Loss of the N-acetylgalactosamine side chain of the GPI-anchor impairs bone formation and brain functions and accelerates the prion disease pathology

Abstract

Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases. Glycosylphosphatidylinositol (GPI) is a posttranslational glycolipid modification of proteins that anchors proteins in lipid rafts on the cell surface. Although some GPI-anchored proteins (GPI-APs), including the prion protein PrPC, have a glycan side chain composed of N-acetylgalactosamine (GalNAc)−galactose−sialic acid on the core structure of GPI glycolipid, in vivo functions of this GPI-GalNAc side chain are largely unresolved. Here, we investigated the physiological and pathological roles of the GPI-GalNAc side chain in vivo by knocking out its initiation enzyme, PGAP4, in mice. We show that Pgap4 mRNA is highly expressed in the brain, particularly in neurons, and mass spectrometry analysis confirmed the loss of the GalNAc side chain in PrPC GPI in PGAP4-KO mouse brains. Furthermore, PGAP4-KO mice exhibited various phenotypes, including an elevated blood alkaline phosphatase level, impaired bone formation, decreased locomotor activity, and impaired memory, despite normal expression levels and lipid raft association of various GPI-APs. Thus, we conclude that the GPI-GalNAc side chain is required for in vivo functions of GPI-APs in mammals, especially in bone and the brain. Moreover, PGAP4-KO mice were more vulnerable to prion diseases and died earlier after intracerebral inoculation of the pathogenic prion strains than wildtype mice, highlighting the protective roles of the GalNAc side chain against prion diseases. Addition of glycosylphosphatidylinositol (GPI) to proteins is a posttranslational modification of proteins widely conserved among eukaryotes (1Kinoshita T. Biosynthesis and biology of mammalian GPI-anchored proteins.Open Biol. 2020; 10: 190290Google Scholar, 2Liu Y.S. Fujita M. Mammalian GPI-anchor modifications and the enzymes involved.Biochem. Soc. Trans. 2020; 48: 1129-1138Google Scholar). Approximately 150 types of mammalian proteins, including Thy1, 120-kDa neural cell adhesion molecule (NCAM120), alkaline phosphatases (ALPs), and prion protein (PrPC), are modified by GPI and expressed in lipid rafts (usually defined as a detergent-resistant membrane [DRM]) on the cell surface through GPI (3Ferguson M.A.J. Hart G.W. Kinoshita T. Glycosylphosphatidylinositol anchors.in: Varki A. Cummings R.D. Esko J.D. Stanley P. Hart G.W. Aebi M. Darvill A.G. Kinoshita T. Packer N.H. Prestegard J.H. Schnaar R.L. Seeberger P.H. Essentials of Glycobiology. Cold Spring Hobor Laboratory Press, Cold Spring Harbor, NY2015: 137-150Google Scholar). These GPI-anchored proteins (GPI-APs) fulfill significant roles in early development, synaptic formation, and immunological regulation. Mammalian GPI is composed of a common core and are and (1Kinoshita T. Biosynthesis and biology of mammalian GPI-anchored proteins.Open Biol. 2020; 10: 190290Google Scholar, 2Liu Y.S. Fujita M. Mammalian GPI-anchor modifications and the enzymes involved.Biochem. Soc. Trans. 2020; 48: 1129-1138Google Scholar). mammalian GPI core structure modified various side modification is the of the to the an P. a to and glycosylphosphatidylinositol in Biol. the is the of the GalNAc side is composed of to the structure of the GalNAc side chain is and GalNAc are and structure of the membrane of Scholar, anchors of the and prion proteins Scholar, A. T. T. A. T. Kinoshita T. of acid to a GPI and an GPI in prion Biol. 2020; Scholar). These side to of GPI in the and are to a by and GPI are by of the GPI to proteins by GPI GPI-APs (1Kinoshita T. Biosynthesis and biology of mammalian GPI-anchored proteins.Open Biol. 2020; 10: 190290Google Scholar, 2Liu Y.S. Fujita M. Mammalian GPI-anchor modifications and the enzymes involved.Biochem. Soc. Trans. 