Published:2013-11-28 Hits:1064

1、Biological Functions of Glycosylation in Cancer
Aberrant glycosylation in cancer cells has been recognized for many years, but we still know little about its mechanism. The glycosylation of protein is regulated by glycotransferases. We focus on the studies of glycosyltransferases, try to reveal the role it plays in cancer.
We have discovered a novel ER-localized protein, ppGalNAc-T18 (T18), which regulates protein O-glycosylation. We found that T18 only existed in vertebrates and lacked classical GalNAc-transferase activity, it can specifically enhance the activity of other ppGalNAc-Ts, and affect the cell surface glycosylation and cell growth. This study challenged the classical concept that O-GalNAc glycosylation only occurs in Golgi, and suggested that there maight be a new mechanism regulating O-GalNAc glycosylation in ER. This result is published in Glycobiology (Glycobiology 2012, 22(5): 602-615).

Figure 1. (A) Veterbrate-specific T18 localized in ER rather than Golgi. (B) T18 lacks catalytic activity, but can selectively enhance the activity of other ppGalNAc-Ts. (C) Knockdown of T18 inhibited the cell growth, changed the cell morphology, and altered the cell O-glycosylation level.

2、Proteomics, Metabolomics & Glycomics Studies in Translational Medicine

1)Up-regulation of Type I Collagen During Tumorigenesis of Colorectal Cancer Revealed by Quantitative Proteomic Analysis 
Our study is to find some serum biomaker candidates of CRC using proteomic approach. We investigated the biological behavior of collagen I duing tumor development in serum and tissue from the same group of CRC patients by relative quantitative proteomics and molecular technologies. We found the serum levels of C-terminal telopeptide of collagen I (CTx) are correlated with staging and poor disease-free survival of CRC patients, suggesting it may provide additional information for prognosis of CRC. Our study also indicates that the degradation fragments in serum generated by some tumor-specific proteases is important biomarker candidates, and provides evidence for the further clinical application of serum proteomics. This result is published in Jounal of Proteomics (Jounal of Proteomics,2013,94:473-485).

Figure 2. Up-regulation of type I collagen during tumorigenesis of colorectal cancer revealed by quantitative proteomic analysis

2)Distinct Urinary Metabolic Profile and Serum Metabolite Markers of Human Colorectal Cancer
We totally have identified 249 serum metabolites in CRC patients based on metabonomics. A robust OPLS-DA model based on these identified metabolites was able to distinguish all of the CRC patients including all the TNM-I stage patients from healthy controls. Six typical differential metabolites concerning the key metabolic pathways have been selected as potential biomarkers for CRC diagnosis. In this study, we systematically observed the metabolite disorder in CRC patients. Notably, it was the first time to observe the increased synthesis and oxidation of fatty acids in CRC metabolism, which would help us in understanding the Warburg effect from a new perspective. Furthermore, we also observed the abnormal gut flora metabolism in CRC patients. All the results provide new clue to understand the pathogenesis of CRC, as well as a potential noninvasive diagnostic method for the early detection of CRC. This result is published in Journal of Proteome Research (Journal of  Proteome Research,2012,11(2): 1354-1363, Journal of  Proteome Research,2013,12(6): 3000-3009. )

Figure 3. Distinct urinary metabolic profile and serum metabolite markers of human colorectal cancer

3)Systematic Approach for Glycomics Studies
The first step of O-GalNAc glycosylation is catalyzed by ppGalNAc-T family. Human ppGalNAc-T family contains 20 members, 15 of them have catalytic activity. ppGalNAc-T family members display tissue-specific expression and substrate-specific activities. However, only a handful of O-glycosylated proteins have been reported as substrates of ppGalNAc-T(s). Thus, to decipher the substrate specificity of ppGalNAc-Ts, we have developed a strategy to identify potential substrates of ppGalNAc-Ts globally by taken advantage of the high-throughput capability of proteome microarray and the high specificity of click chemistry.We used UDP-GalNAz as an analog of natural donor for glycosylation on human proteome microarray, and used the alkynyl fluorescent probe to detect the glycosylated proteins. We took ppGalNAc-T2 as an example and successfully identified 226 candidates. Memberane proteins were highly enriched in the candidates while cytoplasmic and nuclear proteins were also found. We selected some candidates from p53 and tyrosin kinase related pathway, and all of them are validated in vitro. The in vitro O-glycosylation site on p53 was identified by Mass Spectrometry and O-glycosylation of p53 was also found in cell. However, the function of this modification remained to be studied. This strategy could be applied for other ppGalNAc-Ts, and this will greatly facilitate the construction of the complete ppGalNAc-T-substrate network for all ppGalNAc-Ts, and eventually help us to unveil the function of ppGalNAc-T systematically.

Figure 4. (A) Enzymatic reactions are performed ‘on-chip’ by ppGalNAc-T2 using UDP-GalNAz (left track) or UDP-GalNAc (right track) as donor substrate. After click chemistry reaction, GalNAz-modified proteins conjugated by alkyne-fluorophore are then detected by a fluorescence slide reader. (B) 226 proteins were identified as substrate candidates of ppGalNAc-T2. (C) 17 selected proteins from p53 network and Tyrosine kinase network were validated by HPA lectin blot in vitro. (D) The O-glycosylation site on p53 was identified by Mass Spectrometry.

Disulfide- and terminal alkyne-modified magnetic silica particles(DA-MSPs) were synthesized and used to covalently capture and reductively release azido glycopeptides via click chemistry and dithiothreitol treatment. Using DA-MSPs, an efficient and specific enrichment method for separating azido glycopeptides has been developed. This result is published in ChemComm(ChemComm,2012,48(47):5907-5909.)

Figure 5. Capture and release of azido glycopeptides with DA-MSPs.

3、A Novel diagnosis technology study for the glycan biomarker of human disease
Glycan is involved in diverse significant life progress, including cell recognition, cell differentiation, development, signal transduction, and immune response. A large number of studies have shown that abnormal glycosylation of tumor cells is closely related to tumor development. The glycan is expected to become an important target in the diagnosis and treatment of cancer. We use lectin microarray for the enrichment of glycoprotein and mass spectrometry for the identification of glycosylation sites and the structure of glycan. Thus we could find the signature glycoprotein and glycan closely related to tumor progression by bioinformatics. After in vitro and in vivo validation, these signatures could be applied to large-scale clinical serologic test for early detection of diseases.

Research group of Shanghai ICP 11031250 -1 2013 Shanghai Jiao Tong University pollution control

Support by: Wei Cheng