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Biosynthesis and biology of mammalian GPI-anchored proteins

Biosynthesis and biology of mammalian GPI-anchored proteins Biosynthesis and biology of mammalian GPI-anchored proteins Taroh Kinoshita royalsocietypublishing.org/journal/rsob Yabumoto Department of Intractable Disease Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan TK, 0000-0001-7166-7257 Review At least 150 human proteins are glycosylphosphatidylinositol-anchored pro- teins (GPI-APs). The protein moiety of GPI-APs lacking transmembrane Cite this article: Kinoshita T. 2020 domains is anchored to the plasma membrane with GPI covalently attached Biosynthesis and biology of mammalian GPI- to the C-terminus. The GPI consists of the conserved core glycan, phospha- anchored proteins. Open Biol. 10: 190290. tidylinositol and glycan side chains. The entire GPI-AP is anchored to the http://dx.doi.org/10.1098/rsob.190290 outer leaflet of the lipid bilayer by insertion of fatty chains of phosphatidyl- inositol. Because of GPI-dependent membrane anchoring, GPI-APs have some unique characteristics. The most prominent feature of GPI-APs is their association with membrane microdomains or membrane rafts. In the Received: 3 December 2019 polarized cells such as epithelial cells, many GPI-APs are exclusively expressed in the apical surfaces, whereas some GPI-APs are preferentially Accepted: 12 February 2020 expressed in the basolateral surfaces. Several GPI-APs act as transcytotic transporters carrying their ligands from one compartment to another. Some GPI-APs are shed from the membrane after cleavage within the GPI by a GPI-specific phospholipase or a glycosidase. In this review, I will summarize Subject Area: the current understanding of GPI-AP biosynthesis in mammalian cells and biochemistry/cellular biology/genetics discuss examples of GPI-dependent functions of mammalian GPI-APs. Keywords: glycosylphosphatidylinositol, post-translational modification, biosynthetic pathway, GPI 1. Introduction deficiency, protein shedding At least 150 human proteins are glycosylphosphatidylinositol-anchored pro- teins (GPI-APs) [1]. GPI-APs are integral membrane proteins present on the cell surface. The protein moiety of GPI-APs is basically hydrophilic lacking Author for correspondence: transmembrane domains, and is anchored to the plasma membrane (PM) Taroh Kinoshita with GPI moiety covalently attached to the C-terminus (figure 1). Sizes of the e-mail: tkinoshi@biken.osaka-u.ac.jp proteins range widely from only 12 amino acids (CD52 [2]) to greater than 200 kDa (alpha-tectorin [3]). Their functions also vary, including hydrolytic enzymes, receptors, adhesion molecules, protease inhibitors, complement regulators and prions (table 1). The GPI moiety of GPI-APs consists of the conserved core glycan, phosphatidylinositol (PI) and glycan side chains. The structure of the core glycan is EtNP-6Manα2-Manα6-(EtNP)2Manα4-GlNα6-myoIno-P-lipid (EtNP, ethanolamine phosphate; Man, mannose; GlcN, glucosamine; Ino, inositol) [4] (figure 1). The GPI is linked to the C-terminus via an amide bond generated between the C-terminal carboxyl group and an amino group of the terminal EtNP [4]. The entire GPI-AP is anchored to the outer leaflet of the lipid bilayer by insertion of hydrocarbon chains of PI (figure 1). GPI-anchoring is a post-translational modification. The core GPI is assembled on the endoplasmic reticulum (ER) membrane and is transferred en bloc to the precursor proteins immediately after their ER translocation (figure 2). The nascent GPI-APs, in which GPI structure is immature, undergo several remodelling steps in the ER and the Golgi apparatus (figure 3). For many GPI-APs, a side chain glycan is added in the Golgi. Finally, GPI-APs are transported to the PM (figure 3). © 2020 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited. royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 protein EtN glycan ±± ± P EtN GPI lipid plasma membrane P EtN P glucosamine ethanolamine phosphate phosphatidylinositol sialic acid galactose N-acetylgalactosamine mannose Figure 1. Mammalian GPI-APs. The conserved core glycan of mammalian GPI, which consists of EtNP attached to the protein, three Mans, EtNP attached to Man1, and GlcN, is linked to the lipid moiety, which is PI. In some GPI-APs, the core glycan is modified by Man4 and/or GalNAc side chains. The GalNAc side chain can be elongated by Gal and Sia. The entire GPI-AP is anchored to the outer leaflet of PM only by hydrocarbon chains of PI. Because of GPI-dependent membrane anchoring, 2. Biogenesis of GPI-APs: post-translational GPI-APs have some unique characteristics. A prominent feature of GPI-APs is their association with membrane modification of proteins with the GPI microdomains or membrane rafts [5,6]. The membrane mediated by GPI transamidase microdomains are dynamic domains of 20–100 nm width [7]. Sphingolipids and cholesterol are enriched in the outer 2.1. Translocation of the precursor proteins into the ER leaflet of the lipid bilayer in the microdomains. GPI-APs are thought to associate with glycosphingolipids and choles- The precursor proteins of GPI-APs, preproproteins, have terols via lipid–lipid and glycan–glycan interactions. For an N-terminal signal peptide for ER translocation and a lipid–lipid interactions, saturated lipid chains of both sphin- C-terminal signal peptide for GPI attachment (figure 4) golipids and GPI anchors are critical [8,9]. Within the [30]. The N-terminal signal peptide consisting of about 20 membrane microdomains, GPI-APs form dimers with a hydrophobic amino acids is similar to those of other secretory half-life of 100 ms [10]. For GPI-AP homodimer formation, proteins [30]. The C-terminal GPI attachment signal peptide protein–protein interactions are critical [10,11]. GPI- spans 20–30 amino acids starting from the ω+1 amino acid APs-containing microdomains are regulated by cortical (the amino acid to which GPI is attached is termed the ω actin [12]. For the regulation by cortical actin, transbilayer site amino acid [30]). The GPI attachment signal peptide con- interaction between lipid moiety of GPI-APs in the outer sists of a stretch of about 10 hydrophilic amino acids and a leaflet and phosphatidylserine in the inner leaflet is critical stretch of about 20 hydrophobic amino acids. Amino acids [13]. Src family tyrosine kinases [14] and other proteins at the ω site are always small ones, including Ser, Asn, Asp, such as phospholipase Cγ (PLCγ) are associated with mem- Ala, Gly, Cys and Thr, and the ω+2 amino acid is also small brane microdomains in the inner leaflet. Upon ligation, [31–33]. In some GPI-APs, more than two ω sites were ident- GPI-APs make bigger clusters that lead to activation of Src- ified [33]. The