Principal Investigator: Yoshiko Murakami
Grant Title: A novel structure of GPI anchored proteins and its physiological role
Abstract
Objectives: Glycosylphosphatidylinositol (GPI) is a glycolipid that anchors 150 or more proteins tothe mammalian cell surface. GPI precursor has three mannoses and all of them are modified byethanolamine-phosphate (EthN-P). It has been believed that EthN-P linked to the third mannose isalways used as a bridge to the protein whereas one linked to the second mannose is removed after GPI
is attached to proteins. The EthN-P seemingly transiently linked to the second mannose is importantbecause mutations in PIGG, which catalyzes this modification, cause inherited GPI deficiency
characterized by neuronal dysfunction. However, a role of this EthN-P has been unclear because GPIanchored proteins (GPI-APs) such as CD59 and DAF are normally transported and expressed with thenormal structure at normal level in mammalian PIGG-knockout (KO) cells. We tried to elucidate themechanism of neuronal abnormalities in the PIGG deficiencies.
Methods: We established the HEK293 cells highly expressing HFGF-CD59 (CD59 fused with epitope
tags and GST for easy purification) and knocked out PIGB in these cells. HFGF-CD59 proteins weresolubilized by PI-PLC-treatment from the wild type and the PIGB-KO cells, affinity-purified from thesupernatants and subjected to SDS-PAGE and in-gel trypsin digestion. The C-terminal peptide linked
to GPI glycan was determined by LC-ESI-mass spectrometry (MS). Similarly, HA-NT5E and HANTNG2, candidate GPI-APs with the novel structure, expressed on the Expi293F cells, were purifiedand analyzed.
Results: We found that PIGB-KO cells lacking the third mannose and PIGO-KO cells lacking EthNP linked to the third mannose expressed low levels of GPI-APs and that those residual GPI-APs werelost by further knockout of PIGG, indicating that the EthN-P on Man2 can be used to bridge CD59
and DAF in a low efficiency. In the MS sample from the PIGB-KO cells, precursor ions corresponding
to the C-terminal 11 amino acid peptide linked to GPI having two Man and a HexNAc side chain werefound. MS/MS analysis showed not only the GPI diagnostic fragments but also the fragments that are
diagnostic of peptide-attachment to Man2- linked EthN-P. These fragment ions were also found in
approximately 10% of precursor ions which have three Man in wild type cells. These results indicatethat approximately 10% of CD59 protein is expressed with this novel structure in wild type cells. We
next 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 and analysis by flow cytometry, NTNG2 and NT5E (CD73) were picked up and HA tagged
NT5E and NTNG2 were affinity purified to demonstrate attachment of these proteins to Man2-linked
EthN-P by LC-MS/MS. MS/MS analysis of precursor ions 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 NT5Eand NTNG2 expressed in wild typeERHEK293 cells has the preference toPIGG dependent structure. Therefore,
reduced levels of these specific GPI-APs
may result in the clinical symptoms
transported to Golgi
associated with PIGG-IGD.
GPI transamidase

PIGO P PIGG P
PIGF
PIGF P

PPPPPPPPPGAP1GPI transamidase

NT5E.
NTNG2etc.

CD59,
DAF,etc.

PIGK
GPAA1PIGS
PIGT
PIGUP

PGAP5PPPPP

PIGKGPAA1PIGSPIGTPIGUPPPPPGAP1PPPPPPP(PGAP5)PP