The Effect of Fucoidan from the Brown Alga Fucus evanescence on the Activity of α- N-Acetylgalactosaminidase of Human Colon Carcinoma Cells

Abstract

α-N-acetylgalactosaminidase (EC 3.2.1.49) (alpha-NaGalase) catalyzes the hydrolysis of N-acetamido-2-deoxy-α-d-galactoside residues from non-reducing ends of various complex carbohydrates and glycoconjugates. It is known that human cancer cells express an alpha-NaGalase, which accumulates in the blood plasma of patients. The enzyme deglycosylates the Gc protein-derived macrophage activating factor (GcMAF) and inhibits macrophage activity acting as an immunosuppressor. The high specific activity 0.033 ± 0.002 μmol mg^−1 min^−1 of the enzyme was found in human colon carcinoma cells DLD-1. The alpha-NaGalase of DLD-1 cells was isolated and biochemical characterized. The enzyme exhibits maximum activity at pH 5.2 and temperature 55 °C. The K_m is 2.15 mM, V_max⁻0.021 μmol min^−1 mL^−1, k_cat⁻1.55 min^−1 and k_cat/K_m⁻0.72 min^−1 mM^−1 at 37 °C, pH 5.2. The effects of fucoidan from the brown alga Fucus evanescence on the activity of alpha-NaGalase in human colon carcinoma DLD-1 cells and on the biosynthesis of this enzyme were investigated. It was shown that fucoidan did not inhibit free alpha-NaGalase, however, it reduced the expression of the enzyme in the DLD-1 cells at IC₅₀ 73 ± 4 μg mL^−1.

Keywords: DLD-1; Fucus evanescence; alpha-NaGalase; brown alga; colon carcinoma cells; fucoidan; macrophage activating factor; α-N-acetylgalactosaminidase.

Figure 1

Purification of alpha-NaGalase from lysate of cancer cells DLD-1: (А) Scheme of enzyme purification; (B) 12% SDS-PAGE of purified alpha-NaGalase (1), molecular mass markers (2).

Figure 2

pH-Dependence of alpha-NaGalase activity of DLD-1 human colon carcinoma cells, 0.05 M sodium citrate buffer, 37 °C.

Figure 3

Effect of temperature on the alpha-NaGalase activity of DLD-1 human colon carcinoma cells, 0.05 M sodium citrate buffer, pH 5.0.

Figure 4

Temperature stability of alpha-NaGalase of DLD-1 human colon carcinoma cells at different exposition time; 1 and 6 correspond to 100% activity and 50% activity of the enzyme, respectively, 0.05 M sodium citrate buffer, pH 5.0; graph 2, 3, 4, 5 correspond to 15, 30, 45 and 60 min of exposition, respectively.

Figure 5

Dependence of p-Nitrophenyl-N-acetyl-α-d-galactosaminide hydrolysis rate on the concentration catalyzing by alpha-NaGalase of DLD-1 cancer cells in double reciprocal Lineweaver–Burk coordinates, 0.05 M sodium citrate buffer, pH 5.0, 37 °C. The inset table shows the results of liner fitting with using the computer program “Orijin 8.0”.

Figure 6

Effect of NaCl on the alpha-NaGalase of DLD-1 human colon carcinoma cells.

Figure 7

Effect of fucoidan from brown alga Fucus evanescence on the production of alpha-NaGalase in colon cancer cells DLD-1 and on the activity of the free alpha-NaGalase, 0.05 M sodium citrate buffer, pH 5.0, the enzyme with fucoidan was incubated for 30 min at 20 °C. Data are shown as means ± standard deviation (SD) of values from three independent experiments. The asterisk (*) indicates a significant decrease in activity of cellular alpha-NaGalase of DLD-1 cells treated with fucoidan compared with the non-treated cells (** p < 0.01, *** p < 0.001.).