GPI attachment signal peptide can direct GPI tyrosine kinases and PLCγ [15,16]. attachment to non-GPI-APs if it is fused to the C-terminus In the polarized cells such as epithelial cells, many GPI- (see the review for further discussion [34]). Upon transloca- APs are exclusively expressed in the apical surfaces [17] tion of a preproprotein into the ER lumen, the N-terminal whereas some GPI-APs are preferentially expressed in the signal peptide is removed from the precursors generating basolateral surfaces [18] (see the reviews for mechanisms of proproteins (figure 4). The C-terminal signal peptide is recog- GPI-APs sorting [19–22]). Several GPI-APs act as transcytotic nized by the GPI transamidase, which cleaves and replaces transporters carrying their ligands from one compartment to it with a preassembled GPI by transamidation, generating another [23–25]. Some GPI-APs are shed from the membrane nascent GPI-APs [30,35,36]. after cleavage within the GPI by a GPI-specific phospholipase Mechanisms of ER translocation of two GPI-APs, the or a glycosidase [26–29]. prion protein and CD59, are different. The prion requires In this review, I will summarize the current understand- Sec62 and Sec63 whereas CD59 does not [37,38], suggesting ing of GPI-AP biosynthesis in mammalian cells and discuss that the prion translocates in a signal recognition particle examples of GPI-dependent functions of mammalian (SRP)-independent manner whereas CD59 translocates in GPI-APs. an SRP-dependent manner [37]. The dependency of the royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 Table 1. Examples of mammalian GPI-APs. 2.2. GPI transamidase GPI transamidase is an ER-resident enzyme complex that functional mediates GPI-anchor attachment to proteins [41,42]. GPI GPI-AP category roles/characteristics transamidase cleaves the GPI attachment signal peptide between the ω and ω + 1 amino acids, generating a sub- alpha-tectorin unknown hearing/largest GPI-AP strate–enzyme intermediate linked by a thioester bond CD52 unknown T-cell CAMPATH-1 between the ω amino acid carboxyl group and a catalytic antigen/smallest cysteine side chain of the enzyme. The thioester bond is attacked by an amino group of the terminal EtN of GPI, GPI-AP completing a transfer of GPI by transamidation [35]. CD55 (DAF) complement self-damage protection GPI transamidase consists of five subunits, PIGK (initially inhibitor termed GPI8) [43], GPAA1 (initially termed GAA1) [44], CD59 complement self-damage protection PIGS [45], PIGT [45] and PIGU [46] (table 2). PIGK, a single transmembrane protein, is a cysteine protease that cleaves inhibitor the C-terminal peptide and makes a carbonyl intermediate CRIPTO-1 co-receptor morphogenesis/ [43]. GPAA1, a multiple transmembrane protein having shedding sequence homology to an M28 family peptide-forming dipeptidase 1 enzyme hydrolysis of various enzyme, seems to catalyse the formation of an amide bond between the ω amino acid and GPI’s EtN [47]. PIGT, a dipeptides single transmembrane protein, associates with PIGK via a folate receptor 1 receptor folate uptake/ disulfide bond, thereby playing a role in complex formation transcytosis [48]. The roles of PIGS and PIGU, both being multiple trans- GP2 receptor mucosal immunity/ membrane proteins, have remained unknown; however, both are essential for the activity of GPI transamidase [45]. transcytosis GPIHBP1 receptor transcytosis of lipoprotein lipase 2.3. Biosynthetic assembly of GPI precursors LY6 K sperm protein fertilization Biosynthesis of GPI is a stepwise sequence of 11 reactions prion unknown prion disease agent (figure 2 and table 2). The pathway is initiated on the cyto- plasmic side of the ER by GPI N-acetylglucosaminyl RECK protease neurogenesis/shedding transferase (GPI-GnT), which catalyses the transfer of inhibitor N-acetylglucosamine (GlcNAc) from uridine diphosphate TEX101 sperm protein fertilization/shedding (UDP)-GlcNAc to the 6-position of inositol to generate Thy1 unknown neuronal protein GlcNAc-PI. GPI-GnT is the most complex monoglycosyl- transferase, consisting of seven subunits, PIGA [49], PIGC uPAR (CD87) receptor binding of urokinase [50], PIGH [51], PIGQ (initially termed GPI1) [52], PIGP plasminogen [53], PIGY [54] and DPM2 [53], of which PIGA is a catalytic activator/shedding subunit. PIGC, PIGH, PIGP and PIGY are essential for the contactins adhesion cell adhesion activity of GPI-GnT although their specific functions are not clear [50,51,53,54]. PIGQ stabilizes a core complex of PIGA, molecules PIGC and PIGH [55], whereas DPM2 enhances the tissue non- enzyme uptake of vitamin B6 GPI-GnT activity by three times [53]. GlcNAc-PI is de- specific and other functions N-acetylated by PIGL, an ER-resident GPI deacetylase, that alkaline generates the second intermediate glucosaminyl (GlcN)-PI [56,57]. GlcN-PI flips into the luminal side, the mechanism phosphatase of flipping being unknown. The 2-position of the inositol ring of GlcN-PI is acylated by acyltransferase PIGW, a mul- prion upon Sec62 and Sec63 is determined by its N-terminal tiple transmembrane protein, to generate GlcN-(acyl)PI [58]. signal sequence [37]. Studies on ER translocation of Saccharo- The functionally important amino acids in PIGW and its myces cerevisiae GPI-APs showed that their ER translocation is yeast orthologue Gwt1p reside on the luminal side, mainly Sec62- and Sec63-dependent [39]. The study further suggesting that palmitoyl-CoA is available on the luminal demonstrated that they are translocated into the ER through side [58,59]. a post-translational mechanism, to which the C-terminal PI, GlcNAc-PI and GlcN-PI in mammalian cells contain GPI attachment signal peptide also contributes [39]. For diacylglycerol, whereas GlcN-(acyl)PI contains 1-alkyl-2-acyl- GPI-APs’ precursor bearing a strongly hydrophobic C-term- glycerol as a major form and diacylglycerol as a minor form, inal peptide, components of the GET pathway, which have suggesting that diacyl to 1-alkyl-2-acyl lipid remodelling a role in ER incorporation of tail-anchored proteins [40], are occurs at the stage of GlcN-(acyl)PI [60]. Fatty acyl chain involved. SRP-dependent co-translational ER translocation analysis demonstrated that the chain composition of diacyl has a minor role relative to a post-translational mechanism glycerol of GlcN-(acyl)PI is clearly different from that of PI, in yeast [39]. Whether ER translocation of mammalian GPI- GlcNAc-PI and GlcN-PI [61]. In the latter, 1-stearoyl-2-arachi- APs, other than the prion protein, is mediated by a post- or donoyl (38 : 4) PI is by far the most abundant form. By co-translational mechanism is yet to be characterized. contrast, less than 30% of GlcN-(acyl)PI had a chain royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 EtN Dol-P