The Effect Of Different Polysialylated NCAM On The Growth And Motility Of1dlD-14Cell

Neural cell adhesion molecule (NCAM) is a glycoprotein expressing on the surface ofneurons, glial cells, bone cells and natural killer cells, and belongs to the immunoglobulinsuperfamily. NCAM plays an important role in the process of cell-cell adhesion, neuronalmigration and signal transduction. There are6N-glycosylation sites on NCAM, and the majorcarbohydrate is polysialic acid (PSA), NCAM is the model protein for studying polysialicacid. The synthesis of PSA is catalyzed by two different polysialyltransferases, ST8Sia II(STX) and ST8Sia IV (PST). Both are members of the sialyltransferases family andGolgi-resident type II transmembrane proteins, and have high similarity in structure andfunction.ldlD-14is a Chinese hamster ovary (CHO) mutant cell line that deficient in the enzymeUDP-galactose (Gal) and UDP-N-acetylgalactosamine (GalNAc)4-epimerase. When ldlD-14cells are grown in glucose-based media, they can not synthesize UDP-Gal and UDP-GalNActo allow normal synthesis of glycans. But all abnormal phenotypes of ldlD-14cells can befully corrected by exogenous Gal and GalNAc. Due to the unique characters, ldlD-14cellsmay be useful for structural and functional studies of many glycoproteins, proteoglycans, andglycolipids containing Gal or GalNAc.In this study, NCAM-140, STX and PST genes from normal murine mammary glandepithelial (NMuMG) cells were cloned into eukaryotic expression vectors pcDNA3.1(+),pIREShyg3and pIRESpuro3, respectively, and transfected into ldlD-14cells. Carbohydratechain of NCAM molecule in stable-transfected ldlD-14cells can be easily manipulated byadding Gal in the serum free medium or not. On this account, the effects of glycosylation onthe NCAM molecular function were further studied.First of all, pCDNA3.1vector containing NCAM-140gene was transfected into ldlD-14cells. The stable transfectant over-expressing NCAM-140was obtained through the G418selection, confirmed by western blotting and named as ldlD-14/N-140. Transfected cellshowed changed morphology when observed under microscope. MTT assay and phagokineticgold sol assay were performed to analyze cell growth and cell motility. Results show thatover-expressed NCAM-140significantly enhanced cell growth and cell migration comparedto the control group. Secondly, STX and PST were transfected into ldlD-14/N-140cells. Thestable transfectants of STX and PST were confirmed by real-time quantitative PCR andwestern blotting, and then cultured in serum-free medium containing onlyInsulin-transferrin-selenium (ITS) or both ITS and Gal. Results of the MTT assay indicatedthat, compared with ldlD-14/N-140cells under these two conditions, the multiplicationcapacity of ldlD-14/NS cells were strikingly reduced, but ldlD-14/NP cells was quite on the contrary. Phagokinetic motility of ldlD-14/NS and ldlD-14/NP cells were all increasedcompared with ldlD-14/N-140cells.