Biochem. J. (1978) 175, 467-477 467 Printed in Great Britain Purification and Characterization of a Galactan-Reactive Agglutinin from the Clam Tridacna maxima (R8iding) and a Study of its Combiniing Site By BRIAN A. BALDO,*II WILLIAM H. SAWYER,t ROBERT V. STICKt and GERHARD UHLENBRUCK§ *Children's MedicalResearch Foundation, Princess Margaret Hospital, Perth, Western Australia, Australia, tRussell Grimwade School ofBiochemistry, University ofMelbourne, Parkville, Victoria, Australia, tDepartment ofOrganic Chemistry, University of Western Australia, Perth, Western Australia, Australia, and§Department ofImmunobiology, Medical University Clinic, Cologne-Lindenthal, Federal Republic ofGermany (Received 13 March 1978) 1. A f-galactosyl-binding lectin was purified from the haemolymph of the clam Tridacna maxima by affinity chromatography using polyleucyl larch galactan, D-galactosamine coupled to epoxy-activated Sepharose or acid-treated Sepharose. Elution with N-acetyl-D- galactosamine or lactose displaced the bound lectin, which appeared homogeneous by sedimentation analysis. On immunoelectrophoresis at pH 8.6 and against rabbit antisera to crude T. maxima haemolymph, the lectin gave one precipitin arc in the a-region. 2. On alkaline polyacrylamide disc gels, one lightly stained band and a broad diffuse band were seen close to the cathode. Isoelectric focusing in solution revealed two peaks of pI4.05 and 4.25 and a shoulder, pI4.0, whereas at least three bands close together (pI3.9-4.3) were seen after electrofocusing in gel. 3. The agglutinin is a glycoprotein with a mol.wt. of 470300±20000. Amino acid analysis revealed no methionine and a significant amount of half-cystine residues. 4. Tridacna lectin is a metalloprotein requiring Ca2+ for its haemagglutinating and precipitating activities. 5. In haemagglutin- ation studies the agglutinin exhibited a broad pH optimum (4.8-10.6). 6. Polysaccharides and glycoproteins with terminal non-reducing fi-D-galactosyl residues reacted with the lectin to form precipitates both in gel and in solution. Inhibition experiments showed that N-acetyl-D-galactosamine was the best inhibitor of the agglutinin combining sites, followed by p-nitrophenyl f-D-galactoside, methyl ,B-D-galactoside, D-galactosamine and 6-0-,8-D-galactopyranosyl-D-galactopyranose. On a molar basis, N-acetyl-D- galactosamine was 20-fold more active than D-galactose and nearly 10-fold more inhibitory than D-galactosamine. 7. Circular-dichroism studies showed that the lectin contains a relatively high proportion of fl-structure. 8. Mercaptoethanol treatment of the agglutinin followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed subunits with approx. mol.wts. of 10000, 20000 and 40000. Lectin-like proteins that react with carbohydrate invertebrate agglutinins (McKay et al., 1969) and structures and that show precipitating and/or their capacity to bind to foreign cells (Tripp, 1966), haemagglutinating properties have been found in has led to speculation that the agglutinins have a a number of invertebrate species (Cushing et al., role in defence of the host (Acton & Weinheimer, 1963; Acton & Weinheimer, 1974; Gold & Balding, 1974). Burnet (1968) has suggested that the pseudo- 1975). Interest in invertebrate agglutinins or lectins immunological capacities of invertebrate agglutinins (Sharon & Lis, 1972) has been sustained for two may have provided the basis from which the main reasons. Although invertebrates lack the vertebrate antibody system evolved. A second major adaptive immune mechanisms found in vertebrates, stimulus for interest in invertebrate agglutinins has the capacity to recognize and distinguish foreignness been the relatively recent recognition of the useful- is well developed throughout the Invertebrata (Hilde- ness oflectins in research and their wide application to mann, 1974). The specificity shown by some manyareas ofthe biomedical sciences (Lis & Sharon, 1977). Lectins mediate a wide range of biological Abbreviation used: SDS, sodium dodecyl sulphate. effects (Nicolson, 1974) and, as a consequence of 11 To whom requests for reprints should be addressed, their ability to bind to both free and cell-bound at Roche Research Institute of Marine Pharmacology, carbohydrate structures, plant and invertebrate P.O. Box 225, Dee Why, N.S.W. 2099, Australia. agglutinins are being increasingly used as affinity- Vol. 175 468 B. A. BALDO, W. H. SAWYER, R. V. STICK AND G. UHLENBRUCK chromatography support media for the purification under 'Methods'. Methyl a-D-galactopyranoside ofpolysaccharides, peptidoglycans and glycoproteins monohydrate, methyl fi-D-galactopyranoside and (Lloyd, 1970; Adair & Kornfeld, 1974; Bridgen 6-0-methyl D-galactopyranose were purchased from et al., 1976; Ross et al., 