Flavio Nguyen posted an update 3 months ago
An inverted microscope equipped with a 106objective, a mercury lamp, a mirror unit consisting of 470-490 nm excitation filter, a 505 nm dichromatic mirror, a 510-550 nm emission filter and a 16-bit thermoelectrically cooled EMCCD were used for epifluorescence measurements. The derivatized GSH was excited with a 470-490 nm light ray through the objective. The fluorescence emitted by these molecules was collected by the same objective and the fluorescence images were acquired by the EMCCD. Image acquisition was controlled by the MetaMorph software. The micrographs of hRBCs without derivatization were observed by the inverted microscope under bright field and epifluorescence illumination conditions. Mannitol is one of the most abundant sugar alcohols in nature. It exists in a wide range of organisms: bacteria, fungi, higher plants and algae. Mannitol acts as an antioxidant, source of reducing power and osmoregulation substance. Similar to sucrose in higher plants, mannitol was proved to be a major primary photosynthetic product in Laminaria sp., Fucus vesiculosus and Eisenia bicyclis. Mannitol metabolism is one of the main traits that distinguish brown algae from other phyla. In vesicular plants, the mannitol synthesis from fructose-6-phosphate is catalyzed by mannose-6- phosphate isomerase, mannose-6-phosphate reductase and mannitol-1-phosphatase. While in algae, bacteria and fungi, mannitol cycle is proposed to be mediated by four enzymes: mannitol-1-phosphate dehydrogenase and M1Pase for synthesis of mannitol and, mannitol-2-dehydrogenase and fructokinase for its degradation. So far, the molecular knowledge on mannitol metabolism in algae is essentially uncharacterized. Based on expressed sequence tag libraries, Moulin et al. proposed a schematic representation of carbon uptake and fixation in Laminaria digitata, in which mannitol metabolism was involved. With the deciphering of Ectocarpus siliculosus genome, mannitol metabolic pathway was illustrated from the points of evolution, metabolic analysis, and functional gene characterization. Nevertheless, except for EsM1PDH and EsM1Pase, no other reports on the molecular mechanism of mannitol cycle were addressed in brown algae so far. Mannitol is widely applied in medicine, food and chemical industries and its global market is more than 13.6 million kg/ y. Generally, it accounts for 10-20% in brown algae depending on different harvesting time. In China, the mannitol yield is mainly from the kelp and other resources with annual output of approximately 8,000 t. In order to explore the mechanism of mannitol metabolism in the kelp, we initiate the study on the key enzyme of M2DH in the mannitol cycle. It is expected to decipher the structure-function relationship of SjM2DH and further benefit the yields and application of mannitol from S. japonica with biotechnical improvements in the future. Mannitol metabolism in marine plants is poorly understood so far. Although carbohydrate metabolism was deduced from genomic analysis of diatoms, no molecular reports were on mannitol cycle. In brown algae, the limited molecular knowledge available comes from M1PDH and M1Pase enzymatic assays in E. siliculosus. With regard to M2DH, no molecular studies were conducted so far except for the release of nucleotide sequence in E. siliculosus. Horizontal Gene Transfer of M2DH in Brown Algae According to the biochemical characters of mannitol-producing or degrading enzymes, the mannitol pathway in algae was considered to be basically similar to that in fungi. Here with the phylogenetic analysis of M2DHs, the SjM2DH was clustered into prokaryotic clade, which is closer to Proteobacteria and Actinobacteria. Although highly conservative residues were identical in Pro- and Eukaryotic species, the closer phylogenetic relationship indicated that SjM2DH was probably acquired from bacterial genome via horizontal gene transfer event. This was consistent with large-scale HGT found in carbon storage and cell wall biosynthesis in E. siliculosus. SjM2DH is a New Member of PSLDR Family Commonly, MDHs of fungi and higher plants belong to SDR and MDR family, respectively. However, gene structural and phylogenetic analysis of SjM2DH favored that SjM2DH is more alike to bacterial M2DHs, which belong to PSLDR family. Unlike SDRs and MDRs needing a triad of conserved Ser-Tyr-Lys residues or metal ion for catalysis, a conserved Lys459 acted as the basic base for SjM2DH activity. A highly conserved KxxxxNxxH motif was verified to be a unique catalytic signature among all PSLDR members. Here in this study, the presence of KLRLLNGGH segment in SjM2DH sequence is in accordance with this feature of PSLDRs. Previously, M2DHs identified from fungi and red algae were Pazopanib moa believed to be NADP -dependent, while bacterial M2DHs exclusively use NAD as cofactor. Here in our study, the presence of Asp234 and absence of Arg231 contributed to the specificity for NAD as cofactor over NADP for SjM2DH. Accordingly, the reduction of fructose by recombinant SjM2DH exclusively uses NADH as cofactor, which favored that SjM2DH is a member of PSLDR family. However, SjM2DH gene encodes a protein of 668 amino acids unexpectedly, which is beyond the length of reported PSLDRs so far. After searching ‘‘mannitol dehydrogenase’’ in NCBI protein database, the extension of N-terminal module was exclusively found in MDHs of brown algae and did not align with the better-characterized MDHs so far. Therefore, it is believed that the specificity of N-terminal domain should have influence on SjM2DH function. The deletion and insertion of b-sheets in SjM2DH spatial structure might be another character distinguishing brown algal M2DHs. Nevertheless, it needs to verify the function of these regions in the future. SjM2DH Functions in Abiotic Stress Tolerance Referred to sub-lethal stress conditions determined for E. siliculosus, we applied 400-1000 mM NaCl, 0-32% salinities to testify the influence of hyper- and hyposaline stress on SjM2DH. Short-term treatment of 2 h for each individual was adopted to avoid cell death. Unlike the up-regulation of EsM1PDH1 and EsM1PDH2 under hypersaline conditions, the transcription of SjM2DH decreased with increasing of NaCl concentrations. As M2DH could catalyze the mannitol oxidation, the decreasing trend implied that the kelp might resist high NaCl concentrations outside via reducing mannitol degradation. The juvenile sporophytes could maintain robust growth in the salinity as low as 0% for 2 h, with some ‘‘bubbles’’ developed owing to absorbing water from outside.