PGAP6, a GPI-specific phospholipase A2, has narrow substrate specificity against GPI-anchored proteins

Abstract

PGAP6, also known as TMEM8A, is a phospholipase A2 with specificity to glycosylphosphatidylinositol (GPI) and expressed on the surface of various cells. CRIPTO, a GPI-anchored co-receptor for a morphogenic factor Nodal, is a sensitive substrate of PGAP6. PGAP6-mediated shedding of CRIPTO plays a critical role in an early stage of embryogenesis. In contrast, CRYPTIC, a close family member of CRIPTO, is resistant to PGAP6. In this report, chimeras between CRIPTO and CRYPTIC and truncate mutants of PGAP6 were used to demonstrate that the CFC (Cripto-1/FRL1/Cryptic) domain of CRIPTO is recognized by an N-terminal domain of PGAP6 for processing. We also report that among 56 human GPI-anchored proteins tested, only glypican 3, prostasin, SPACA4 and contactin-1 in addition to CRIPTO are sensitive to PGAP6, indicating that PGAP6 has a narrow specificity toward various GPI-anchored proteins.

Ethanolamine-phosphate on the second mannose is the preferential bridge for some of the brain GPI-anchored proteins

“…For instance, GPI-APs in red blood cells are completely resistant to PI-PLC as they are not deacylated from inositol by PGAP1 (Roberts et al, 1988). PGAP1 activity is reported to have close association with ER stress (Liu et al, 2018). Under ER stress conditions, misfolded GPI-APs accumulate in the ER and deplete the available PGAP1, resulting in the exit of normal GPI-APs from the ER without being processed by PGAP1 (Liu et al, 2018).…”

“…PGAP1 activity is reported to have close association with ER stress (Liu et al, 2018). Under ER stress conditions, misfolded GPI-APs accumulate in the ER and deplete the available PGAP1, resulting in the exit of normal GPI-APs from the ER without being processed by PGAP1 (Liu et al, 2018). What structure is recognized by PGAP1 and how PGAP1 activity is regulated under non-stressed conditions, as well as the resulting GPI-APs sensitivities to PI-PLC under these conditions, are worth being clarified.…”

“…GPI-APs were HA-tagged by cloning their coding cDNA amplified from the human cDNA library or from Invitrogen plasmids, as described (Lee et al, 2020), into the mammalian expression vector (pME-puro) and co-transfected into the PIGG-KO cells with PIGG cDNA or the empty vector as control. The expression levels of the GPI-APs were determined by flow cytometric analysis…”

Inflammatory Effects of Bothrops Phospholipases A2: Mechanisms Involved in Biosynthesis of Lipid Mediators and Lipid Accumulation

“…These enzymes hydrolyze the acyl ester bond at the sn-2 position of phospholipids, generating free fatty acids, such as arachidonic acid (AA) and oleic acid, and lysophospholipids, such as lyso-PAF [21,22]. Currently, PLA 2 s are classified into close families, including secretory PLA 2 (sPLA 2 ), cytosolic phospholipase (cPLA 2 ), and Ca 2+ -independent PLA 2 (iPLA 2 ); platelet activating factor acetylhydrolase (PAF); and lysosomal PLA 2 (LPLA 2 ), PLA/acyltransferase (PLAAT), α/β hydrolase (ABHD), adipose-PLA 2 (AdPLA), and glycosylphosphatidylinositol (GPI)-specific PLA 2 [23][24][25]. This classification of PLA 2 s is based on the cell location, amino acid sequence, molecular weight, presence of intramolecular disulfide bridges, calcium requirement, and catalytic activity, including the hydrolysis at the sn-2 position of glycerophospholipids [22,26].…”

ER entry pathway and glycosylation of GPI-anchored proteins are determined by N-terminal signal sequence and C-terminal GPI-attachment sequence

“…The amplified sequences were ligated into pIRES2-ZsGreen1 plasmid (Takara) digested with the same enzyme pair. The cDNA sequences of the mature protein part and C-terminal GPI-AS of hPrion were amplified from pME-ssHA-hPrion ( 67 ) as a template using primers (#30 and #29 and #31 and #29), followed by digestion with Pst I and BamH I. The amplified sequences were ligated into pIRES2-ssPrion-hCD59-Prion and pIRES2-ssCD59-hCD59-Prion digested with the same enzyme pairs.…”