B PIGB-KO cells expressing low levels of CD59 and DAF (blue line). Complete removal of residual NTNG2 were affinity purified from the supernatant of PI-PLC treated Expi 293F cells transfected with N-terminally HA tagged NT5E or NTNG2, followed by SDS-PAGE and in-gel trypsin (NT5E) or chymotrypsin (NTNG2) digestion. As for NT5E, by LC-MS analysis, we found precursor ions corresponding to the C-terminal 2 amino acid (FS) peptide linked to GPIs having three Man and a HexNAc side chain (m/z 796.242+; MW 1590.48) as well as some bearing Hex extension (m/z 877.262+; MW 1752.52). MS/MS analysis indicated not only the GPI diagnostic fragments, but also the fragments which are specific to protein attachment to Man2-linked EthN-P. The analysis of NTNG2 showed precursor ions corresponding to the C-terminal 22 amino acid (TGVRCEQPRCDPADDDGGLDCD) peptide linked to GPIs having three Man and a HexNAc chain (m/z 1282.783+; MW 3845.49, m/z 962.344+; MW 3845.36, m/z 770.075+; MW 3845.35) as well as some bearing Hex extension (m/z 1002.854+; MW 4007.40, m/z 1336.803+; MW 4007.40). MS/MS analysis indicated not only the GPI diagnostic fragments, but also the fragments which are specific to the novel structure. These specific fragment ions were present in all precursor ions from NTNG2 and NT5E. These results indicated that NT5E and NTNG2 expressed in wild type HEK293 cells has the preference to PIGG dependent structure (Figure 3). Therefore, reduced levels of these specific GPI-APs may result in the clinical symptoms associated with PIGG-IGD, such as developmental delay, intellectual disability, seizures and hypotonia. Recently, loss-of-function mutations in NTNG2, which is selectively expressed in presynaptic region, were found Figure 1. Complete silencing of residual expression of CD59 and DAF GPI-APs in PIGO-, and PIGB-KO HEK293 cells by knocking out PIGGA PIGO-KO cells expressing low levels of CD59 and DAF (blue line). Complete removal of residual C-terminal 11 amino acid peptide linked to GPI having two Man and a HexNAc side chain (m/z 858.03+ and 1286.42+; MW 2570.87) and those bearing Hex extension (m/z 912.03+; MW 2732.9) were found. MS/MS analysis showed not only the GPI diagnostic fragments (m/z 422+, 447+, 707+, 1421+ and 1663+) but also two fragments corresponding to P-Man-(EthN-P-) Man-GlcN (m/z 689+) and peptide-EthN-P-Man-(EthN-P-) Man (m/z 1948+), that are diagnostic of peptide-attachment to Man2-linked EthN-P (Figure 2A and B). These fragment ions were found in approximately 10% of precursor ions with MW2732.9, which have three Man in wild type cells. These results indicate that approximately 10% of CD59 protein is expressed with this novel structure in wild type cells.expression of CD59 and DAF in PIGO-KO cells by further knockout of PIGG (orange line).expression of CD59 and DAF in PIGB-KO cells by further knockout of PIGG (orange line).Expression of several GPI-APs are PIGG dependent and preferentially bridged through Man2-linked EthN-P.We hypothesized that some GPI-APs are preferentially linked in this way and the expression of these proteins might be lower in PIGG-KO cells than in wild type cells. Through the quantitative proteomics analysis comparing PIGG-KO and wild type cells, and through comparison of expression levels of transfected GPI-APs between PIGG-KO cells and those with PIGG-rescued cells, we found 18 GPI-APs out of 50 as the candidates with this novel structure. Among them, NTNG2 and NT5E (CD73) were picked up to demonstrate attachment of these proteins to Man2-linked EthN-P by LC-MS/MS. HA tagged NT5E and 95
元のページ ../index.html#95