2020; 48: 1129-1138Google Scholar). and of GPI-APs are in the and to GPI (1Kinoshita T. Biosynthesis and biology of mammalian GPI-anchored proteins.Open Biol. 2020; 10: 190290Google Scholar, 2Liu Y.S. Fujita M. Mammalian GPI-anchor modifications and the enzymes involved.Biochem. Soc. Trans. 2020; 48: 1129-1138Google Scholar). Although the of the core GPI is of the GPI-GalNAc side chain for the structure of mammalian including GalNAc in structure of the membrane of Scholar). We to proteins as a GalNAc that the for the GalNAc side chain T. Fujita M. Kinoshita T. of a in the Scholar). to an for and T. Fujita M. Kinoshita T. of a in the Scholar). we as the for the GPI side chain Y.S. A. T. Fujita M. Kinoshita T. of glycosylphosphatidylinositol and 2020; Scholar). to PGAP4, is in of GPI of glycolipid M. T. of that the expression of Biol. Scholar, M. M. T. of of of a Biol. Scholar). We that acid of the GPI side chain of PrPC is an A. T. T. A. T. Kinoshita T. of acid to a GPI and an GPI in prion Biol. 2020; Scholar). These have largely the structure and the of the GPI-GalNAc side of mice of GPI have the physiological of the core structure of GPI and of of GPI-APs GPI phenotypes, including early M. Kinoshita T. A. of by as M. M. Kinoshita T. mice show and Biol. Scholar, M. is by Scholar, in GPI in mouse of and M. T. P. Glycosylphosphatidylinositol and are required for neural development, and neural cell M. M. Kinoshita T. mice show and Biol. Kinoshita T. of roles of acid of GPI and M. glycosylphosphatidylinositol in development, and in a mouse 2020; Scholar, M. M. A. A. M. A. mouse for GPI-anchor and synaptic A. Scholar). have GPI have in GPI including GPI of are and A. M. M. M. M. A. of glycosylphosphatidylinositol by and 10: Scholar, in glycosylphosphatidylinositol Scholar). in are as and of the as and as and and of a in in Scholar, A. T. A. T. Kinoshita T. M. in the of the GPI and Scholar, A. A. M. in an GPI an and in Scholar, P. in the glycosylphosphatidylinositol Scholar, M. A. Kinoshita T. in in and Scholar, M. T. P. of and in Scholar). These of that GPI is for and various physiological and loss of expression GPI These that GPI side including the GalNAc side significant roles in the in vivo functions of the GPI-GalNAc side chain including and in and and in are and A. Scholar, protein Scholar). diseases are by the of the of prion protein of the prion is normal prion protein prion A. Scholar). Although the of PrPC to largely the of PrPC and are and A. Scholar, M. M. A. of in the of the prion A. Scholar). mice of the PrPC, are to the prion after inoculation A. A. P. M. of are to Scholar, A. M. of the prion protein in mice and of A. that the of PrPC is for the of prion diseases. Furthermore, knocking out in after inoculation of strains of PrPC to in and by the A. in prion and Scholar). of in and A. in prion and Scholar). in a against prion diseases for that the of prion diseases the of PrPC to and the and in vivo in Scholar). have that the GPI of PrPC is in the of prion diseases through M. of in and Scholar). the of mice PrPC after inoculation and of M. M. prion protein in Scholar, A. of of prion in of mice of prion Scholar, M. M. of prion of prion protein membrane that of on membrane of through Furthermore, an in that PrPC GPI is to to A. acid on the glycosylphosphatidylinositol cell and prion Biol. Scholar). that mice PrPC GPI have a after of the in and T. M. GPI-anchor PrPC and and on of prion in Scholar). These that the GPI-anchor the of PrPC to of the enzymes for the GalNAc side chain to the GPI-GalNAc side chain is in the of prion diseases in this we to in vivo functions of the GPI-GalNAc side chain in normal and this we PGAP4-KO mice and and we in PGAP4-KO mice and We that PGAP4-KO mice various and of the of prion Thus, the physiological and pathological of the GPI-GalNAc side PGAP4-KO mice were to the physiological and pathological roles of the GPI-GalNAc side We the by of the the Pgap4 to expression and to the Pgap4 to and Pgap4 the Scholar, P. M. in a the and of the Pgap4 the the Pgap4 were