Effects of alginates on legumain activity in RAW 264.7 cells and on prolegumain autoactivation

Alginates belong to a family of linear co-polymers of 1→4-linked β-D-mannuronic acid (M) and α-L-guluronic acid (G). They are mainly found in marine brown seaweed, and their composition and structure are highly variable depending on the source from which they are isolated [1]. Alginates with a high content of M have been shown to induce secretion of TNF-α from the mouse macrophage cell line RAW 264.7 [2]. The tumour-associated protease legumain is synthesized as a zymogen and undergoes pH-dependent, autoactivation of the proform to reach an enzymatically active form [3]. Little is known about the effects of exogenous polysaccharides on cancer-associated proteases and, therefore, the aim is to elucidate possible effects of alginates on the level of legumain activity in macrophages and also the direct interactions between prolegumain and alginates in the extracellular environment. Alginates of different composition and molecular sizes were studied. The proteolytic activity of legumain (dF/s) was measured using a fluorogenic peptide substrate [4]. Here we show that alginates are able to increase the activity of legumain in RAW 264.7 cells, but with different potency; Alginates with 75% M > 60% M ≥50% M > 100% M ≥94% M. Activity is lost upon treatment with alginate lyase and chemical reduction of alginate carboxylic acid groups showing that molecular size as well as the polymer conformation are essential for activity. Heparin and a sulphated alginate were the most potent activators of legumain activity, but the least potent TNF-α inducing agents tested. Alginate with 94% M had the highest TNF-α inducing activity in RAW 264.7 cells. Alginates that may enter the lysosomes via micropinocytosis interact with prolegumain directly. Here we show that alginates through ionic interactions accelerate the autocatalytic activation of prolegumain at pH 4.0 and 5.0. At pH 5.5 only the alginate with 75% M was able to promote prolegumain activation.

Keywords: Alginates, mannuronic acid, guluronic acid, legumain, protease

References:

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[2] Yamamoto, Y., Kurachi, M. et al. (2007). Bioscience, Biotechnology, and Biochemistry, 71(1), 238 – 241.

[3] Li, D.N., Matthews, S.P. et al. (2003). The Journal of Biological Chemistry, 278(40), 38980 – 38990.

[4] Johansen, H.T., Knight, C.G. et al.(1999). Analytical Biochemistry, 273(2), 278 – 283.