Effect of Modification of the Carboxyl Groups of the Sialic Acid Binding Lectin from Bullfrog (Rana catesbeiana) Oocyte on Anti-tumor Activity.

Abstract

The sialic acid binding lectin from Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity.2) cSBL is a multifunctional protein which has pyrimidine base specific ribonuclease activity. 3,4) In respect to RNase, cSBL belongs to the RNase A family, and is a very heat stable enzyme. It remained 100% active after heating at 100°C. 3,5) Four RNases belonging to the RNase A family have been isolated from Rana family frogs to date. Their primary structures are shown in Fig. 1. They have anti-tumor activity, except for that from bullfrog liver. 11)They are a simple protein and constituted from 104 to 111 amino acid residues. The anti-tumor activity of one of them, onconase, obtained from Rana pipiens, has been studied in detail, and is on its way to application as an anti-tumor drug, now in phase III clinical trials. 12,13) On the other hand, the anti-tumor activity of cSBL has been reported by Nitta et al.2) Although the mechanism of the anti-tumor activity of cSBL is not fully elucidated to date, the first step of the action was different from that of onconase. That is, the interaction of cSBL with sialoglycoprotein on the surface of tumor cells is thought to be essential for cSBL action. The interaction enhances the internalization of cSBL into the tumor cells. However, in contrast to cSBL, for onconase, the contribution of sialic acid to the action has not been identified to date.Nitta et al. showed that the chemical modification of cSBL with a water-soluble carbodiimide (EDC) in the presence of glycine methylester increased the anti-tumor activity. The The sialic acid binding lectin from bullfrog Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity. In order to investigate the relationship between the net charge of cSBL and its anti-tumor effect, cSBL was modified with a water-soluble carbodiimide (EDC) in the presence of three kinds of nucleophiles, taurine, glycine methylester and ethylenediamine. cSBL having four carboxyl groups was partially modified (ca. 2 residues). The anti-tumor activity of modified cSBLs was in the order of ethylenediamine-modified cSBLϾglycine methylestermodified cSBLϾtaurine modified cSBLмnative cSBL. The results suggested that anti-tumor activity seems to increase with the increase in positive net charge, possibly enhancing the interaction of cSBL with sialoglycoprotein on the surface of tumor cells. The ribonuclease activity of ethylenediamine-modified cSBL decreased with the progress of the reaction, but the number of internalized molecules in the tumor cell increased. Thus, for antitumor activity, a higher incorporation of cSBL with reasonable RNase activity seems to be more important than total RNase activity.

Effect of Replacing the Aspartic Acid/Glutamic Acid Residues of Bullfrog Sialic Acid Binding Lectin with Asparagine/Glutamine and Arginine on the Inhibition of Cell Proliferation in Murine Leukemia P388 Cells.

“…In the previous report, 12) we reported that a chemically modified form of cSBL with a higher positive net charge was internalized more effectively into P388 cells than the native cSBL. Therefore, we investigated the relative rate of internalization of the mutant cSBLs.…”

“…The results of this report were qualitatively consistent with similar experiments in which the acidic amino acid residues of cSBL were modified chemically with EDC-ethylenediamine. 12,13) The substitutions of E97 with arginine and glutamine most affected the enzymatic activities of cSBL, when RNA was the substrate. This may be due to the fact that E97 is one of the constituents of the B2 base recognition site of the RNase A superfamily, and is conserved in the RNase A superfamily, although we do not know the exact location of this group in the substrate analoguecSBL complex.…”

“…2) In the previous paper, 12) we reported that (i) modification of the carboxyl groups of cSBL with EDC in the presence of nucleophiles enhanced the cytotoxic activity toward P388 cells. The enhancing effect is dependent on the increase in positive net charge, thus the cytotoxicity was greatest in the order of cSBL modified in the presence of ethylenediamine (EDC-ED cSBL)Ͼglycine methylester (EDC-GM cSBL)Ͼ taurine (EDC-TA cSBL).…”

“…On the other hand, Wu, N. Y. reported (cited in ref. 11) that the anti-tumor activity of onconase is not affected by pretreatment with sialidase, in spite of its structural similarity with cSBL.Nitta et al reported that the chemical modification of cSBL with a water soluble carbodiimide (EDC) in the presence of glycine methylester increased its anti-tumor action.2)In the previous paper, 12) we reported that (i) modification of the carboxyl groups of cSBL with EDC in the presence of nucleophiles enhanced the cytotoxic activity toward P388 cells. The enhancing effect is dependent on the increase in positive net charge, thus the cytotoxicity was greatest in the order of cSBL modified in the presence of ethylenediamine (EDC-ED cSBL)Ͼglycine methylester (EDC-GM cSBL)Ͼ taurine (EDC-TA cSBL).…”

Enzymatic Properties of Sialic Acid Binding Lectin from Rana catesbeiana Modified with a Water-Soluble Carbodiimide in the Presence of Various Nucleophiles.

“…The location of the acidic amino acid residues in the primary structure modified with EDC has yet to be determined. 5) To elucidate the fundamental mechanism by which antitumor activities enhanced, we investigated the RNase activity of the modified cSBL. We focused on the effects of modification on double stranded RNase activity and the base specificity of the modified cSBL.…”