PE EtN PE PE Dol-P Dol-P Dol-P PEtN EtN EtN EtN Acyl-CoA PE? (12) P P UDP- PEtN PEtN PEtN PEtN PEtN PEtN P EtN Lumen P P P P P P P P P ER P P P (4) (5) (6) (7) (8) (9) (10) (11) (13) (3) PIGW PIGM PIGV PIGN PIGB PIGF PIGF PIGK (1) (2) PIGX PIGO PIGG GPAA1 PIGA PIGL PIGS PIGC PIGT PIGH PIGU PIGP PIGQ PIGY DPM2 P EtN ethanolamine phosphate N-acetylglucosamine inositol phosphate glucosamine diacyl glycerol saturated chain unsaturated chain mannose 1-alkyl-2-acyl or diacyl glycerol Figure 2. Biosynthesis of mammalian GPI in the ER. The complete GPI precursor competent for attachment to proteins is synthesized from PI by stepwise reactions (1)–(11). The Man4 side chain is attached in the ER to some GPI (step (12)). The preassembled GPI is en bloc transferred to proteins (step (13)). Genes involved in these reaction steps are shown below step numbers. composition of 38 : 4 whereas the rest of GlcN-(acyl)PI had EtNP6Manα2(EtNP)6Manα6(EtNP)2Manα4GlcN-(acyl)PI is a chain compositions of 36 : 4, 38 : 5 and 40 : 5. Therefore, it is mature GPI precursor competent for attachment to proteins. highly likely that the lipid remodelling is diacyl to diradyl The fourth Man (Man4) may further be transferred from remodelling, in which not only 1-alkyl-2-acyl PI but also Dol-P-Man to the 2-position of Man3 in the mature GPI pre- diacyl GlcN-(acyl)PI are the remodelled products [61]. cursor as a side chain to generate Manα2(EtNP)6Manα2 For generation of the 1-alkyl-2-acyl form, 1-alkyl phospholi- (EtNP)6Manα6(EtNP)2Manα4GlcN-(acyl)PI, which is also pid synthetic pathway in the peroxisome is required [61]. competent for attachment to proteins. Transfer of Man4 is Although the exact enzymatic reaction of the lipid remodelling mediated by PIGZ (initially termed SMP3), GPI-MTIV [69]. is unclear, it is conceivable that either diacyl glycerol or diacyl- All four GPI-MTs (PIGM, PIGV, PIGB and PIGZ) are multiple phosphatidyl moiety is replaced by a corresponding diradyl transmembrane proteins bearing catalytic sites within the structure. A donor of diradyl structure is also unknown; how- luminal regions [63]. Three GPI-ETs (PIGN, PIGO and ever, the fatty chain composition of GlcN-(acyl)PI is somewhat PIGG) are also multiple transmembrane proteins bearing cat- similar to that of diradyl phosphatidylethanolamines (PE), alytic sites within the luminal regions [67]. The bioinformatic suggesting their possible contribution [61]. study identified a common GPI recognition region in trans- Two mannoses (Man1 and Man2) are sequentially trans- membrane domains of PIGW, PIGM, PIGV, PIGB, PIGZ, ferred from dolichol-phosphate-mannose (Dol-P-Man) to PIGN, PIGO and PIGG, and suggested a gene family GlcN-(acyl)PI to generate Manα6Manα4GlcN-(acyl)PI. PIGM comprising them [70]. and PIGV are GPI-mannosyltransferase (MT) I and II, respect- ively, that catalyse these reactions [62,63]. PIGM functions in association with PIGX, which stabilizes PIGM [64]. GPI- 3. GPI-AP maturation pathway and ER-to- ethanolaminetransferase I (ETI), PIGN, transfers EtNP from PE to the 2-position of the first, α4 linked Man generating Golgi transport of GPI-APs Manα6(EtNP)2Manα4GlcN-(acyl)PI [65]. The third Man (Man3) is then transferred from Dol-P-Man by PIGB, which 3.1. GPI remodelling in the ER and ER-to-Golgi is GPI-MTIII, to generate Manα2Manα6(EtNP)2Manα4GlcN- transport (acyl)PI [66]. Two EtNPs are sequentially transferred to the 6-positions of Man3 and then Man2 by PIGO [46] and The GPI moiety in the nascent GPI-APs is immature in its PIGG (initially termed GPI7) [67], catalytic subunits of structure and undergoes remodelling reactions to become GPI-ETII and GPI-ETIII. Both PIGO and PIGG are stabilized mature during transport to the PM (figure 3 and table 2). by association with PIGF [46,67,68]. The resulting Soon after generation of the nascent GPI-APs, the acyl royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 UDP- UDP- 5 CMP- Acyl-CoA EtN EtN EtN EtN EtN EtN EtN EtN EtN EtN EtN P P P P P P P P P P P COPII vesicle P EtN P EtN P EtN P EtN P EtN P EtN P EtN P EtN P EtN P EtN P EtN P EtN P EtN P EtN P P P P P P P P P P P (16) Raft ER Golgi PM (13) (14) (15) (17) (18) (19) (20) (21) PIGK PGAP1 PGAP5 PGAP3 PGAP2 PGAP4 B3GALT4 GPAA1 PIGS PIGT PIGU P EtN glucosamine ethanolamine phosphate saturated chain mannose 1-alkyl-2-acyl or diacyl glycerol unsaturated chain galactose sialic acid N-acetylgalactosamine Figure 3. Maturation of mammalian GPI-APs during ER–PM transport. Nascent GPI-APs generated by the transfer of GPIs to proteins (step 12) undergo two reactions, inositol-deacylation (step 14) and removal of the EtNP side chain from Man2 (step 15) in the ER. The ER–Golgi transport of GPI-APs is mediated by COPII-coated vesicles (step 16). In the Golgi apparatus, GPI-APs undergo fatty acid remodelling (steps 17 and 18). Some GPI-APs is modified by the GalNAc side chain (steps 19–21). The mature GPI-APs are transported to the PM where they are associated with raft microdomains. Genes involved in these reaction steps are shown below step numbers. chain linked to the inositol ring is usually removed by GPI- 3.2. Fatty acid remodelling in the Golgi deacylase PGAP1 (for Post GPI Attachment to Proteins 1), an ER-resident multiple transmembrane protein [71]. In As described in the previous section, the PI moiety of GPI- some cases, the acyl chain remains in the mature GPI-APs APs is initially derived from cellular PI and is subjected to [72]. For example, GPI-APs in erythrocytes maintain the ino- lipid remodelling at the stage of GlcN-(acyl)PI, in which the sitol-linked acyl chain [73,74]. GPI-APs that have (acyl)PI, original diacyl PI moiety is remodelled to a mixture of which has three hydrocarbon chains, associate with the diacyl and 1-alkyl-2-acyl PIs, with the latter being the major membrane more stably than those that have PI, which has form [60,61] (step 5 in figure 2). The fatty chain composition two hydrocarbon chains. The former structure might be of the remodelled diradyl PI should correspond to that in the useful for maintaining levels of GPI-APs during the very donor lipid, possibly PE, used in the lipid remodelling reac- long life of erythrocytes by reducing spontaneous release tion [61]. The alkyl chain and the 1-acyl chain in the from the PM. diradyl PIs are mostly saturated C18 or C16 alkyl and C18 While still in the ER, EtNP linked to Man2 is removed by acyl chains, whereas the 2-acyl chains are unsaturated fatty a phosphodiesterase PGAP5/MPPE1 [75]. PGAP5, a mem- acids of the C16–C24 chains [61]. In the Golgi apparatus, brane protein bearing two transmembrane domains, exists the unsaturated 2-acyl chain is replaced with a saturated near the ER exit sites in the ER and in the ERGIC [75]. fatty acid, mostly stearic acid by fatty acid