are carbohydrate-binding substances which do not exhibit enzymic activity nor are they produced as a result of specific immunological response. Lectins can agglutinate a variety of cell types and this agglutination may be inhibited by specific carbohydrates. A variety of microorganisms, plants, invertebrates and vertebrates possess lectins, indicating wide occurrence. Among invertebrates, lectins have been found in the haemolymph of Octopus, Limulus, Carcinoscopius, Tridacna, Aplysia, Tachypleus, Chelicerata and Dolabella. Other tissues may contain lectins, for example, gonads of Aplysia, the albumen gland of Helix, eggs of Anthocidaris and tentacles of Cerianthus.

Various functions have been ascribed to lectins. Haemolymph 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 Aplysia lectin may bind fertilized eggs to the capsule.

Lectin-binding specificity varies. For example the agglutinins of Limulus and Carcinoscorpius bind to 2-keto-3- -deoxy-octonic acid, a specific component of Gram negative bacteria. The haemolymph lectin of Ilyvsia binds to material containing N-acetyl amino sugars.

In particular, at the present time horseshoe crab (Limulus) lectin is employed for example in determining Gram negative bacterial presence in clinical samples and for the measurement of the bacterial endotoxin content of a variety of materials.

WO 88/04303 PCT/SE87/00577 2 However, use of this lectin is subject to several drawbacks 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 digestive glands of a scallop selected from the family Pectinidae and the genus Haliotis, for example the sea scallop, Placopecten magellanicus, for example by treatment wjthn Tris buffer, and b) purifying the resulting lectin-containing extract.

The purification can be effected by a variety of methods, including a) heat treatment followed by ammonium sulfate precipitation, gel chromatography, chromatofocussing and chromatography on hydroxylapatite; b) heat treatment, ammonium sulfate precipitation, gel chromatography and isoelectric focussing; and c) heat treatment, .ammonium sulfate precipitation and affinity chromatography. Other combinations of these techniques are also feasible.

Another aspect of the invention provides 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 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 Spinobacter and Synechococcus: j i F_ i W WO 88/04303 PCT/SE87/00577 d) marked ability to agglutinate Gram negative bacteria particularly those having accessible surface endotoxin, of the species Aqrobacterium tumefaciens, Escherichia coli and Serratia marcescens: e) an isoelectric point in the range of about pH 4-5, in particular about 4.52; f) an amino acid composition resembling that given in the following Table A; and g) resistance to the proteolytic enzymes proteinase K, pronase and trypsin.

Thus, in accordance with the method of the invention, scallop lectin is extracted from the digestive gland of e.g.

Placopecten magellanicus. The yield of lectin is generally approximately 20 mg from thirty large scallop digestive glands having a wet weight of about one pound. Although digestive glands are the prime source of the lectin, other tissues 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 harvested annually in Eastern Canada and a similar quantity of digestive glands would be available for extraction. The potential 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 homogenizing the glands in Tris buffer containing sodium chloride or Tris buffer supplemented with n-aminocaproic acid, EDTA, benzamidine, dithiothreitol, phanylmethylsulfonyl fluoride, sodium azide and sodium chloride. n-Aminocaproic acid, EDTA, benzamidine, and phenylmethylsulfonyl 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- WO 88/04303 PCT/SE87/00577 4 nity chromatography.

Using standard protein purification procedures, namely, heat treatment, ammonium sulfate precipitation, gel chromatography, chromatofocussing (or isoelectric focussing) and chromatography on hydroxylapatite, a lectin preparation can be obtained which gave a single band following SDS-PAGE electrophoresis. 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 composed of subunits which may assemble into aggregates of higher molecular weight, The purified lectin can be labelled 14 C) by reductive methylation 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 extract 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 contains 2-keto-3-deoxy-octonate (KDO) or sialic acid in polymeric 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, Luderitz and Westphal, European J. Biochem. 9: 245-249 (1969).