1976). Koch-Light. All other monosaccharides, oligo- Clams of the family Tridacnidae are specialized saccharides and glycosides were from Sigma. bivalves that inhabit the shallower waters of coral Polysaccharides and glycopeptides were prepared or reefs. Tridacna maxima (Roding), the most ubiquitous obtained as previously described (Baldo & Uhlen- species of the family, is distributed widely in the bruck, 1973, 1975b; Uhlenbruck et al., 1975; Pacific and Indian Oceans within an area ranging from Eichmann et al., 1976; Baldo et al., 1977). East Africa to Eastern Polynesia (Rosewater, 1965). Human erythrocytes were supplied by the Red Haemolymph from T. maxima contains a Cal+- Cross Blood Transfusion Service, Perth, Western dependent protein, tridacnin, that strongly aggluti- Australia, Australia. Erythrocytes from other species nates some animal erythrocytes and that precipitates were obtained fresh as needed by bleeding animals with a number of polysaccharides and glycopeptides directly into Alsever's solution. (Baldo & Uhlenbruck, 1975a,b; Uhlenbruck et al., 1975). Although tridacnin has been isolated (Baldo Methods & Uhlenbruck, 1975c), characterization of the purified agglutinin and detailed combining-site Haemolymph. Clams obtained from Israel were studies were not carried out. opened by immersion in C02-treated sea water and In the present study we describe three different haemolymph was collected after careful dissection of methods for the purification of tridacnin, together the clams with a knife. Before being used in purifica- with quantitative precipitin-inhibition studies de- tion experiments, haemolymph was dialysed exten- signed to explore the specificity and size of the sively against water and freeze-dried. lectin-combining sites. Results of electrophoretic, Purification of tridacnin. Tridacnin was isolated molecular-weight, amino acid- and carbohydrate- from previously dialysed and freeze-dried haemo- composition, circular-dichroism and isoelectric- lymph by affinity chromatography using the focusing studies are also presented and, from following support media and procedures. investigations of the subunit composition of the 1. Polyleucyl-arabinogalactan. Larch arabino- protein, a tentative model is proposed for the struc- galactan (Serva, Heidelberg, Germany) was dis- ture of the agglutinin. solved in 0.07M-NaHCO3 at 4°C and co-polymerized with an equal quantity of the N-carboxyanhydride of L-leucine (Tsuyuki et al., 1956; Kaplan & Kabat, Experimental 1966). The precipitate was recovered by centri- Materials fugation at 30000g for 40min and washed twice with 0.07M-NaHCO3 and twice with water. For column Sepharose 2B, 4B and 6B and epoxy-activated chromatography, five parts of polyleucyl-arabino- Sepharose 6B were from Pharmacia, Uppsala, galactan was mixed with two parts of Sepharose 4B Sweden. L-Leucine N-carboxyanhydride (lot no. in 0.9 % NaCl containing 0.01 M-Ca2+ (saline/Ca2+), LE 45) was obtained from Miles-Yeda, Rehovoth, left at 4°C for 24h and then poured into small columns Israel. Acrylamide was from Eastman Organic (1cmx 10cm). After washing with saline/Ca2+, Chemicals, Rochester, U.S.A. and NN'-methylene- T. maxima haemolymph extract (10-20mg in lml bisacrylamide, NNN'N'-tetramethylethylenediamine, of saline/Ca2+) was added to the column and eluted sodium dodecyl sulphate and ninhydrin from Sigma with the same vehicle. Fractions were collected and Chemical Co., MO, U.S.A. Agar (Noble) and examined for A280 and for haemagglutinating Freund's complete adjuvant were obtained from activity against human blood-group-O erythrocytes. Difco Laboratories, Detroit, MI, U.S.A. Ampholines Bound agglutinins were eluted from the column by pH 3.5-5.0 and pH 3.5-10 were supplied by LKB- adding 0.025M-N-acetyl-D-galactosamine in saline/ Produkter A.B., Bromma, Sweden. Ca2+. Fractions showing haemagglutinating activity Cytochrome c (horse heart) and ribonuclease were *and/or absorbance at 280nm were pooled, dialysed from Schwartz-Mann, Orangeburg, NY, U.S.A., against frequent changes of water, concentrated by pepsin (from pig stomach mucosa; three-times- ultrafiltration and freeze-dried. crystallized) from Koch-Light Laboratories, CoIn- 2. Acid-treated Sepharose. Sepharose 2B, 4B or brook, Bucks., U.K., lysozyme (hen's egg white) 6B was treated with 0.2M-HCI at 50°C for 2h as and bovine serum albumin from Sigma Chemical Co. described by Ersson et al. (1973). The acid-treated and fl-galactosidase from Worthington Biochemical beads were packed into a column, washed with Corp., Freehold, NJ, U.S.A. saline/Ca2+ and used as an affinity support medium The disaccharide 6-O-f)-D-galactopyranosyl-D- for the preparation of tridacnin. Elution of bound galactopyranose was synthesized as described below lectin