remodelling [79] After the remodelling of the GPI by PGAP1 and PGAP5, (steps 17 and 18 in figure 3). In the first step of the fatty GPI-APs are recruited into the COPII-coated transport ves- acid remodelling, the unsaturated 2-acyl chain is removed icles that are directed towards the Golgi apparatus [75,76]. by a GPI-specific phospholipase A PGAP3, a Golgi-resident A complex of p24α2, p24β1, p24γ2and p24δ1ofthe p24 multiple transmembrane protein, generating a lyso form family of proteins acts as a cargo receptor for recruitment intermediate [79]. In the second step, a saturated chain is of GPI-APs into the transport vesicles at the ER exit sites transferred back to the sn2-position. PGAP2, a multiple trans- [76–78]. The remodelling reactions by PGAP1 and PGAP5 membrane Golgi protein, is required for the reacylation [80]. are important to generate the proper structure of the GPI Whether PGAP2 is the acyltransferase itself remains unclear. for association with the cargo receptor [76]. A defect in As described in the Introduction, GPI-APs are typical com- PGAP1 or PGAP5 results in slowed transport of GPI-APs ponents of membrane microdomains or rafts on the cell surface. from the ER to the Golgi. After arrival at the Golgi, GPI- In the outer leaflet of the PM, GPI-APs interact with glyco- APs are released from the cargo receptors under acidic sphingolipids and cholesterol forming dynamic membrane conditions. microdomains of 20–100 nm. Presumably, microdomains are royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 N-terminal signal 6 C-terminal signal w w+1 preproprotein N C ER translocation N w proprotein GPI attachment nascent GPI-AP N GPI GPI maturation mature GPI-AP N Figure 4. Steps in biogenesis of GPI-APs. Preproprotein of GPI-AP has the N-terminal signal peptide for ER localization (brown box) and the C-terminal signal peptide for attachment of GPI (yellow box). The ω site is the amino acid residue, to which GPI is attached. Upon translocation into the ER, the N-terminal signal peptide is cleaved off, generating proprotein. Preassembled GPI is attached to the ω site by replacing the C-terminal signal peptide by GPI transamidase, generating nascent GPI-AP. Nascent GPI-AP undergoes maturation reactions to become mature GPI-AP. formed through lipid–lipid interactions. The fatty acid remodel- tandem transmembrane domain might also be involved in ling that forms GPI-APs bearing two saturated fatty chains is binding the lipid moiety of the GPI [82]. critical for raft association of GPI-APs [79]. Recently, the Gal transferase was identified to be B3GALT4 [88], the Golgi-resident Gal transferase previously known to synthesize GM1 from GM2 [89,90]. B3GALT4 also synthesizes GA1 and GD1 from GA2 and GD2, respectively 3.3. Side chain modification [89]. In these substrate glycosphingolipids, position 3 of In many GPI-APs, βGalNAc is transferred to the 4-position of GalNAc β1–4-linked to Gal in the lactosylceramide (LacCer) Man1 as a side chain [81]. This modification occurs in the moiety is the acceptor for βGal. Similarly, position 3 of Golgi after fatty acid remodelling (figure 3) [82]. The GalNAc β1–4-linked to Man1 is the acceptor in the GPI GalNAc side chain can be elongated by β1–3Gal and Sia for βGal. Whereas UDP-Gal and GM2 are sufficient for (figure 3) [81]. Certain GPI-APs, such as Thy1 [4], always B3GALT4 to generate GM1, the presence of LacCer is have non-elongated GalNAc as a side chain whereas other required for Gal transfer to the βGalNAc side chain of the GPI-APs, such as the prion and dipeptidase, have mixed GPI [88]. Because LacCer is a common partial structure of GalNAc side chains containing those without elongation the acceptor substrates of B3GALT4 (GM2, GA2 and GD2), and those elongated by Gal or by Gal and Sia [83,84]. The LacCer might directly bind to B3GALT4 and might modulate physiological and functional roles of the GalNAc side substrate specificity towards GPI. Sia transferase mediating chains have remained largely unknown. For the prion, a Sia transfer to the Gal-GalNAc-side chain of the GPI has study reported by one group indicated that Sia in the side not been identified. chain is involved in the conversion of PrP to pathogenic sc PrP [85]. Transfer of GalNAc is mediated by PGAP4/TMEM246, a 4. Free, non-protein-linked GPI Golgi-resident GPI-specific GalNAc transferase [82]. PGAP4 is widely expressed in various tissues/organs with higher Protozoan parasites such as Toxoplasma gondii, Plasmodium expression in the brain. All Golgi-resident glycosyltrans- falciparum, Trypanosoma cruzi and Leishmania species have ferases (GTs) are type 2 single transmembrane proteins (i.e. non-protein-linked GPIs as free glycolipids on the cell surface they have a short cytoplasmic segment at the N-terminus, fol- (see the reviews for more details [81,91]). In mammalian cells, lowed by a transmembrane domain and a luminal catalytic there have been few reports about the expression of the un- GT domain [86,87]). By contrast, PGAP4 has three transmem- linked GPI on the cultured cell surface [92–95]. Recently, brane domains [82]. Similar to other type 2 GTs, PGAP4 has a this issue was revisited [96] using a monoclonal antibody short cytoplasmic segment at the N-terminus that is followed T5_4E10 that was originally generated against T. gondii free by the first transmembrane domain and a GT domain. The GPI [97]. T5_4E10 mAb recognizes the non-protein-linked GT domain of PGAP4, of the GT-A type, is split into two GPI bearing the Man1-linked GalNAc side chain without regions, and between the regions there are tandem trans- Gal elongation [82]. Because the T5_4E10 antibody does not membrane domains linked by a short stretch of hydrophilic bind to the protein linked GPI, it is useful to detect the free residues. The tandem transmembrane domains inserted into GPI bearing non-elongated GalNAc side chain (free GPI- the GT domain locate the GT domains close to the membrane, GalNAc from now on) in mammalian cells by flow cytometry which may facilitate the interaction of the GT domain with or western blotting [96]. Relatively higher levels of free GPI- the substrate GPI, which is inserted into the membrane. The GalNAc were expressed in the pons, medulla oblongata, EtN EtN P P P EtN P EtN P EtN P P royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 Table 2. Mammalian proteins involved in GPI -AP biogenesis. step protein function step protein function 1 PIGA GPI-GnT, catalytic 13 PIGK GPI transamidase, catalytic 1 1 PIGC GPI-GnT 13 GPAA1 GPI transamidase, catalytic 2 1 PIGH GPI-GnT 13 PIGS GPI transamidase 1 PIGQ GPI-GnT 13 PIGT GPI transamidase 1 PIGP GPI-GnT 13 PIGU GPI transamidase 1 PIGY GPI-GnT 14 PGAP1 inositol-deacylase 1 DPM2 GPI-GnT, regulatory 15 PGAP5/MPPE1 EtNP phosphodiesterase 