About the Lectin

The hemolymph of the American horseshoe crab contains several lectins that react with sialic acid and related proteins, and other lectins that react with GlcNAc moieties or monosaccharides unique to bacterial lipopolysaccharides. ^1,2 The lectin isolated from the horseshoe crab has been used extensively in the study of sialic acid containing glycoconjugates on cells and tissue sections. ³ The fluorescently labeled lectin has been used to obtain differential staining patterns between the colonic mucosa of normal rats and the mucosa adjacent to colon tumors, and it has been used in the study of glycosylation patterns associated with chemically induced tumors ⁴ . Purified LPA has a specificity similar to that of the lectin from Limax flavus, but it has been reported that LPA binds to N-acetyl-D-glucosamine and D-glucuronic acid to some degree. In addition to the carbohydrate binding sites, the purified lectin has a distinct binding site for phosphorylcholine. Another sialic-acid binding lectin does not react with phosphoryl esters. ¹ However, a C-reactive protein that lacks sialic acid binding is the major phosphoryl ester-binding protein in the hemolymph. The protein with both activities (apparently the true LPA) also exhibits hemolytic activity toward agglutinatable cells, and the hemolysis is inhibited by sialic acid. LPA is similar to many other lectins in that it has a greater affinity for glycopeptides than for free sialic acid. LPA requires the addition of calcium in order to bind sialic acid. The lectin is so sensitive to the presence of calcium that the use of a wash buffer lacking calcium will remove specifically bound LPA in histochemical staining experiments. This is also the case when using immobilized LPA for the purification of glycoconjugates. In order to use free sialic acid as a competitive inhibitor, calcium must be included in the working buffer. The absolute dependence on calcium for binding permits three procedures to be used as controls for specificity. One procedure is to incubate the lectin with sialic acid in a calcium containing buffer. Another procedure is to use a buffer lacking calcium or one in which a chelating agent such as EDTA has been added. The third procedure is to digest the sample with neuraminidase prior to experimentation. The lectin is somewhat sensitive to pH; agglutination is greatest at pH 8.0. Lectin activity decreases when the pH is below 7.5. LPA is perhaps the largest lectin purified, with molecular weights reported between 350,000 and 500,000 Da. The subunit structure is not clearly defined, with some reports of as many as 20 distinct subunits ^5,6 . This complex structure leads to a fairly unstable protein. The subunits tend to dissociate, resulting in decreased activity of the lectin. Dissociation is accelerated in the presence of low concentrations of detergents and by freeze-thaw cycles. High ionic conditions can also lead to dissociation and loss of lectin activity. EY Laboratories produces relatively small batches of purified material at a time to guarantee that only fresh material is used in preparing the various conjugates. This provides the researcher with the longest possible shelf life for the product.

REFERENCES

1. Armstong, P.B., et al. (1996) J. Biol. Chem. 271 : 14717-14721.
2. Seito, T., et al. (1997) J. Biol. Chem. 272 : 30703-30708.
3. Muresan, V., et al. (1982) J. Histochem. Biochem. 30: 938-946.
4. Freeman, H. J. (1983) J. Histochem. Cytochem. 31: 1241.
5. Robey, F. A. and Liu, T.-Y. (1981) J. Biol. Chem. 256 : 969-975.
6. Roche, A. C. and Monsigny, M. (1974) Biochim. Biophys. Acta. 371: 242-254.