FUNCTIONAL CHARACTERIZATION OF NATURAL SUNSCREEN COMPOUND MYCOSPORINE-2-GLYCINE FROM EXTREMOPHILIC CYANOBACTERIUM Halothece sp. PCC 7418 IN MACROPHAGE CELL LINE AND FRESH WATER CYANOBACTERIUM

Abstract

Mycosporine-2-glycine (M2G) is a rare UV-screening compound in a group of mycosporine-like amino acids (MAAs). This molecule is found in only two cyanobacteria; Euhalothece sp. LK-1 and Aphanothece halophytica (Halothece sp. PCC 7418). The structure of M2G composed of 4-deoxygadusol as a core structure, attached by 2 molecules of glycine at C1 and C3 positions. M2G has a maximal adsorption at 331 nm and a better antioxidative activity than other MAAs. Thus, it is interesting in determination of other activities for further cosmeceutical and pharmaceutical applications. In this study, a high purity M2G was successfully extracted and purified from Halothece sp. PCC 7418 by using strong cation exchange chromatography, and reverse phase chromatography, respectively. The obtained M2G has a very low concentration of endotoxin (0.004 EU/mL). 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay found that M2G exhibited the highest radical scavenging activity at pH 6. In this study, anti-inflammatory activity of M2G in RAW 264.7 murine macrophage cell line at the transcriptional level under lipopolysaccharide inflammatory-induction was also examined. It revealed that iNOS, encoding inducible nitric oxide synthase enzyme, was highly suppressed by M2G (75±2 %). This is consistent with a decrease in nitric oxide level. COX-2 expression was also suppressed at < 10 µM M2G treatment. Anti-oxidative capability of M2G was inspected in transcriptional level under oxidative stress induced by H2O2. It was found that 5 µM of M2G suppressed sod1, which encodes the radical scavenging enzyme Cu/Zn SOD. Other antioxidant-related genes; cat, Hmox1, and Nrf2, were upregulated by the supportive of M2G as well. Heterologous expression of M2G biosynthetic gene cluster (Ap3858-Ap3855) in a fresh water cyanobacterium Synechococcus elongatus PCC 7942 revealed a higher H2O2-induced oxidative stress tolerance than that of the control cells. IC50 of H2O2 in the transformant cells harboring M2G genes and the control cells were 2.293±0.06 and 1.523±0.05, respectively. Transcriptional level of antioxidant genes in expressing cells revealed that cat, sodB, and tpxA, were 4.5±0.4, 2±0.2 and 5±0.2 times upregulated, respectively, under H2O2 oxidative stress.