The latter procedure is simpler and for this reason is preferred. Electrophoresis of LPS preparations indicated the LPS preparation contained a single band of high mobility as detected by silver staining Trifluoroacetolysis of the LPS preparation yielded a pentasaccharide consisting of 3 molecules of KDO (2-keto-3-deoxy-octonate) and 2 molecules of glucosamine. The pentasaccharide is purified by extraction with ether, deacetylation (ammonium hydroxide in methanol, 11, 1 hour, 20 0 chromatography on Sephadex G-25 and HPLC using a column of porasil and ethyl acetate: acetic acid: water as solvent. The free amino group of glucosamine WO 88/04303 PCT/SE87/00577 is the reactive group used to attach the ligand (KDO) to affigel 15 (Biorad Laboratories Ltd.). The extract of the digestive glands was heated (1000C for i hour), cooled to and centrifuged (9000xg, 20 min). Ammonium sulfate was added to the supernatant fluid and proteins precipitated between 40-60% saturation collected by centrifugation (9000xg, min). The protein fraction was dissolved in distilled water, dialyzed and freeze dried. An aliquot (ig) was dissolved in Tris buffer (0.02 M, pH 7.6, 10 ml) containing sodium chloride and sodium azide The solution was passed through a KDO ligand-affigel 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 buffer (5 ml) containing the KDO-glucosamine pentasaccharide (4mg) followed by washing with Tris buffer (20 ml). Protein- -containing fractions were pooled and placed in an Amicon Ultrafiltration cell equipped with a UM 10 filter. The pentasaccharide was recovered by eluting the ultrafiltration cell with distilled water and chromatographing the KDO-containing sugars in the filtrate on Sephadex G-25. The lectin was recovered from the ultrafiltration cell by freeze drying.

Amino acid analysis carried out on samples of the scallop lectin gave the following results: WO 88/04303 PCT/SE87/00577 6 TABLE 1 Amino acid composition of P.-Maqellanicus lectin.

Molar ratio Amino acid i Analysis 12 Analysis II 3 Alanine 9.5 Arginine 2.4 2.4 Aspartic acid 7.4 8.6 Cysteic acid 2.2 Glutamic acid 7.4 7.3 Glycine 8.7 13.5 Histidine 2.0 Isoleucine 6.0 Leucine 6.0 Lysine 3.2 3.3 Methionine 1.0 0.4 Phenylanine 2.9 2.7 Proline 4.6 3.9 Serine 10.7 9.4 Threonine 20.5 17.4 Tyrosine 3.0 1.1 Valine 4.5 5.4 i Several unidentified peaks were detected in each analysis.

2 Lectin was hydrolyzed by 6N HC1 at 10 0 C for 24 hours.

3 Lectin was oxidized with performic acid, then hydrolyzed by 6N HC1 at 100 0 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.

WO 88/04303 1 WO 8804303PCT/SE87/00577 TABLE 2 Cell type Specific activi.ty units/Irn) 512 lectin bound 2 2 I Erythrocytes sheep II Bacteria A. Picoplankton Spinobacter sp.

Synechococcus bac il1lar is Synechococcus sp.(WHl 7803) Synechococcus sp.CWH- 7805) B.Grarn positive Bacillus cereus Bac illus megateriuri Bacillus subtilis Brev ibac ter jur linens Staphylococcus aureus C.Gram negative Aeromonas hydrophila Aeromonas punctata 2 Agrobacterium turnefaciens 32 Chromobacteriun violacem 4 Citrobacter freundii 2 Enterobacter aerogenes 8 Erwinia amulovora 4 Escherichia coli B 128 Escherichia coli (heptoseless )64 Klebsiella pneumoniae 4 Proteus vulgaris 8 Providentia alcalifaciens 4 Pseudomonas aerogenosa 8 Pseudomonas fluorescens 2 Serratia iarcescens 64 WO 88/04303 PCT/SE87/00577 cont. Table 2 Shigella flexneri 4 Vibrio anguillarum 4 Yersinia enterocolitica 4 Zymomonas mobilis 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 agglutinate, therefore 1 4 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 compromised 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 agglutinated by scallop lectin were Aeromonas hydrophila and Zymomonas mobilis. These bacteria have LPS which does not contain KDO, suggesting the feature of LPS recognized by scallop lectin contains KDO. This was confirmed by affinity chromatography as more than 70% of the labelled (1 4 C) lectin bound to an affinity column which had a KDO-containing pentasaccharide as the ligand. A portion of the bound lectin 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 WO 88/04303 PCT/SE87/00577 9 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, thyroglobulin and human acid glycoprotein all contain sialic acid (N-acetylneuraminic acid) and were able to block agglutination of sheep erythrocytes by scallop lectin. Proteins which do not contain sialic acid were unable to block agglutination of sheep erythrocytes. Sialic acid is similar in structure to KDO. Sialic acid-containing material could elute bound lectin from the affinity column described above, however, the small quantity of lectin eluted suggests the lectin has a much higher affinity for the KDO-containing pentasaccharide than for sialic acid.

The scallop lectin of the present invention possesses several unique features of advantage, including firstly heat stability. For example, agglutination titer was not diminished by heating a solution of the lectin for 1 hour at 100 0

C.

