Research On Liver Glycogen To Reveal The Binding Mechanism And Pathological Structure In Type-2 Diabetes

Liver glycogen is an important energy reservoir in mammals. Glycogen exists widely in our body, liver and muscle is the two main deposition in human. Liver glycogen helps in balancing the blood-sugar homeostasis. Structurely, glycogen is a hyper-branched polysaccharide composed by glucose monomers, it can be divided into three different structural levels: the first level refers to glucose monomer forming linear chains via α-(1,4) bonds and branching atα-(1,6) bonds, there were average 12 glucose monomers in very chain; second level refers to β-particles composed by this linear and branching chains, whose diameter is around 20 nm; the third level refers to α-particles composed by β-particles, its diameter is around 100 nm.This research focuses on the third level, aims at identifying the key protein(s) combining small glycogen β-particles into large α-particles. Relationship between the structure difference of glycogen from db/db mice(a type-II diabetes animal model) and healthy mice were studied, as well as the correlation between the difference and the qualitative and quantitative behavior of the key protein(s) to search a novel drug target for diabetes.This thesis is composed by four parts.In the first part, the methodology of purifying glycogen from its associated protein is explored. a. trypsin was used to digest the proteins on the surface of glycogen into peptides, which was excluded by the use of molecular weight cut off ultra spin tube(≤10kDa). Analytical SEC was used to test the possibility of trypsin degrading glycogen at the same time. b. preparative SEC was applied to separate and purify glycogen according to the molecular size between glycogen and proteins. After that, α-amylase was used to digest and release the proteome containing both intrinsical and external proteins associated with glycogen. On this proteome, we employed high resolution MS instrument----triple-TOF to identify proteins, and decide which method has the most efficient purification capacity. lots of glycogen unrelated proteins are still binding with glycogen after trypsin purification method through proteomic analysis; while SEC purification shows excellent ability that only one glycogen related protein existsglycogenin, the others are all contaminants during the procedure. It suggests that glycogen after SEC purification has the most stringent and accurate proteome.In the second part, the possible “binding glue” identified previously was expressed through transgenic technique, and validated by in vitro experiments. That could help to confirm the postulate that small β-particles would form large α-particles via the binding protein. The gene expressing the target protein was integrated into the vector from E.coli and transfected into it. Once the target protein was obtained, multiple method was employed to test its sugar binding activity. Glycogen extracted from mice was incubated with this sample in vitro and its size was observed, to provide direct evidence of glue binding glycogen molecule. Glycogenin shows mild polysaccharides binding activity, its incubation with glycogen molecule is still undergoing.In the third part, qualitative proteomics was used to explore the possible key proteins combining small β-particles into large α-particles followed with the confirmation of glycogen proteome purification method. Quantitative proteomic method was applied to explore the feasibility of identification of trace amount of proteins and finalise the quantitative methodology to reveal protein difference between the diabetic mice and healthy mice. A pathological model need to be proposed about the fragile db/db glycogen mechanism, and validated using the technique of SEC and FACE. All this indicate that quantitative proteomic research find that “glue” protein in β-particles from diabetic glycogen and healthy glycogen have sigficant difference: glycogenin in side of diabetic β-particle was accessible by trypsin even though the glycogen was not degraded by α-amylase, while the glycogenin in healthy β-particle could be protected. a postulate of the model stating that relatively loose structure of glycogen β-particle is the key to fragility in diabetic glycogen(fig 20) is proposed.In the forth part, the whole thesis was summarized from the aspect of meaningless of liver glycogen purification methodology, enlightment of glycogen structural research on diabetes and plan on next move of the research.