2 PIGL GlcNAc-PI-deacetylase 16 p24α2 cargo receptor 3? flipping 16 p24β1 cargo receptor 4 PIGW GlcN-PI-acyltransferase 16 p24γ2 cargo receptor 5 ? lipid remodelling 16 p24δ1 cargo receptor 6 PIGM GPI-MTI, catalytic 17 PGAP3 fatty acid remodelling, PLA2 6 PIGX GPI-MTI, regulatory 18 PGAP2 fatty acid remodelling, reacylation 7 PIGV GPI-MTII 19 PGAP4/TMEM246 GPI-GalNAcT 8 PIGN GPI-ETI 20 B3GALT4 GPI-GalT 9 PIGB GPI-MTIII 21 ? GPI-SiaT 10 PIGO GPI-ETIII, catalytic 10/11 PIGF GPI-ETII/III, regulatory 11 PIGG GPIETII, catalytic 12 PIGZ GPI-MTIV spinal cord, testis, epididymis and kidney of adult mice and mammalian PGAP2 [101,102], whereas the C-terminal part Neuro2a and CHO cells [96]. In cells defective in GPI transa- of CWH43 corresponds to the PGAP2-interacting protein. midase, high levels of free GPI-GalNAc are expressed on the Although the latter is termed the PGAP2-interacting protein, cell surface. Studies using mutant CHO cells, defective in GPI there is no evidence for interaction with PGAP2. PGAP2 is transamidase and one of the genes involved in GPI maturation localized in the Golgi whereas the PGAP2-interacting protein reactions, demonstrated that free GPIs follow the same struc- is localized in the ER [80]. Whether the PGAP2-interacting tural remodelling pathway as do protein linked GPIs [96]. protein has any functional relation to mammalian GPI bio- Therefore, non-protein-linked GPIs exist as glycolipids of genesis, for example, involvement in lipid remodelling at some mammalian cell membranes. The physiological roles of the stage of GlcN-(acyl)PI, is yet to be determined. the free GPIs are yet to be clarified. By contrast, the pathological Yeast defective in ARV1 (for ARE2 required for viability effects of abnormally accumulated free GPIs in cells defective in 1) accumulates GlcN-(acyl)PI, suggesting that the first man- GPI transamidase have been demonstrated in patients with nosylation of GPI is defective [103]. ARV1 is an ER PIGT mutations (see below) [98]. membrane protein involved in the homeostasis of sterols [104] and sphingolipids [105]. Proper conditions for Dol-P- Man-dependent mannose transfer to GlcN-(acyl)PI might 5. Comparison of mammalian and yeast not be generated when ARV1 does not function. Mammalian ARV1 cDNA complemented ARV1-defective yeast, which GPI biosynthesis shows the functional role of mammalian ARV1 [105,106]. In yeast S. cerevisiae, the GPI undergoes two types of lipid Recently, patients with biallelic loss-of-function mutations in remodelling reaction that occur in the ER after attachment ARV1 have been reported [107,108]. The affected children to proteins. Fatty acid remodelling of the GPI occurs to the had similar symptoms to those with inherited GPI sn2-linked acyl chain of diacyl PI, in that the C16/C18 deficiencies, such as developmental delay and seizures, chain is exchanged with the C26 chain by PER1- and which is consistent with mammalian ARV1 having a putative GUP1-dependent reactions [99,100]. PER1 is orthologous to role in GPI biosynthesis. PGAP3 [79], whereas GUP1 is not orthologous to PGAP2 Gpi18p, the yeast PIGV homologue, requires Pga1p for but is a member of the MBOAT family of acyl transferases GPI-MTII activity [109]. Gpi18p and Pga1p form a complex. [100]. The other lipid remodelling of the GPI is its change The PGA1 homologue is not found in the mammalian from a diacylglycerol form to a ceramide form. CWH43 is genome. The reason for Gpi18p, but not PIGV, requiring a required for this lipid remodelling [101,102]. The ceramide partner protein is not known. form of the GPI is not known in mammalian cells; however, Yeast has two homologues of PGAP5: TED1 and CDC1. parts of CWH43 are homologous to two mammalian pro- Like PGAP5, TED1 is involved in the removal of EtNP teins. The N-terminal part of CWH43 corresponds to from Man2 [110] whereas CDC1 is required for the removal royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 of EtNP from Man1 [111]. In mammalian cells, EtNP linked to like 1, on the same cell, which in turn shuts down Notch sig- Man1 remains as a conserved side chain in mature GPI-APs. nalling into the neighbour neuronal progenitor cell, leading By contrast, some yeast GPI-APs do not have EtNP on Man1. to initiation of neuronal differentiation [26,123]. There is a hypothesis that the removal of EtNP from Man1 is The urokinase receptor (uPAR), a GPI-AP, mediates degra- necessary for the incorporation of GPI-APs into the cell wall dation of the extracellular matrix through protease recruitment (see [112] for further discussion). and enhances cell adhesion, migration and invasion. Cell sur- face activity of the uPAR is negatively regulated by its shedding from the cell surface, which is mediated by GDE3, another member of the glycerophosphodiester phosphodiester- 6. Quality control of GPI-APs ase family [27]. GDE3 mediates cleavage and release of the When the precursor proteins of GPI-APs are not processed by uPAR by its GPI-specific phospholipase C activity. Interest- GPI transamidase for GPI attachment, they are retrotranslo- ingly, the uPAR is resistant to GDE2 [27], suggesting that cated for degradation by ubiquitin- and proteasome- substrate specificities of GDE2 and GDE3 are dependent dependent ER-associated degradation (ERAD) [113]. It is upon the protein portions of RECK and the uPAR, respectively. likely that the GPI attachment signal sequence is recognized PGAP6/TMEM8A is a GPI-specific phospholipase A2 as an unfolded region, which leads to ubiquitination. expressed on the surface of some cells, including embryonic By contrast, when protein folding fails after attachment of cells. PGAP6 has sequence similarity to PGAP3, a Golgi-resi- the GPI, the misfolded GPI-APs are mainly transported out of dent GPI-specific phospholipase A2 involved in fatty acid the ER for degradation in the lysosomes [114,115]. The pro- remodelling of the GPI [28]. PGAP6 and PGAP3 belong to cess is termed RESET (for rapid ER stress-induced export) the CREST (for alkaline ceramidase, PAQR receptor, Per1, [114]. When the ER stress is induced by thapsigargin, the SID-1 and TMEM8) superfamily of hydrolases [124]. The RESET pathway functions efficiently. The ER exit of the mis- substrate of PGAP6 is CRIPTO-1 [28], a GPI-anchored co-re- folded GPI-APs is facilitated by p24 family proteins, which ceptor of TGFβ receptors [125]. CRYPTIC, a GPI-AP closely act as cargo receptors of GPI-APs. The misfolded GPI-APs related to CRIPTO-1, is resistant to PGAP6, suggesting that appear transiently on the cell surface before degradation PGAP6 is a CRIPTO-1-specific phospholipase A2 [28]. [114]. During the transport through the secretory pathway, When CRIPTO-1 and PGAP6 are expressed on the same misfolded GPI-APs are escorted by ER-derived