Limulus lectin, which is presently used for some of the purposes 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, proteinase K, pronase and trypsin were unable to diminish the titer of a lectin preparation during overnight incubation thereof with each of the above proteolytic enzymes. This attribute 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-containing materials and the degree to which Limulus lectin and scallop lectin have similar binding sites is not at present fully known.

A number of other lectins which recognize sialic acid-contai- WO 88/04303 PCT/SE87/00577 ning materials are known, although Limulus lectin appears to be the only lectin commercially available.

The scallop lectin of the invention is useful in agglutinating 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 antigens for ligands for antibody production/purification of known LPS specificity.

For example, scallop lectin purified by affinity chromatography 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 glycoproteins.

3) Scallop lectin may be used to remove LPS from vaccine preparations or other injectable pharmaceuticals. The lipid A moiety of LPS elicits many biological activities in mammals. Consequently in recognition of these toxic properties LPS is also called endotoxin. The prefix "endo" indicates 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 WO 88/04303 PCT/SE87/00577 11 lectin attached to a suitable supporting gel would serve the same purpose but would have the advantage of increased specificity. Non-specific binding of other components besides endotoxin may sometimes be a problem with Detoxi-Gel particularly when protein is present.

4) The scallop lectin may alsc find utility in measurement of endotoxin levels in injectable pharmaceuticals and foods, which is presently accomplished using Limulus amebocyte lysate (LAL) assay.

While the above disclosure is primarily concerned with extraction of lectin from the sea scallop, P. magellanicus, useful lectins can also be extracted and employed from other molluscs of the family Pectinidae, for example Chlamys islandica, Chlamys rubida and Arctica islandica. The following Table 3 shows the activity of several other molluscs of interest.

TABLE 3 Lectin activity of representative molluscs in addition to Placopecten magellanicus.

NAME

Group Scientific Common Lectin Heat Inhibiactivi resis- ted by ty l u- tant 2 LPS3 nits/mg) Scallops Argopecten Bay scal- 1 Yes Yes irradians lop Chlamys Iceland 4 Yes Yes islandica scallop Chlamvs -1 Yes Yes hostata L WO 88/04303 WO 8804303PCT/SE87/00577 cont. Table 3, Chiamys rubida Yes Clams, mus s e I s5 and oysters Gras sadoma gigantea Myt ilus Calif ornic Us Spi sula Pa ivuryma Cystodr ia sy 1lopua Arct ica is landica Crassostrea aigas Big scallop Cali~f ornia mu ssel1 Stimpson' s surf clam Porpe 11cr clam Yes Yes, Yes Yes Yes Yes Yes Yes Yes Ocean quahaug Yes Yes Japanese oyster Yes Limpets Lucapina sp. Key hole limpet Yes Yes Yes Snails Moon snail Yes In contrast to the results given in Example I for extraction 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.mapellanicus.

Lectin activity was measured after heating the extract to 1001C for 30 minutes. Lectin activity was tested in the presence of E.coli'LPS (serotype 055).

WO 88/04303 PCT/SE87/00577 13 Of the molluscs extracted other than P. magellanicus the lectin from Chlamys islandica was studied most intensively. C.islandica lectin was similar to lectin from P.maqellanicus with respect to heat resistance, resistance to proteolytic enzymes, agglutination blockage by bacterial LPS and sialic acid-containing 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 diameters in the range of 10-20 cm were obtained from Mushaboom, Nova Scotia (44051';62034') by divers. The live scallops were kept in sea water at 2-6 0 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 EDTA (0.005 benzamidine (0.005 dithiothreitol (0.001 phenylmethylsulfonyl fluoride (0.001 and sodium azide and sodium chloride using a Polytron ultrasonic-mechanical homogenizer for 2 minutes. The homogenized extract was centrifuged (10.000xg, 60 minutes) and the supernatant fluid was dialyzed against distilled water at 4 0 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 extract 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 (10.000xg, minutes) and the supernatant fluid was dialyzed against distilled water at 4 0 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 (10.000xg, 30 minutes). Ammonium sulfate was added to the supernatant fluid in increments of 20% from 0 to 100% saturation. The precipitates which formed at each 20% increment were removed WO 88/04303 PCT/SE87/00577 14 by centrifugation (10.000x.g, 60 minutes). The five precipitates were dialyzed against distilled water at 4 0 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 saturation) was chromatographed on Fractogel 65F (2.5x42 cm) developed with the same buffer used for lectin extraction at a flow rate of 50 ml/h. 10 ml fractions were collected and tested for lectin activity. Active fractions were pooled, dialyzed against distilled water at 4 0 C for 24 hours and freeze dried.

