The Functional Study Of N-glycosylation On CD147and Its Role In Cancer Progression

Glycosylation of glycoproteins and glycolipids is one of many molecularchanges that accompany with malignant transformation. Changes inpost-translational modification of proteins are closely associated with theadhension, invasion and metastasis of tumor cells. The studies on the structureand founction of tumor associated glycosylation provide valuable clues to tumordiognosis and therapy.CD147is a transmembrane glycoprotein that widely expressed on numerouscells. It plays roles in many physiological and pathological processes, especiallyin cancer progression. CD147is highly expressed on many tumor cells andtissues. CD147is highly expressed in various human carcinoma tissues and celllines, correlating with tumor progression under experimental and clinicalconditions. The best characterised function of CD147is its ability to induce theexpression of MMPs including MMP-1, MMP-2, MMP-9and in stromal cells. A significant biochemical property of CD147is its high level ofglycosylation. N-glycosylation contributes to almost half of the size of themature CD147molecule. Many studied have demonstrated that theglycosylation of CD147contribute to its MMPs induction activity. However,largely is unknown when concerned to the glycosylation of CD147, especiallythe tumor associated glycan structures of CD147and its role in cancerprogression. The study on the nature of cancer-associated glycans present onCD147and their functions would create opportunities for the development oftargeted therapies for carcinoma.The main objects of this study including: identify the feature of N-glycanstructure on CD147; founctional study of N-glycans by site mutation threeglycosylated sites of CD147; founctional study of β1,6-branching N-glycans onCD147; revealing the regulation of CD147glycan forms（HG-CD147andLG-CD147）by EGFR signaling pathway.Part1: Purification of native CD147from human lung cancer tissue andresolution of glycan structures of purified native CD147by MS. We purifiednative CD147from human lung cancer tissue by immuno-affinitychromatography. deglycosylation treatment using peptide-N-glycosidase Frevealed that the purified eukaryotic CD147was N-glycosylated.Endoglycosidase F3treatment indicating that eukaryotic CD147containedasparagine-linked bi-antennary and tri-antennary complex oligosaccharides.Mature form of CD147contains β1,6-branched polylactosamines, which is atumor associated glycan structure. Resolution of glycan structures of purifiednative CD147by Complex Carbohydrate Research Center in the University ofGeorgia revealed that CD147contains a series of high mannose and the complextype N-glycans, and the tumor associated structure core fucose and sialic acid was also found.Part2: The oligomeric state of purified native CD147and its biologicalactivity. By comparing the oligomeric state of native eukaryotic CD147andnonglycosylated prokaryotic CD147, we found that native glycosylated CD147existed exclusively as oligomers in solution in solution, whereas theoligomerisation of prokaryotic CD147was much weaker than that of nativeeukaryotic CD147. The following functional studies confirmed that nativeglycosylated CD147directly stimulated MMPs production more efficiently thannonglycosylated prokaryotic CD147. The results suggesting that glycans onCD147preserve the stability of the tertiary and quaternary protein structure andhelp maintain an active molecular conformation, which is essential for CD147’sactivity.Part3: Glycosylation site mutation study to reveal the founction of CD147N-glycosylation. The glycosylation site mutation results indicated that amongthree N-glycan attachment sites （N44Q、N152Q、N186Q）, the N152Q mutantswere retained in the endoplasmic reticulum and unfolded protein responsesignaling was activated. The improper intracellular accumulation impaired itsMMP-inducing activity. Furthermore, the N186Q mutants suppressed theoligomerization of CD147.Part4: Functional study of β1,6-branching N-glycans on CD147. Increasedβ1,6-branching of N-glycans as a result of over expression of GnT-V in cancerplays an important role in tumor metastasis. We found that overexpression ofGnT-V resulted in elevated level of CD147on plasma membrane and in cellconditioned medium, thereby increased the induction of MMPs. Whereasswainsonine treatment suppressed this process.Part5: The regulation of CD147glycosylation by epidermal growth factor receptor signaling pathway. We found the gene and protein expression ofCD147is increased after the stimulation of EGF and AR. PD153035andZD1839are specific and potent inhibitor of the epidermal growth factor receptor(EGFR) tyrosine kinase. We found that the treatment of PD153035and ZD1839inhibited the conversion of the LG form of CD147to the HG form. Theintracellular CD147was increased after PD153035and ZD1839treatment.In conclusion, by investigating the structure and fouction of N-glycans onCD147, this study revealed that glycosylation is essencial for the muturation,plasma membrane translocation and oligomerization of CD147. Furthermore,N-glycosylation of CD147contributes to its MMP-inducing activity. Thepresent study reveals that post-translational modification throughN-glycosylation is a mechanism that modulates the functions of CD147incancer progression and that it would be valuable for the development of targetedtherapies for carcinoma.