chaperones, cell, CRIPTO-1 is processed by PGAP6’s phospholipase A2 such as BiP and calnexin, and p24 proteins [116]. Most GPI- activity, loses one fatty acyl chain (lyso form of GPI) and is APs have at least one N-glycan and their folding is mediated spontaneously released from the cell surface. When GPI- by N-glycan-dependent calnexin cycle [117]. Calnexin also specific phospholipase D (GPI-PLD) is present in the sur- binds to PGAP1 and facilitates PGAP1-mediated removal of rounding medium or is expressed in the cell, the lyso form the acyl chain from inositol [117]. When the calnexin cycle of CRIPTO-1 is cleaved and more efficiently released [28]. is inefficient, GPI-APs bearing inositol-linked acyl chain The released CRIPTO-1 possessed its activity as a co-receptor appear on the cell surface. of TGFβ receptors [126]. CRIPTO-1 is involved in various In yeast, misfolded GPI-APs are targeted to ERAD to steps in embryogenesis. In an early mouse embryonic stage some extent, while a sizable fraction of the misfolded GPI- (6.5 days post coitum), Cripto-1 is essential in the generation APs exit the ER for degradation in vacuoles [115,118]. For effi- of the anterior–posterior axis [127]. A fraction of PGAP6 cient ER–Golgi transport of GPI-APs mediated by p24 cargo knockout embryos do not form the anterior–posterior axis, receptors, remodelling of both lipid and glycan moieties is confirming the critical role of PGAP6-dependent shedding required [119]. These GPI remodelling reactions occur even of CRIPTO-1 in Nodal signalling regulation in vivo [28]. when the protein moiety is misfolded, allowing efficient A complex of two GPI-APs, LY6 K and TEX101, is recruitment into COPII-coated transport vesicles. In this required for sperm migration into the oviduct. Males of regard, quality control of GPI-APs in the ER is limited and LY6 K knockout mice and TEX101 knockout mice are infer- the misfolded GPI-APs pass other compartments in the tile. Their sperm are defective in binding to an egg’s zona secretory pathway [115]. pellucida in vitro as well as in migration into the oviduct in vivo [128]. A testis form of angiotensin-converting enzyme (ACE) is essential for the maturation of spermatids. Similar 7. Shedding of GPI-APs mediated by GPI to LY6 K knockout and TEX101 knockout male mice, ACE knockout male mice are infertile and their sperm does not cleaving/processing enzymes (GPIases) pass through the uterotubal junction in vivo and does not A prominent characteristic of GPI-APs is their shedding from bind to the zona pellucida in vitro. ACE cleaves GPI by its the cell surface. There are several pairs of GPI-APs and GPI putative endomannosidase activity, which seems to reside cleaving/processing enzymes, GPIases, that are responsible in a different site from the well-characterized carboxy dipep- for their shedding. RECK (for reversion-inducing-cysteine- tidase catalytic site for conversion of angiotensin [129]. rich protein with Kazal motifs) is a GPI-anchored inhibitor TEX101 is a specific target for the GPIase activity of ACE of matrix metalloproteinases, such as MMP-9 and ADAM10 [29]. Shedding of TEX101 by ACE is critical for spermatozoa [120]. GDE2 is a member of the glycerophosphodiester phos- to become fertilization competent [29,128]. phodiesterase family, having six transmembrane domains Pairs of the GPI-AP substrates and GPIases described and an extracellular phosphodiester domain [121]. There is above are examples of the protein specific cleavage/proces- evidence that GDE2 has RECK-specific GPI phospholipase sing of the GPI. By contrast, GPI-PLD has been known for C activity [26]. RECK suppresses ADAM10 on certain neur- a long time as a GPIase that is active against a wide variety onal progenitor cells [122]. When RECK is released by of GPI-APs [130,131]. However, GPI-PLD requires a detergent GDE2, ADAM10 cleaves a Notch ligand, such as the Delta- for its GPIase activity towards the cell surface GPI-APs [132]. royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 As described above, GPI-PLD cleaves the lyso form of GPI- chromosome is sufficient to cause the phenotype. Because APs in the absence of a detergent, facilitating their shedding PIGA mutation exists only in the affected blood cells but from the cell surface [28]. Further, GPI-PLD is able to cleave not in germ cells, PNH is not an inherited disease. PIGA GPI-APs within the intracellular secretory pathway [133]. somatic mutations found in patients with PNH cause com- These results suggest that when GPI-APs are associated with plete loss or a severe reduction of GPI-APs on the cell the membrane microdomains, they are resistant to GPI-PLD. surface. It should be pointed out that the somatic PIGA Studies with GPI-PLD knockout mice suggested that GPI- mutation alone does not cause PNH because the generation PLD generates diacylglycerol by cleaving GPI within hepato- of one GPI-AP-defective HSC among many hundreds of cytes where GPI-PLD expression is most abundant [134]. HSCs should not affect the blood system. Indeed, similar Diacylglycerol might activate PKCε, which in turn binds to PIGA somatic mutations are found in blood granulocytes the insulin receptor and inhibits its tyrosine kinase activity, from healthy individuals [139]. When the mutant haemato- leading to insulin resistance. GPI-PLD knockout ameliorated poietic stem cell exhibits clonal expansion and generates glucose intolerance and hepatic steatosis under a high-fat large numbers of GPI-AP-defective blood cells, clinical and high-sucrose diet through a reduction of diacylglycerol PNH is manifested (see reviews for further discussion and a subsequent decrease of PKCε activity [134]. about clonal expansion [137]). Another form of GPI deficiency is inherited GPI deficiency (IGD) (table 3). The first inherited form of GPI deficiency was reported in 2006 [140]. A homozygous hypo- 8. Transcytotic endocytosis of GPI-anchored morphic mutation in PIGM located in chromosome 1q was receptors found in three children from two consanguineous families. The affected children suffered from seizures and thrombosis Several GPI-anchored receptors act as transcytotic receptors in the hepatic or portal vein. The mutation was within the that transport ligands from one membrane domain to another promoter region of the PIGM gene and disrupted an Sp1- in endothelial and epithelial cells. GPIHBP1 (GPI-anchored binding site important for PIGM transcription in some cell heparin-binding protein 1) binds lipoprotein lipase (LPL) on types, such as blood granulocytes, B-lymphocytes and fibro- the basolateral surface of capillary endothelial cells and trans- blasts. In those cells, the cell surface levels of some GPI-APs port it to the apical (vascular) surface [23]. On the apical are reduced. surface of capillary