to early mice were wildtype mice to PGAP4-KO mice. were and the of the mice were by PGAP4-KO mice were and and and mice were that is for and various to the of Pgap4 We that Pgap4 expression to including brain, and the expression in GPI-APs are expressed (3Ferguson M.A.J. Hart G.W. Kinoshita T. Glycosylphosphatidylinositol anchors.in: Varki A. Cummings R.D. Esko J.D. Stanley P. Hart G.W. Aebi M. Darvill A.G. Kinoshita T. Packer N.H. Prestegard J.H. Schnaar R.L. Seeberger P.H. Essentials of Glycobiology. Cold Spring Hobor Laboratory Press, Cold Spring Harbor, NY2015: 137-150Google Scholar). that the GalNAc side chain have functions in the brain, expression of PGAP4, by expression in mice, widely of for and for and that is expressed in and and in vivo functions of the GalNAc side chain were by protein expression of GPI-APs in the brain. expression levels of and in is the PrPC, and in mouse were in that the GalNAc side chain is for the expression of GPI-APs in we investigated the GalNAc side chain to the association of GPI-APs lipid this we the and the as PrPC, and Thy1, were in the and the levels were and that the GalNAc side chain to raft association of GPI-APs. We that in of PrPC and than proteins and that of proteins in mice. the of proteins is to an of we the to after the and and that GPI on proteins, are in mice. We PrPC membrane to the GPI in the mouse and and by and were by spectrometry analysis a PrPC We the of by a and We of in PrPC, and the GalNAc side chain and to on analysis of and PrPC anchors of the and prion proteins Scholar, A. T. T. A. T. Kinoshita T. of acid to a GPI and an GPI in prion Biol. 2020; we GPI the of PrPC we and for mass spectrometry analysis to PrPC and GPI of the GalNAc side were in PrPC and that loss of the of the GalNAc side chain in the brain. of in in PrPC that the GalNAc side chain is a in brain. that and the GalNAc and are expressed in the we and mRNA by and in and in the mouse expression expression were These that and are expressed in the and the GalNAc side chain is by in the brain. the of in GalNAc to GPI in we the of GPI that the and this the T. Fujita M. Kinoshita T. of a in the Scholar, T. Fujita M. Kinoshita T. on mammalian cell Biol. Scholar). GPI the GalNAc side chain the GalNAc is the of the GPI to highly expressed T. Fujita M. Kinoshita T. on mammalian cell Biol. we a in mice, the in in in the of the as in the were in the mouse we that is the GalNAc for the GPI-anchor of GPI-APs and GPI in the and loss of the GPI-GalNAc side chain the expression and raft association of GPI-APs. the in vivo functions of PGAP4, a blood that of the including and were in mice that the and functions and of and were of by in were normal in mice the of blood that of in mice that the cell surface of is decreased in mice. Moreover, the blood in mice These that bone in mice is required for of M. roles of and alkaline phosphatase in Scholar, A. alkaline phosphatase and cell membrane are of bone A. Scholar, phosphatase mice the and of Scholar). and of bone were in mice and We investigated the of the by Although in the of including and in mice of and were and Furthermore, the in mice than that in mice These that the GPI-GalNAc side chain is required for normal bone We and Although PGAP4-KO mice to in were the that in PGAP4-KO mice the in mice. the cell in mice, and in and cell the of cell types in mice. on the expression of Pgap4 in the brain, we is required for functions and various the the by mice especially in the of the and and the were These that PGAP4-KO mice have of and to were and mice that that decreased in mice that of mice and These that the GPI-GalNAc side chain is required for normal locomotor We a mice more a the by mice a mouse in the mouse and mice that loss of the GPI-GalNAc side chain in to We a and in the to the and mice that PGAP4-KO mice show a We loss of the GPI-GalNAc side chain by and in and are to of and to and Scholar). mice were an a in the the on the mice were in