Proteomic characterization and identification of murine liver and small intestine proteins modulated by tea (Camellia sinensis) consumption

Abstract

The oral intake of green tea, black tea and some of the tea constituents has been demonstrated to protect against various diseases and show protective effects on the cell. Tea is able to regulate gene expression, regulate enzymes, regulate the cell cycle, cause cell cycle arrest, induce apoptosis, and inhibit the proteasome. The aim of this study was to find proteins by a proteomic approach that may be modulated in mice drinking tea in contrast to control animals that receive water. Three groups; control, low dose, and a high dose tea group were chosen to determine the effect of tea on protein regulation of C57BL/6 male mice. Daily liquid consumption was measured, and even though the high dose group consumed less liquid they still ingested more tea than the low dose group at the end of the study. Weight gain was measured for all the groups but no significant differences were found. Some differences were found in organ weights of the low and high dose groups. There was no dose dependent effect for the liver and small intestine, while the colon showed a positive and the pancreas a negative dose dependent effect. Small intestine and liver proteins were separated by one and two dimensional gel electrophoresis. No significant differences were found for the small intestine and liver when the proteins were separated by tricine SDS PAGE. However some significant differences were found on the glycine SDS PAGE gels of both the small intestine and the liver. The small intestine had three significant bands at 66kDa, 45kDa and 10kDa. The three significant liver bands were at 110kDa, 66kDa and 14kDa. HPLC analysis of the liver 66kDa band showed that the band consisted of only one protein while the 14kDa band consisted of possibly two proteins. MS analysis of the 14kDa band identified the proteins as hypothetical protein XP_358319 (15 190Da) and immunoglobulin Ą chain (13 140Da). Although the identified proteins match the molecular weight of the 14kDa band these results will need to be confirmed by MudPIT. Thirty 2DE spots of the small intestine were regulated by tea. Ten of these spots were analyzed by MALDI TOF MS, but only seven of these proteins were identified. These proteins were S-phase kinase associated protein p19, hypothetical protein XP_903753, unnamed protein product, adenylyl cyclase-associated protein 2, developmental control protein, lysosomal acid phosphatase, and cytochrome P450 (CYP2D13). All seven the small intestinal proteins will need to be confirmed by de novo sequencing, to ensure the positive identification of the proteins. Currently there is no 2DE map in literature of the small intestine. This study will provide the first 2DE map of the murine small intestine proteins. Thirty three 2DE spots of the liver were regulated by tea. Twenty of these were analyzed by MALDI TOF MS, but only fifteen of these proteins were idenitifed. These regulated proteins are: superoxide dismutase, and glutathione peroxidase that are antioxidant enzymes to counteract oxidative stress, detoxification enzymes like glutathione S-transferase mu-1, glutathione peroxidase theta-1 and cytochrome b5. Annexin A4 is able to help stabilize plasma proteins and the cytoskeleton and may induce apoptosis, keratin 8 may help with network formation and reinforcement of cellular membranes, malate dehydrogenase for energy expenditure and ketohexokinase in carbohydrate metabolism, while ubiquitin conjugating enzyme E2 plays a role in protein turn over. Other identified proteins include inosine-triphosphate-pyrophosphatase, triosephosphate-isomerase, and myoglobin. This study provides a novel 2DE map for liver protein regulation by tea. This was the first study that has taken a proteomic approach to the identification of the overall regulation of proteins by tea. The aim of this study was met by identifying the tea regulated proteins and elaborating on the protective effects and possible cancer chemo preventative effects of tea.