endothelial cells, LPL acts on lipoproteins In 2010 when the application of whole-exome sequencing to liberate fatty acids to fuel tissues (see the review for further to rare inherited diseases became feasible, biallelic (homozy- discussion [135]). Folate receptor 1, a GPI-anchored type of gous or compound heterozygous) mutations in PIGV located folate receptors, binds 5-methyltetrahydrofolate (5MHF) on in chromosome 1p were found in several affected children the vascular (basolateral) side of the epithelial cells of the with Mabry syndrome, also known as hyperphosphatasia choroid plexus in the brain and is endocytosed. The folate with mental retardation syndrome (HPMRS) [141]. The affected receptor 1 and 5MHF complexes are released from the children inherited loss-of-function mutations from their parents apical surface into the cerebrospinal fluid as exosomes, who were heterozygotes of wild-type and mutant alleles. GPI- which then pass the ependyma and enter the brain parench- APs in blood cells and fibroblasts from affected individuals yma. This is a critical route of 5MHF incorporation from the were partially lost by these biallelic PIGV mutations. blood to the brain parenchyma [24]. Glycoprotein 2 (GP2) is To date, pathogenic germ line mutations causing IGD selectively expressed on the surface of M cells, cells in the have been found in 21 out of 27 genes in GPI biosynthesis mucosal lymphoid tissue Peyer’s patch. GP2 is involved in [140–157] and the GPI-AP maturation pathway [158–161] sampling foreign antigens into the mucosal immune system (table 3). Inheritance of IGDs caused by mutations in autoso- (see the review for further discussion [136]). Botulinum mal genes, such as PIGB-IGD [151] and PIGC-IGD [147], is toxin produced by the food-borne botulism-causing bacteria autosomal recessive. Inheritance of IGD caused by germ Clostridium botulinum in the intestine binds to GP2 on the line mutations of PIGA (PIGA-IGD) is X-linked recessive, in apical surface of M cells and traverses the intestinal epithelial which all affected individuals are boys [145]. Their mothers layer via transcytosis [25]. who were heterozygous carriers of a PIGA mutation, were phenotypically mosaic at the cell level (blood cells were mix- tures of normal and GPI-AP-deficient cells) but did not have 9. Lessons from GPI deficiencies clear symptoms of IGD [162]. More recently, cases of PNH caused by mutations of PIGT 9.1. Molecular genetics of GPI deficiencies were found [98,163,164]. These cases did not have the somatic Defective biosynthesis of the GPI is caused by several differ- PIGA mutation found in the affected blood cells from patients ent genetic mechanisms. The first GPI deficiency that was with PNH, but instead had biallelic loss-of-function clarified at the molecular genetics level is paroxysmal noctur- mutations of the PIGT gene. One PIGT allele had germ line nal haemoglobinuria (PNH) (table 3) [137]. GPI deficiency in mutations whereas the other PIGT was lost by deletion of PNH is caused by somatic mutations in PIGA that occur in the 8–24 Mb region of chromosome 20q, which somatically the haematopoietic stem cells (HSCs) [138]. PIGA is the occurred in a haematopoietic stem cell [98]. Therefore, GPI- only X-linked gene among all genes involved in GPI biosyn- APs were lost by a combination of germ line mutation and thesis. Because of its X-linkage, one loss-of-function somatic somatic mutation. The somatic mutation is always caused mutation in PIGA causes a GPI defective phenotype [138]. by a Mb scale deletion because simultaneous deletion of the This is true for both male and female cells because one X myeloid common deleted region [98], implicated in clonal chromosome in female cells was inactivated during embryo- expansion of the HSCs [165,166], is required for clinical genesis and hence a mutation in PIGA in the active X manifestation of PIGT-PNH. royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 Table 3. Diseases caused by loss-of-function mutations in PIG and PGAP genes. a b step gene disease symptoms patients Chr mutation c d e 1 PIGA PNH haemolysis, thrombosis many Xp S in HSC f g h i j k IGD /MCAHS Sz , DD/ID , Hpt 26 G 1 PIGC IGD Sz, DD/ID, Hpt 2 1q G 1 PIGH IGD Sz, DD/ID, Hpt 2 14q G 1 PIGP IGD Sz, DD/ID, Hpt 2 21q G 1 PIGQ IGD Sz, DD/ID, Hpt 3 16p G 1 PIGY IGD Sz, DD/ID, Hpt 4 4q G 2 PIGL IGD, CHIME syndrome Sz, DD/ID, Hpt 15 17p G 4 PIGW IGD/HPMRS Sz, DD/ID, Hpt 3 17q G 6 PIGM IGD Sz, thrombosis 7 1q G, promotor 7 PIGV IGD/HPMRS Sz, DD/ID, Hpt 18 1p G 8 PIGN IGD/MCAHS, Fryns syndrome Sz, DD/ID, Hpt 22 18q G 9 PIGB IGD/HPMRS Sz, DD/ID, Hpt 12 15q G 10 PIGO IGD/HPMRS Sz, DD/ID, Hpt 13 9p G 11 PIGG IGD Sz, DD/ID, Hpt 7 4p G 13 GPAA1 IGD Sz, DD/ID, Hpt 10 8q G 13 PIGS IGD Sz, DD/ID, Hpt 7 17p G 13 PIGT PIGT-PNH haemolysis, thrombosis, infla 4 20q G + S in HSC IGD/MCAHS Sz, DD/ID, Hpt 28 G 13 PIGU IGD Sz, DD/ID, Hpt 5 20q G 14 PGAP1 IGD Sz, DD/ID, Hpt 8 2q G 17 PGAP3 IGD/HPMRS Sz, DD/ID, Hpt 45 17q G 18 PGAP2 IGD/HPMRS Sz, DD/ID, Hpt 23 11p G Numbers of published patients as of December 2019. Chr, chromosome location. PNH, paroxysmal nocturnal haemoglobinuria. S, somatic mutations. HSC, haematopoietic stem cell. IGD, inherited GPI deficiency. MCAHS, multiple congenital anomalies-hypotonia-seizures syndrome. Sz, seizures. DD/ID, developmental delay/intellectual disability. Hpt, hypotonia. G, germline mutations. HPMRS, hyperphosphatasia mental retardation syndrome/Mabry syndrome. infla, inflammation. partial but severe reduction in GPI biosynthesis would also 9.2. Genotype–phenotype relationship cause embryonic lethality. A homozygous PIGC mutation Twenty-one genes whose mutations caused IGD cover associated with a family with recurrent foetal loss seems almost all steps in the biosynthesis of the core GPI, its trans- highly likely to be such a case [168]. If the reduction in GPI fer to proteins and maturation of the GPI, for which genes biosynthesis is less severe, the outcome would be IGD. In are known except a step mediated by PGAP5 [140– fact, hypomorphic mutations of PIGC have been associated 146,149,151,153,158,161]. It seems, therefore, that every with epilepsy and intellectual disability [147]. As summar- step in GPI-AP biogenesis is critical for human health. ized in recent articles, the most prominent clinical There has been no report of pathogenic mutations in symptoms of IGD are neurological ones, including seizures, genes involved in side chain modifications (i.e. PIGZ and developmental delay/intellectual disability, cerebral atrophy PGAP4); therefore, it is unknown what roles the side and hypotonia [169,170]. Many GPI-APs, such as contactins, chains of the GPI play in human health. tissue non-specific alkaline phosphatase (TNAP) and Thy1, Complete loss of GPI biosynthesis