the the and the We that mice the and the of mice that of the significant that mice have impaired the mice were in the the after the and the the and Although the the of mice that of mice These that loss of the GPI-GalNAc side chain impaired especially in impaired functions in PGAP4-KO mice, we the of PGAP4-KO mouse brains. and of and in PGAP4-KO mouse and that the GPI-GalNAc side chain is for cell and We investigated the expression levels of proteins for cell protein and protein for neurons, and protein for molecule for and protein and for expression levels of and in mice were in mice and the of neurons, and in mice. the expression of the in mice, significant in the expression of and expression of analysis the mouse of in to this that loss of the GPI-GalNAc side chain the of of we the pathological roles of the GPI-GalNAc side that of PrPC GPI a of A. acid on the glycosylphosphatidylinositol cell and prion Biol. Scholar, T. M. GPI-anchor PrPC and and on of prion in Scholar). that acid in GPI on the GalNAc side is that the GalNAc side chain is in of prion diseases. we loss of GalNAc modification the of mice. We prion M. M. of to and prion R.L. in mice by inoculation Scholar). and mice were and We that mice after inoculation types of strains and the of in mouse that in mice, as by and and and in the mouse were in the that levels were These that of its is in PGAP4-KO mouse brains. the we inoculation and the of an early of inoculation We that of in PGAP4-KO mice mice that loss of the GPI-GalNAc side chain the of PrPC to this we the in vivo roles of the GPI-GalNAc side chain by mice for its initiation enzyme, to the expression of GPI (3Ferguson M.A.J. Hart G.W. Kinoshita T. Glycosylphosphatidylinositol anchors.in: Varki A. Cummings R.D. Esko J.D. Stanley P. Hart G.W. Aebi M. Darvill A.G. Kinoshita T. Packer N.H. Prestegard J.H. Schnaar R.L. Seeberger P.H. Essentials of Glycobiology. Cold Spring Hobor Laboratory Press, Cold Spring Harbor, NY2015: 137-150Google Pgap4 mRNA expressed in including the brain, and GPI-APs brain, and were to modified the GalNAc side chain structure of the membrane of Scholar, A. T. T. A. T. Kinoshita T. of acid to a GPI and an GPI in prion Biol. 2020; Scholar, M. T. A. of glycosylphosphatidylinositol on mouse cell structure of the GPI glycan and Scholar, A. of the anchors of and membrane on the and of the glycan core Biol. Scholar). expression of that the GPI-GalNAc side chain functions of of the GPI-GalNAc side chain the expression and association of GPI-APs in the and and T. Fujita M. Kinoshita T. of a in the in particularly in bone and brain. the that the core GPI structure is and the GalNAc side chain is required for the of some GPI-APs in the of the GPI-GalNAc side is to this side chain and of GPI-APs. in PGAP4-KO mice, including elevated blood bone formation, activity, and impaired memory, are for in glycosylphosphatidylinositol Scholar). as a of of largely on the of have and PGAP4-KO mice the that the show elevated blood levels bone blood of a expressed on the cell highly elevated in PGAP4-KO mice association of impaired by loss of the GalNAc side in the of blood mice, in is impaired Kinoshita T. of roles of acid of GPI Scholar, T. Kinoshita T. of protein in lipid rafts for the of Biol. of the blood Kinoshita T. of roles of acid of GPI and a in a in GPI-anchor a of Scholar). of GPI the and levels in some is that of the GPI in the the of the GPI of to its A. Kinoshita T. for of alkaline phosphatase by glycosylphosphatidylinositol in Biol. Scholar). in the T. Fujita M. Kinoshita T. of a in the loss of the GalNAc side chain of by a Thus, is to are GPI-APs is by association elevated and in the blood bone and of bone were in PGAP4-KO mice, and of and were in PGAP4-KO mice is for of in the bone through the of levels A. alkaline phosphatase and cell membrane are of bone A. that impaired membrane association of in PGAP4-KO mice the of bone We that some phenotypes, as and mass in PGAP4-KO