Although NCT undergoes complex glycosylation processing

Although NCT undergoes complex glycosylation processing for maturation through trafficking from endoplasmic reticulum to Golgi or additional compartments, the effect of NCT glycosylation on γ-secretase activity and substrate specificity is still unknown. The present study focused on glycosylation status of NCT on γ-secretase activity and substrate specificity in a panel of mutant CHO Lec (Lec-1 and Lec-2) cells defect in different steps of glycosylation processing. CHO Pro-5 was used as parental cells (WT), which lacks β-1,4 galactosyltransferase 6 gene expression. Proteins expressed in CHO Lec-1 cells carry mannose rich oligosaccharides due to the lack of glucosaminyltransferase-I (GnT-I) activity. CHO Lec-2 cells lack cytidine 5′-monophosphate (CMP)-sialic hsp90 inhibitors transporter, and are incapable of transporting CMP-sialic acid from the cytosol to the Golgi, thereby producing sialic acid-deficient complex oligosaccharides [13,14]. In this study, we demonstrated that the complex N-glycosylation including the terminal sialic acid residues on NCT glycan positively regulate the γ-secretase activity and absence of complete glycosylation of NCT (Lec-1) selectively reduced γ-secretase mediated C99 processing. These results highlight the functional importance of NCT glycosylation on γ-secretase activity and substrate specificity, providing new line of research for the development of improved biomarkers and treatment methods for AD.
Materials and methods
Results
Discussion AD is the most common cause of dementia in elderly people. Currently 46.5 million people are suffering from dementia worldwide and the number is going to be doubled every 20 years [22]. Posttranslational protein modification including glycosylation, a relatively unexplored topic, would be an alternative arena of AD research since most of the AD related proteins are glycosylated and protein glycosylation is altered in AD [9,10]. This study demonstrates that complex glycosylation of NCT including terminal sialylation is critical for γ-secretase activity. We also found that immature NCT as shown by CHO Lec-1 preferentially reduced Aβ generation in both cell-based and biochemical assays (Fig. 1, Fig. 2B, respectively). Pioneer study reported that γ-secretase activity was not influenced by glycosylation status of NCT and kifunensine, a mannosidase I inhibitor, did not inhibit Aβ generation [7], which is discrepant with our observation. To interpret this discrepancy, we revisited the issue and found that kifunensine did not inhibit Aβ generation in C99 transfected HEK293 cells (data not shown). This is possibly because immature like NCT generated by kifunensine treatment exhibits characteristics like mature NCT including trypsin resistance, thus immature like NCT is capable of generating Aβ like mature NCT since trypsin resistance of the NCT ectodomain is associated with γ-secretase activity, assembly and maturation [23]. A.R. Lopez and colleagues, using γ-secretase isolated from CHO Lec-1 overexpressing γ-secretase components, reported that glycosylation deficient γ-secretase exhibited activity comparable to that of fully glycosylated γ-secretase in vitro [24]. In contrast, our study demonstrated that mutant CHO Lec-1 without overexpression of γ-secretase components displayed reduced activity in cell-based and that γ-secretase assay using endogenous enzyme level also exhibited reduced Aβ production from CHO Lec-1 and Lec-2 (Fig. 1, Fig. 2B). It is possible to interpret that overexpression of γ-secretase components in cells resulted in saturated enzymatic activity of γ-secretase even in CHO Lec-1 in the pioneer study. CHO Lec-2 containing oligosaccharides deficient in terminal sialic acid showed reduced activity in cell-based assay whereas in biochemical assay Lec-2 partly restored its activity. Nonetheless, subcellular distribution of γ-secretase subunits was not changed in mutants as apparent in sucrose density gradient analysis (Fig. S1.), suggesting that increased activity of Lec-2 in biochemical assay may be due to enzyme concentration variation because we can not expect perfectly similar level of enzyme concentration between WT and mutants in the real reaction conditions. Intriguingly, the level of functional γ-secretase complex in mutants is higher or equivalent to that of parental cells as apparent in blue native gel analysis (Fig. 3A and B), strongly suggests that mutant γ-secretase either fails to recognize substrate or may have defect in their cleavage efficiency. Coimmunoprecipitation study demonstrated that mutant γ-secretase can recognize substrate like parental γ-secretase still they exhibited lower activity, suggesting that glycosylation deficiency of NCT induce cleavage defect in γ-secretase rather than substrate recognition. Mutagenesis including deeper glycobiology study will be significant to identify γ-secretase activity and substrate specificity modulating glycosylation site of NCT, and to see how AD pathology is regulated by the glycosylation status of NCT.