in the whole body of are expressed on neurons, oligodendrocytes and other glial the mouse caused early embryonic lethality, as demonstrated cells. Partial reduction of these GPI-APs impairs neurological by knockout of the Piga gene [167]. Complete GPI deficiency development and functions, highlighting the importance of would also cause a similar phenotype in a human being. A GPI-APs in the neuronal system. One example is a causal royalsocietypublishing.org/journal/rsob Open Biol. 10: 190290 role of defective expression of TNAP in seizures [171,172]. blood cells, the wild-type allele of PIGT was lost somatically TNAP is involved in cellular uptake of pyridoxal phosphate, as described above. This combination of a nearly null germ active vitamin B6, where TNAP dephosphorylates pyridoxal line mutation and somatic loss of the normal allele caused a phosphate to generate membrane permeable pyridoxal. Once nearly complete lack of GPI-APs, resulting in PNH [164]. in the cytoplasm, pyridoxal is reverted to pyridoxal phos- The two children with IGD did not have PNH symptoms, phate by pyridoxal kinase and is used by many B6- such as intravascular haemolysis, because their blood cells dependent enzymes including GABA (γ-aminobutyric acid) had nearly normal levels of GPI-anchored complement synthetic enzyme. Therefore, the reduction of cell surface regulators, CD59 and CD55 [175,176]. TNAP might result in reduced GABA synthesis, leading to Patients with PIGT-PNH had typical complement- the easy occurrence of seizures. mediated intravascular haemolysis and, in addition, recurrent Concerning genes required for the biosynthesis of the core autoinflammatory symptoms, such as urticaria, arthralgia and GPI, many mutations found in individuals with IGD are null noninfectious meningitis, associated with elevated IL18 and or nearly null [173]. Heterozygotes of such null alleles in the serum amyloid A [98]. Unlinked free GPIs were expressed same families are healthy, suggesting that 50% of normal on affected erythrocytes, monocytes, granulocytes and levels of GPI biosynthesis may be sufficient for not causing B-lymphocytes. Both intravascular haemolysis and autoinflam- clinical symptoms. When the null allele is combined with a matory symptoms were controlled by anti-complement C5 hypomorphic allele, GPI levels further decrease, resulting in antibody drug eculizumab, suggesting the involvement of symptoms of IGD. terminal complement activation for both. Studies comparing Concerning the PGAP1 and PGAP3 genes involved in the PIGT- and PIGA-knockout monocyte/macrophage cell lines maturation of the GPI, homozygous null alleles do not cause showed that accumulated free GPI on PIGT-knockout cells embryonic lethality, but do cause IGD [158,161]. The cell sur- caused enhanced complement activation and subsequent IL1β face levels of GPI-APs do not decrease, or only slightly secretion. Therefore, although free GPI is normal component decrease, in PGAP1 or PGAP3 knockout cells, whereas struc- of certain cells, abnormally accumulated free GPI is pathogenic. tures of GPI moiety in GPI-APs are different from those in It is conceivable that when free GPI levels are abnormally high, wild-type cells. In PGAP1 knockout cells, the inositol-linked interactions with some unknown protein(s) involved in comp- acyl chain remains in GPI-APs [71]. In PGAP3 knockout lement activation, such as lectin pathway components, may be cells, the fatty acid remodelling does not occur (i.e. the sn2- enhanced due to multivalent binding. linked fatty acid remains as unsaturated fatty acid [79]). These structural abnormalities are probably causally related to the functional impairment of certain GPI-APs, resulting in 10. Concluding remarks clinical symptoms. Recently, it was demonstrated that fatty acid remodelling of GPI-APs is required for the generation of Biogenesis of mammalian GPI-APs have been studied well, nanoclusters of GPI-APs, which is critical for activation of but some points remain to be clarified: mechanisms of ER β1-integrins on lymphocytes and their subsequent spreading translocation of preproproteins of mammalian GPI-APs; and migration [174]. The role of GPI fatty acid remodelling genes involved in flip of GlcN-PI into the luminal side of in cellular physiology might be causally related to clinical the ER; mechanisms and genes involved in lipid remodelling symptoms of IGD caused by PGAP3 mutations. of GlcN-(acyl)PI; identification of the sialyl transferase for Concerning genes of GPI transamidase, such as PIGT, elongation of GalNAc-Gal side chain; and functions of indi- mutations that cause complete or nearly complete loss of vidual components of GPI-GnT and GPI transamidase. GPI transamidase activity of a cell lead not only to complete Physiological roles of Man4 and GalNAc side chains need or nearly complete lack of GPI-APs but also to the accumu- to be studied. Physiological roles of unlinked free GPI lation of unlinked free GPI in the cell. By contrast, mutations found in some tissues by T5_4E10 monoclonal antibody that cause partial loss of GPI transamidase activity lead to par- need to be understood. Because T5_4E10 antibody binds tial reduction of cell surface GPI-APs. The same germ line only to free GPI bearing GalNAc as a side chain, probes for PIGT mutation has been found in IGD [175,176] and PNH other forms of potentially expressed free GPIs, such as [164]. PIGT bearing c.250G>T, p.E84X is a genetic variant those lacking a side chain and those bearing GalNAc side found in the East Asian population at an allele frequency of chain with elongation by Gal and Sia, are required to eluci- 0.00023. PIGT E84X has very weak activity due to read date an entire picture of free GPIs. There might be more through of the nonsense codon. Two Japanese children with examples of biologically important GPI-AP shedding IGD, who had no familial relationship, inherited this variant mediated by GPIases that are specific to the target GPI-APs. PIGT and different PIGT variants from their parents. Both of the other variant PIGTs had partially reduced activities Data accessibility. This article has no additional data. [175,176]. Their cells had subnormal levels of GPI-APs Competing interests. I declare I have no competing interests. [175,176]. A Japanese adult patient with PIGT-PNH had the Funding. This work was funded by Ministry of Education, Culture, same E84X variant in the germ line [164]. In his clonal PNH Sports, Science and Technology (grant no. KAKENHI 17H06422). References 1. UniProt C. 2015 UniProt: a hub for protein 2. 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Biosynthesis and biology of mammalian GPI-anchored proteins

Open Biology , Volume 10 (3) – Mar 11, 2020

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