Purification and characterization of a galactan-reactive agglutinin from the clam Tridacna maxima (Röding) and a study of its combining site

Abstract

1. A beta-galactosyl-binding lectin was purified from the haemolymph of the clam Tridacna maxima by affinity chromatography using polylecyl 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 pH8.6 and against rabbit antisera to crude T. maxima haemolymph, the lectin gave one precipitin arc in the alpha-region. 2. On a alkaline polyacrylamide disc gels, one lightly stained band and a broad diffuse band were seen close to the cathode. Ioselectric 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 electrofusing 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 haemagglutination studies the agglutinin exhibited a broad pH optimum (4.8-10.6). 6. Polysaccharides and glycoproteins with terminal non-reducing beta-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 beta-D-galactoside, methyl beta-D-galactoside, D-galactosamine and 60O-beta-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 beta-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.

Structure of the N‐Linked Oligosaccharides from Tridacnin, a Lectin Found in the Haemolymph of the Giant Clam Hippopus Hippopus

“…The presence of two protein bands (25 and 45 kDa) on SDSl PAGE in the purified tridacnin preparation is similar to that observed with the tridacnin from Triducrzu urzuxima (Baldo et al, 1978). We have demonstrated that both of these bands have the same N-terminal amino acid sequence indicating that the disulfide bonds may be incompletely reduced resulting in dimerisation (Puanglarp, Skewes and Yellowlees, unpublished data).…”

“…The haemolymph (340 ml) was collected following excision of the adductor muscle and centrifuged (10000Xg, 30 min, 4°C) before concentrating to 50 ml by ultrafiltration on a YMl00 membrane (Amicon). The concentrate was applied to a column ( 3 0 x 2 cm) of acid-treated Sepharose 4B (Baldo et al, 1978) and washed with buffer A (50 mM Tris/HCI pH 7.2, 150 mM NaCl and 1 0 mM CaCI2) until no further protein was eluted from the column. The tridacnin (30 mg) eluted from the column following addition of buffer A supplemented with 20 mM EDTA.…”

Immunorecognition in the gastropod molluscs with particular reference to the freshwater snailPlanorbarius corneus(L.) (Gastropoda, Pulmonata)

“…The molluscan agglutinins that seem to act similarly to antibodies are protein or glycoprotein (Hammarström & Kabat, 1969;Jenkin& Rowley, 1970;Pauleyeffl/., 1971a;Hardy et al, 1977;Baldo et al, 1978;Renwrantz & Stahmer, 1983). They may also have, opsonic properties (Pauley et al, 1971b;Harm & Renwrantz, 1980; van der Knaap et al, 1982;Renwrantz & Stahmer, 1983), and need divalent cations (in particular, Ca ++ ) to manifest biological activity (Hammarström & Kabat, 1969;Baldo et al, 1978;Renwrantz & Stahmer, 1983).…”

“…They may also have, opsonic properties (Pauley et al, 1971b;Harm & Renwrantz, 1980; van der Knaap et al, 1982;Renwrantz & Stahmer, 1983), and need divalent cations (in particular, Ca ++ ) to manifest biological activity (Hammarström & Kabat, 1969;Baldo et al, 1978;Renwrantz & Stahmer, 1983). They can also mediate chemotaxis (Schmid, 1975) and bind specific carbohydrates (Renwrantz, 1983).…”

Characterization of the Trimeric, Self‐Recognizing Geodia cydonium Lectin I

“…The protein concentration in the extract was 5.2 mg/ml. The lectins were isolated by affinity chromatography using acid-treated Sepharose 2 B (Pharmacia) [14]. The column (1 x 20 cm), equilibrated with Ca2+-free and Mg2+-free artificial sea water [5], was loaded with 20ml of crude extract (104mg protein) and washed thoroughly.…”