The dried material was chromatofocussed on an ion exchange 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 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 determined. Active fractions were pooled, dialyzed and freeze dried.

Analysis of the freeze dried material by gel electrophoresis indicated the presence of low molecular weight material 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 and chromatographed on a hydroxylapatite column 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 chromatography 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-10 was added to the light and dense solutions to give a final concentration of 20% The freeze dried lectin-containing material (600 mg) from gel chromatonegative bacterial presence in clinical samples and for the measurement of the bacterial endotoxin content of a variety of materials.

1 WO 88/04303 PCT/SE87/00577 graphy was dissolved in the light solution (215 ml). After filling the column, the voltage was adjusted to keep the power input at 6 Watts. After 36 hours at 5°C, focussing was terminated and the column emptied in 10 ml fractions. Fractions 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 agglutination 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) fic ac- acti- puritivity vity fica- (units/ (units, tion mq) x10 3 Crude extract 1470 512 752 1 Heat treatment 1308 512 669 89 1

(NH

4 2 S0 4 pre- 550 1,024 563 75 2 cipitation Fractogel 65F 310' 2,048 634 84 4 Chromatofo- 18 18,125 326 41 cussing hydroxylapatite Polyacrylamide gel electrophoreses (PAGE) was carried out using a gradient gel (PAA 4/30, Pharmacia Ltd.) equilibrated with buffer containing Tris (0.09 boric acid (0.08 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 in acetic acid 2 hours) or PAS stain for carbohydrate. In the latter procedure, parallel gels were treated with aqueous periodic acid, Schiff's reagent then destained with 1 M HC1 containing potassium meta- WO 88/04303 PCT/SE87/00577 16 bisulphite SDS-PAGE electrophoresis of proteins was carried out using a gradient gel (PAA 4/30, Pharmacia Ltd.) equilibrated with buffer containing Tris (0.04 sodium acetate (0.02 M) EDTA (2mM) and SDS at pH 7.4. Samples were heated (100 0 C, 10 minutes) in Tris buffer (10mM, pH EDTA (1 mM) with SDS and mercaptoethanol and applied to the gel. An electrical potential of 300 volts was applied for 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 electrophoretically at 24 volts for 30 minutes in 25% isopropanol, containing 10% acetic acid. Gels were stained with Coomassie blue in acetic acid 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 sulfate 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 molecular weight as illustrated in the accompanying Figure 1.

Chromatofocussing indicated that the lectin was eluted at pH 3.8-4.2 as seen from Figure 2..This step substantially increased specific activity, however, gel electrophoresis detected the presence of contaminating material of high mobility which probably originated from the polybuffer used for chromatofocussing. This was removed by chromatography on hydroxylapatite as shown by Figure 3. The final product had a specific activity of 18 x 10 3 units/mg which represented a purification.

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- -i *i 4 WO 88/04303 PCT/SE87/00577 17 Gel electrophoresis with and without SDS indicated the presence of one major diffuse band. The bands were also stained by the PAS procedure indicating the presence of'carbohydrate. Gel electrophoresis without SDS demonstrated the presence of high molecular weight aggregates. The molecular weight of these aggregates decreased to about 14.000 in the presence of SDS. Replacing the chrsmatofocussing purification step with isoelectric focussing resulted in a 64-fold purification 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 obtained in Example 1 was assessed by two methods. Firstly, the tissues were exposed to fluorescein isothiocyanate labelled E.coli LPS (1 mg/ml, 10 minutes, serotype 055:5B, Sigma Chemical Co.) and unbound LPS was removed by repeated washing with saline. The tissue was examined by fluorescent microscopy (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 otherwise received identical treatment.

Secondly, tissues were extracted as previously described and the extracts analyzed for lectin activity. Haemocytes were collected from haemolymph by centrifugation (5.000xg, 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 Model 200 at 15 Watts. Cell debris was removed by centrifugation (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 WO 88/04303 PCT/SE87/00577 18 did not bind LPS at all. Tissue extraction verified the presence 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 indicate the presence of lectin in these tissues in a form not extractable by Tris buffer. Disruption of haemocytes released haemagglutination 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 TABLE 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 Heart Muscle Crystalline style Hind gut N.D. Plasma ND. 4 1 N.D. not determined; no fluorescence or no agglutination.

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.

)L I WO 88/04303 PCT/SE87/00577 19 EXAMPLE 3 To assess the ability of scallops to filter picoplankton from sea water, a suspension of S.bacillaris (4L, approximately 4x10 7 cells/ml) was divided equally into two 4L beakers. 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 suspension was determined by direct count using a Petroff-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 in flowing sea water at 5 0 C for one month, then dividing the scallops into two groups. One group of 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 107 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 addition of S.bacillaris. 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 ir the following Table 6.

