Recombinant Glycoprotein N-glycosylation Of Cd44s Be Lsectin Recognition Mechanism Exploration

CD44 proteins are class I transmembrane glycoproteins mediating the response of cells to their cellular microenvironment. CD44 proteins participate in many cellular processes, including the regulation of growth, survival, differentiation and motility. The altered expression or dysfunction of CD44 proteins contributes to numerous pathological conditions. The adhesive qualities of the CD44 can lead to interaction between two different cells or between a cell and its pericellular matrix. CD44 mediates cell-cell and cell-matrix interactions, in large part through its affinity for hyaluronan (HA), a glycosaminoglycan of extracellular matrices (ECMs), but also potentially through its affinity for other ligands such as osteopontin, collagens. Furthermore the interaction of CD44 with these cellular proteins involved in motogenesis and proteolysis is a determinant factor in cell migration and invasion. The smallest CD44 isoform, which is known as CD44 standard (CD44s), is ubiquitously expressed in vertebrates in developing and adult organisms. The mRNA of this isoform contains no variant exons. Recently, more and more studies found the N-glycosylation of CD44s was involved in the regulation of its function. One primary function of CD44 is binding to HA. Metastasis is a multistep process that includes de-adhesion, migration, adhesion, and invasion. CD44 and its specific interaction with HA have been implicated in many of these processes. Reagents blocking the CD44/HA interaction have been found to prevent primary tumor growth and metastasis. Moreover, recent evidence suggests that glycosylation may be a major mechanism controlling the interaction between CD44 and HA. Some studies also reported that the N-glycosylation of CD44s is associated with the interaction between CD44s and L-selectin, P-selectin, fibrin (ogen). However, the glycosylation situation of CD44s including the types of the glycans, the sites occupancy of the glycosylation has never been comprehensively studied before.LSECtin is a member of the C-type lectins that is found predominantly on sinusoidal endothelial cells of the liver and lymph nodes, suggesting that it plays a specific role in these cells. It was reported that the interaction between LSECtin and glycoproteins on some kind of virus may concentrate certain viral pathogens in liver and lymph nodes and augment infectious. Recent study found that LSECtin can recognize the N-glycan of CD44s on breast cancer cells. A large number of studies show that breast cancer cells will metastasize to endothelial cells of lymph node on the opposite side. So it was spectulated that the recognition between LSECtin and CD44s may play an important role in the tumor metastasis from breast to lymph node, and the specific glycoform of CD44s is the keypoint of the recognition. In order to study the mechanism of the incident, comprehensive characterization of the N-glycosylation of CD44s is fundamental and very important.Glycosylation is one of most common post-translational modification. But the study of glycosylatin is lagged far behind the analysis of protein. It is because their biosynthesis occurs in a non-template controlled manner and mature structures arise through the activities of a series of enzymatic reactions. So the glycans on one site are mixtures of variants (named microheterogeneity), which makes the analysis for them very complicated. The characteristics of glycan structure determine that they do not have UV absorption, and have lower response in the mass spectrometry than peptides. So the derivatization of glycan or enrichment of glycopeptides before MS analysis should be carried on in the analysis of glycosylation. The process is multistep and complicated, especially in the situation of the available amount of samples is little, the analysis of its glycosylation is particularly difficult.In this thesis, the N-glycosylation of CD44s expressed in NS0 cells was characterized completely for the first time. We used the PNGase F deglycosylation in H218O to label the N-glycosylation sites and then analyzed the peptides and glycopeptides by LC-LTQ MS. As a result, all five predicted N-glycosylation sites were confirmed to be occupied by an oligosaccharide moiety. Then the N-glycans released by PNGase F digestion were analyzed by permethylation-MALDI-TOF MS and 2-AB labeled-ESI-Q-TOF MS. The profile of CD44s'N-glycans and the relative quantity were obtained. The glycopeptides and peptides of CD44s were separated by HILIC-UPLC system at the peptide level and detected online by a Q-TOF mass spectrometer to identify the information of site heterogeneity. The glycoforms on Asn5 are bi-, and triantennary complex type, little with fucose and NeuGc. The glycoforms on Asn37, Asn80 and Asn90 are also bi-, and triantennary complex type, all of them are core fucoslated, some structure carrying sialic acid. On Asn100, there are high mannose type structures having from five to seven mannose residues and no core fucosylation. A comprehensive study on the glycosylation status of CD44s was performed for the first time, which will facilitate future understanding about the roles of oligosaccharides from CD44s in related biological process.On the basis of completely characterization of CD44s'glycosylation, we made preliminary studies on the structure bases of the recognition between LSECtin and CD44s. We used the immbolized LSECtin to capture the specific glycopeptides of CD44s which was then detected by the LTQ-MS. The results showed that the Asn80 is the very important site recognized by LSECtin. The result was first validated by the mutation of N-glycosylation sites. When Asn80 was mutated to Ser, the interaction between the LSECtin and CD44s decreased clearly. It was reported that LSECtin can bind to GlcNAc-1-2Man with very high affinity. Then we treated the CD44s with three exoglycosidases and detected the interaction with LSECtin by ELISA. The results showed that the GlcNAc at the terminal of the glycan play the important role in the interaction between LSECtin and CD44s. The analysis of three dimentaional structure of CD44s reported by reference also indicated that the Asn80 is in a palce that can interact with other molecular easily. These findings have the important research significance in the understanding the mechanism of the recognition between LSECtin and CD44s. And it may provide new perspective to develope the drug to repress the metastasis of breast cancer.The N-glycosylatin of the CD44s expressed in CHO cells was also analyzed by the same way above. All five consensus sequences for N-glycosylation were found to be glycosylated. The profile of CD44s'N-glycans are detected by permethylation- MALDI-TOF MS and exoglycosidase treatment. The glycan forms of CD44s expressed in NS0 and CHO cells are compared. The glycans in NS0 cells are more fucosylated than in CHO cells. There are Gal (α1-3) Gal in NS0 cells andβform in CHO cell just like in human cells. They all have NeuGc in glycans, which is NeuAc in human cells. The differences of N-glycans between the two cells confirmed our deduction that recognition between the LSECtin and CD44s was not affected by the amount of fucose, linkage form of Gal, type of sialic acid.To solve the widespread problem in glycoproteomics studies, such as lower response of the glycan in mass spectrometry, a rapid and convenient derivatization method of oligosaccharides was developed. The hydrazones were generated by the reaction of the reducing ends of oligosaccharides and the hydrazide of CPH. The derivatives can be directly analyzed by MALDI-TOF-MS without any treatment. An approximately 10-fold increase in detection sensitivity was observed. The new developed method was applied to the analysis of N-glycans on CD44s expressed in CHO cells and satisfactory results were obtained. The method can be used to analyze the N-glycans of glycoprotein especially when only small amount of sample is available.