Exploring Of The Methods On The Research Of Protein O-GlcNAcylation

O-linked attachment of N-acetyl-glucosamine (O-GlcNAc) is apost-translational modification on serine and threonine residues of nuclear andcytoplasmic proteins. O-GlcNAc ubiquitously presents in higher eukaryotes. SinceO-GlcNAcylation is discovered, a multiple of experiments have demonstrated thatO-GlcNAcylation plays an important role in modulating protein folding,transportation, activity, stability and so on. To date, more than one thousand proteinshave been found to be O-GlcNAcylated. These proteins participate in almost all thephysiological processes. Abnormality of O-GlcNAcylation closely correlates withcancer, diabetes, cardiovascular disease, and so on. Cellular O-linkedN-acetylglucosamine (O-GlcNAc) modification is modulated by only two enzymes:uridine diphosphate-N-acetyl-D-glucosamine: polypeptidyl transferase (OGT) andO-GlcNAcase (OGA).There are numerous evidences that O-GlcNAcylation plays an important role inmultiple physiological and pathological processes. More and more researchers payattention to O-GlcNAcylation. However, the study of O-GlcNAcylation has laggedfar behind other post-translational modifications, such as phosphorylation andN-linked glucose modification. This is because low O-GlcNAcylation level, unstableglycosidic bond, dynamic modification and low abundance target proteins. So it ishard to establish research methods of O-GlcNAcylation. Nowadays, a variety ofmethodological issues of O-GlcNAcylation must be resolved. This study investigateshow to obtain O-GlcNAcylated proteins and site mapping of O-GlcNAcylation.To study the physiological and pathological functions of the O-GlcNAcylation,it is essential to obtain highly O-GlcNAcylated proteins. However, in eukaryoticcells, proteins have a variety of post-translational modifications. It interferes with theresearch of O-GlcNAcylation. In addition, it’s hard to obtain a large number ofhighly O-GlcNAcylated proteins in eukaryotic cells. Nowadays, theO-GlcNAcylation of proteins in prokaryotic cells and in vitro are efficient ways tosolve this problem. The first chapter is to study preparation of O-GlcNAcylatedproteins.To establish a method of O-GlcNAcylation of recombinant proteins in prokaryotic cells, we choose Sp1, which is perhaps one of the best studiedO-GlcNAc-modified proteins, as a positive protein, and constructs a co-expressionsystem of Sp1and OGT in E.coli BL21(DE3). It verifies Sp1could beO-GlcNAcylated by co-expression with OGT. In this way, a method ofO-GlcNAcylation of recombinant proteins in prokaryotic cells is established. p120ctnhas prominent roles in cellular activities associated with cancer, including cell growth,adhesion, motility, and morphology. Our laboratory found that O-GlcNAcylation ofp120might play roles in cancer formation and metastasis. So we use the establishedmethod to study p120, and find that p120could be O-GlcNAcylated with this method.But it also finds that p120is O-GlcNAcylated at a higher level in eukaryotic cells thanin prokaryotic cells, so it explores the factors influencing O-GlcNAcylation of p120.The results show that there is no prominent effect to O-GlcNAcylation of p120byextending induction time, changing salt ion and nutrition component or addingglucose or its analogues. It suppose this is because some proteins in prokaryotic cellsinterfere with interaction of p120and OGT. So it uses Sp1to establish a method ofO-GlcNAcylation of recombinant proteins in vitro. It also finds that p120could beO-GlcNAcylated with this method. And O-GlcNAcylation of p120is at a higher levelin vitro than in prokaryotic cells.O-GlcNAcylation of different sites of proteins has different functions. Somapping O-GlcNAcylation sites of proteins has great significance. Massspectrometry has become the preferred method to map the O-GlcNAcylation sitesbecause of its simplicity and reliability. The second chapter is a methodologicalstudy of site mapping of O-GlcNAcylation with mass spectrometry.In this study, the process of site mapping of O-GlcNAcylation with massspectrometry is as follows: Firstly, the purified protein becomes peptides byenzymolysis of trypsin. Secondly, cysteine is blocked by oxidation or alkylation ofpeptides. Thirdly, O-GlcNAcylation is replaced with dithiothreitol (DTT) byBEMAD (β-elimination and Michael addition of DTT). Lastly, DTT-modifiedpeptides were enriched with the thiol column and detecting with mass spectrometry.This study investigates the process of pre-treatment to determine the way to blockcysteine, the condition of BEMAD and the feasibility of BEMAD and thiol columnenrichment.(This paper investigates the process of pre-treatment, including the wayto block cysteine, the condition of BEMAD, and feasibility of BEMAD binding thethiol column enrichment. It provides useful methods for sites mapping of proteins O-GlcNAcylation.In summary, this paper is a methodological study of preparation ofO-GlcNAcylated proteins and site mapping of O-GlcNAcylation with massspectrometry. It establishes methods of O-GlcNAcylation of recombinant proteins inprokaryotic cells and in vitro, and initially establishes a method of site mapping ofO-GlcNAcylation with mass spectrometry. The study will provide an effective toolto study of O-GlcNAcylation of proteins.