TABLE 6 Filtering of S.bacillaris from sea water by scallops Treatment Time Cell count I (number/ml) Control (without scal- 0 3.7 0.4 x 107 lops) 18 4.4 0.3 x 107 24 4.8 0.8 x 107 With scallops 0 4.2 0.4 x 107 18 1.8 0.3 x 107 24 1.6 0.2 x 107 1 Average of five determinations.

c I' WO 88/04303 PCT/SE87/00577 It can be seen from Table 6 that scallops removed approximately 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 scallops 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 I 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 bacterial LPS from solution, an affinity gel was constructed as follows. Lectin from P.magellanicus was attached to Affigel using the method recommended by the manufacturer. The resulting 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 pg KDO) was placed on the column. The column was washed with Tris buffer (0.02 M, pH 7.6 containing 0.9% NaC1 and 0.02% Na azide). The eluant contained 941kg KDO indicating that approximately 30% of the LPS (404g 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 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 1000C for 30 minutes. E.coli units were measured using E.coli B which had grown for 24 hours at 37°C on Tryptic Soy Broth (Difco). Cells were washed three times with i WO 88/04303 PCT/SE87/00577 saline and then suspended in saline to give an optical density 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 mq units/mg mg units/mg Haliotis rufescens 800 0 200 800 Haliotis pulgens 683 0 171 1365 Haliotis cracne- 800 0 200 1600 rodii Haliotis corrugata 656 0 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 (100il, 0.02 M, pH 7.6) supplemented with sodium chloride and sodium azide was added. The material to be assayed was dissolved in saline (20 mg/ml), insoluble material removed by centrifugation (5,000 x g, 10 min) and the supernatant fluid (100 41) was added to the first well, mixed and the mixture (100 pl) 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 erythrocytes (15 41, 0.35 absorbance at 620 nm or 1.1 x 106 cells/ml) or a suspension of E.coli cells (15~l, optical absorbance of 0.5 at 690 nm) was added. The microtiter plate was gently shaken and incubated at 4 0 C for 24 hours. The ti- WO 88/04303 PCT/SE87/00577 22 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 activities were determined with sheep erythrocytes.

To determine agglutination activity of fractions obtained during purification of lectin, an aliquot (100il) of each fraction was tested for activity as outlined above.

The E.coli agglutinating lectin of Haliotis kamtschatkama was shown to have a molecular weight of between 10.000 and 20.000 by SDS-PAGE electrophoresis under denaturing conditions. Isoelectric focussing indicated that the agglutinating activity had an isoelectric point between 4.0 and in particular at a value of 4.6.

dmw-s ad

Claims (14)

1. A method for manufacturing a scallop lectin-haemag- glutinin possessing activity against Gram-negative bacteria 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, and b) purifying the resulting lectin-containing extract so as to obtain a product having. 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 Aqrobacterium tumefaciens, 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.

2. A method according to claim 1, wherein the scallop is selected from the family Pectinidae, such as the sea scal- lop, Placopecten magellanicus.

3. A method according to claim 1, wherein the extrac- tion is effected by treatment with a suitable buffer.

4. A method according to claim 3, wherein the buffer is Tris buffer or phosphate buffer.

A method according to claim 1, 3 or 4, wherein puri- fication is effected by heat treating the lectin-containing 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 lectin-containing extract, followed by ammonium sulfate precipitation, gel chromatography and isoelectric focussing. I _OA PCT/ SE 8 7 0 0 5 7 7 24 1989 -02- 2 3

7. A method according to claim 1, 3 or 4, wherein puri- fication is effected by heat treating the lectin-containing 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 with one or more reagents selected from n-aminocaproic acid, EDTA, benzamidine, dithiothreitol, phe- nylmethylsulfonyl fluoride, sodium azide and sodium chloride.

A method according to claim 1, which comprises: a) extracting a lectin-containing product from the diges- tive glands of the sea scallop, Placopecten macellanicus 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 Acrobacterium tumefaciens, Es- cherichia coli and Serratia marcescens; e) an isoelectric point in the range of about pH 4-5; and Lo iuu-u IOr su minutes. Lectin activity was utou6u i presence of E.coli LPS (serotype 055). LI II I~T;C' r I: A -gP PCT/SE 87/00577 1989 -02- 23 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 in claim 12 or 13, ha- ving an amino acid composition resembling that given in Table 1 hereinabove.