The Characterisation And Application Of Galactose-Binding Lectin From The Seeds Of Champedak (Artocarpus Integer).

Author

MARIATI BINTI ABDUL RAHMAN

Date

2002

Keyword

Artocarpus integer, 'champedak', galactose binding lectin, glycoproteins

Abstract

When previously described champedak lectin-C was subjected to SDS-PAGE separation, two sharp bands were resolved with Mr of approximately 15,000 and 17,000. In the isoelectric focusing experiments, the lectin displayed a broad and diffused pattern within the pI range of 5.8-7.9. Separation of lectin-C by reverse phase high performance liquid chromatography generated three peaks, which corresponded to three distinct types of protein subunit. The two smaller subunits, each comprising of 21 amino acid residues, demonstrated minor sequence variability and were generally comparable to the ßchains of a few other galactose-binding Artocarpus lectins. The N-terminal sequence of the first forty-seven residues of the large subunit demonstrated at least 95% homology to the a chains of the other Artocarpus lectins. When used as a probe for human serum glycopeptides that were separated by two-dimensional gel electrophoresis, lectin-C demonstrated strong interaction with several glycopeptides such as lgA1, hemopexin,α2-HS glycoprotein, α1-antichymotrypsin and a few unknown glycoproteins. Similar interactions were also observed with the serum of a pregnant subject and the serum of an ovarian tumour patient. Lectin-C was also used . as an affinity column by coupling it to Sepharose. Subjecting normal human serum to the immobilised lectin affinity  chromatography  was  able  to   retain  the   lectin-C-reactive  serum glycoproteins. The column was also capable of retaining similar serum glycoproteins from the serum of a pregnant subject but with less intensity when compared to the lectin-C-reactive serum glycoproteins from the normal serum. The interaction of lectin-C with neuraminidase-treated purified serum glycoproteins and hCG was also investigated. For lgA1 and hCG, the binding of lectin-C to these glycoproteins were similar before and after the removal of sialic acid by neuraminidase. Interaction of lectin-C with α2-HS glycoprotein and ai-antichymotrypsin was reduced after treatment with neuraminidase. The interaction of lectin-C with hCG was further investigated with 2DE and Western blotting. Out of the two groups of spots that were tested, only one group interacted with lectin-C. These isoforms from the group are believed to be the (3 subunits of hCG based on its carbohydrate moieties and their Mr. Champedak lectin-C also demonstrated interaction with two SDS-PAGE protein bands from crude urine extract of urine of a pregnant subject. In another related study, a comparison was made on the expression of O-glycosylated serum glycoproteins that were detected with lectin-C between the sera of normal healthy individual with pregnant subjects or ovarian tumour patients. There were no significant differences in the mean volume contributions of hemopexin, lgA1, α2-HS glycoprotein, α1-antichymotrypsin,  Ua (hemopexin-like) and  Uc (haptoglobin-like) when a comparison was made between serum samples of normal individuals to pregnant subjects or tumour samples. The results from another study demonstrated no significant differences in the percentages of isoform volume contribution of various isoforms of lgA1, hemopexin, α2-HS glycoprotein, of antichymotrypsin, Ua (hemopexin-like) and Uc (haptoglobin-like) when compared between serum samples of normal individuals to pregnant subjects or tumour samples.