Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
  • G01N33/56911—Bacteria
  • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
  • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • G01N2400/50—Lipopolysaccharides; LPS

Definitions

• 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 maqellanicus, 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 lmmunological response. Lectins can agglutinate a variety of cell types and this agglutination may be inhibited by specific carbohydrates.
• lectins have been found in the haemolymph of Octopus, Limulus, Carcmoscopius, Tridacna, Aplvsia, Tachypleus, Chelicerata and Dolabella.
• Other tissues may contain lectins, for example, gonads of Aplvsia, the albumen gland of Helix, eggs of Anthocidaris and tentacles of Cerianthus.
• 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
• the Aplysia lectin may bind fertilized eggs to the capsule.
• Lect in-binding specificity varies.
• the agglutinms of Limulus and Carcinoscorpius bind to 2-keto-3- -deoxy-octonic acid, a specific component of Gram negative bacteria.
• the haemolymph lectin of APlvsIa binds to material containing N-acetyl ammo sugars.
• horseshoe crab (Limulus) 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 materlals.
• 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.
• 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 with 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, chromatof ocussing 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: d) marked ability to agglutinate Gram negative bacteria particularly those having accessible surface endotoxin, of the species Agrobacterium tumefaciens, Escherichia coli and Serrat la marceseens: e) an isoelectric point in the range of about pH 4-5, in particular about 4.52; f) an ammo acid composition resembling
• 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.
• digestive glands are the prime source of the lectin, other tissues may also be extracted and yield lectin.
• 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-ammocaproic acid, EDTA, benzamidme, dithiothreitol, phenylmethylsulfonyl fluoride, sodium azide and sodium chloride.
• n-Aminocaproic acid, EDTA, benzamidme, 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 nity chromatography.
• 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 (14C) 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.
• 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.
• KDO 2-keto-3-deoxy-octonate
• the ligand used for affinity chromatography is obtained by trif luoroacetolysis of lipopolysacchar ide (LPS) from a heptoseless mutant of E. coli.
• LPS lipopolysacchar ide
• the LPS can be extracted by the method of Darveau and Hancock (1983) or Galanos, Luderitz and Westphal, European J. Biochem. 9: 245-249 (1959). The latter procedure is simpler and for this reason is preferred.
• LPS preparations contained a single band of high mobility as detected by silver staining.
• Trif luoroacetolysis 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, 1M, 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 glucosamine is the reactive group used to attach the ligand (KDO) to affigel 15 (Biorad Laboratories Ltd.).
• KDO ligand
• the extract of the digestive glands was heated (100°C for 1 hour), cooled to 2°C and centrifuged (9000xg, 20 min). Ammonium sulfate was added to the supernatant fluid and proteins precipitated between 40-60% saturation collected by centrifugation (9000xg, 20 min). The protein fraction was dissolved in distilled water, dialyzed and freeze dried. An aliquot (1g) was dissolved in Tris buffer (0.02 M, pH 7.6, 10 ml) containing sodium chloride (0.9%) and sodium azide (0.02%).
• 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-glucosamme pentasaccharide (4mg) followed by washing with Tris buffer (20 ml).
• Protein- -contammg fractions were pooled and placed in an Amicon Ultrafiltration cell equipped with a UM 10 filter.
• the pentasaccharide was recovered by elutmg the ultrafiltration cell with distilled water and chromatographmg the KDO-containing sugars in the filtrate on Sephadex G-25.
• the lectin was recovered from the ultrafiltration cell by freeze drying.
• 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.
• 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 ( 14 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 O, A, B and AB.
• Fetuin, thyroglobulm and human acid glycoprotein all contain sialic acid (N-acetylneurammic 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-contammg 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 dimmished by heating a solution of the lectin for 1 hour at 100°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.
• protemase 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 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.
• 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 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 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.
• Affinity purification of LPS from most Gram negative bacteria is possible using scallop lectin to yield highly purified LPS preparations.
• 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.
• 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.
• KDO 2-keto-3-deoxy-octonate
• Scallop lectin recognizes sialic acid-containing glycoproteins and, consequently, scallop lectin could be used for affinity purification of sialic acid containing glycoproteins.
• 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.
• 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 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.
• the scallop lectin may also 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. magellameus, useful lectms can also be extracted and employed from other molluscs of the family Pectinidae. for example Chlamvs lslandica. Chlamys rubida and Arctica islandica. The following Table 3 shows the activity of several other molluscs of interest.
• LAL Limulus amebocyte lysate
• 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).
• E. coli LPS serotype 055
• C . lslandica lectin was similar to lectin from P .magellamcus with respect to heat resistance, resistance to proteolytic 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 homogenized extract was centrifuged (10.000xg, 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.
• 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 centrifugat ion (10.000xg, 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 centrifugat ion (10.000xg, 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 (10.000xg, 60 minutes).
• the five precipitates 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 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°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 (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 determined. Active fractions were pooled, dialyzed and freeze dried.
• 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-100 ® was added to the light and dense solutions to give a final concentration of 20% CV/V).
• the freeze dried lectin-containing material 600 mg 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.
• PAGE Polyacrylamide gel electrophoreses
• the anatomical distribution of the purified lectin obtamed in Example 1 was assessed by two methods. Firstly, the tissues were exposed to fluorescem 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 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 otherwise received identical treatment.
• tissues were extracted as previously described 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 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.
• S. bacillaris 4L, approximately 4x10 7 cells/ml
• 3 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 m 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 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.
• an affinity gel was constructed as follows. Lectin from P.magellanicus was attached to Affigel 15 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 ⁇ 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 94 ⁇ g KDO indicating that approximately 30% of the LPS (40 ⁇ g 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 Tryptic Soy Broth (Difco). Cells were washed three times with saline and then suspended in saline to give an optical density of 0.5 at 690 nm.
• RBcs sheep erythrocytes
• E. coli cells 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 saline and then suspended in saline to give an optical density of 0.5 at 690 nm.
• Lectin activity was measured using sheep erythrocytes and the picoplankton, Synechococcus bacillaris.
• Tris buffer 100 ⁇ l, 0.02 M, pH 7.6 supplemented with sodium chloride (0.9%) and sodium azide (0.02%) 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 ⁇ l) was added to the first well, mixed and the mixture (100 ⁇ l) was transferred to the second well. This process was continued giving a serial dilution of the preparation being assayed.
• 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 5.0, in particular at a value of 4.6.

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