mice, were in Although the for this the were in various in a analysis of in mammalian Scholar). PGAP4-KO mice exhibited various as activity, in an and impaired despite the of mice in the the of mice in the to that of mice and that the of mice of is in in glycosylphosphatidylinositol and protein mice M. glycosylphosphatidylinositol in development, and in a mouse 2020; of are GPI-APs in the brain, including the of cell adhesion and PrPC, and (3Ferguson M.A.J. Hart G.W. Kinoshita T. Glycosylphosphatidylinositol anchors.in: Varki A. Cummings R.D. Esko J.D. Stanley P. Hart G.W. Aebi M. Darvill A.G. Kinoshita T. Packer N.H. Prestegard J.H. Schnaar R.L. Seeberger P.H. Essentials of Glycobiology. Cold Spring Hobor Laboratory Press, Cold Spring Harbor, NY2015: 137-150Google Scholar, cell adhesion and in and 10: Scholar). These proteins are in and synaptic A. T. of GPI-anchored protein in lipid rafts of Scholar, T. J.H. A. of in the through Scholar, Biol. Scholar, A. of as a for in Scholar, P. on and loss of GPI-APs synaptic and in the M. M. A. A. M. A. mouse for GPI-anchor and synaptic A. Scholar, cell adhesion and in and 10: Scholar, M. P. of membrane protein to impaired synaptic and Scholar). the of more of GPI-APs the in PGAP4-KO mice. GPI of GPI-APs for and PrPC have is to GPI of the of functions in PGAP4-KO mice. PGAP4-KO mice a of prion diseases than mice by of of PrPC to in PGAP4-KO mice. the of a to an to T. protein a that 2020; the of a in PGAP4-KO mice, as by the elevated expression that the of and in PGAP4-KO mice that in mice and the of in mice is more types of in PGAP4-KO mice, more required to the of in PGAP4-KO mice we that the is in PGAP4-KO mice. by the that of on the GalNAc side the of PrPC to A. acid on the glycosylphosphatidylinositol cell and prion Biol. Scholar). to the of the GPI-GalNAc side chain of PrPC the to is that the GalNAc side chain of PrPC, as by a T. M. GPI-anchor PrPC and and on of prion in Scholar). the GPI-GalNAc side chain the of PrPC as to protein M. protein and Biol. Scholar). and of GPI-anchored PrPC are required to the of the side chain in PrPC mouse a T. M. GPI-anchor PrPC and and on of prion in is that of GPI-GalNAc side chain and have protective prion diseases. PrPC GPI is more to in A. T. T. A. T. Kinoshita T. of acid to a GPI and an GPI in prion Biol. 2020; than mouse PrPC on the GPI-GalNAc side chain an for prion diseases. of the structure and of the GPI-GalNAc side chain of the of prion diseases and the of on the structure of the GPI-GalNAc side we have investigated the in vivo functions of the GPI-GalNAc side chain and that this side chain is required for bone and functions and a protective against prion diseases. These that the GPI-GalNAc side chain is for and of the of the GPI-GalNAc side chain to the of for prion diseases. is to the of the GPI-GalNAc side chain the functions of GPI-APs and the of the GPI-GalNAc side chain is in were in the for and and and for of and in by the of and in by the and of and to were for and the and mice were were in inoculation to mice were prion strains and the mice were a the a expression to mouse Pgap4 is a the by by of the a were by mouse as a and and and and and by and by the and by and PGAP4-KO mice were by of M. M. of mouse as A. T. M. 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Kinoshita T. of acid to a GPI and an GPI in prion Biol. 2020; Scholar). to PrPC were and by were to the of PrPC in the were as A. T. T. A. T. Kinoshita T. of acid to a GPI and an GPI in prion Biol. 2020; Scholar). were and were in on a a 150 to an mass mass for mass for for of of acid and were by a of to mass to mass spectrometry analysis by and mass spectrometry were to the were in acid for were in on a an 150 to the mass were the by the as and were by a of for by a of for of mass spectrometry as mass were by and were by the for modification and modifications were to and of of to for prion R.L. in mice by inoculation by prion M. M. of to in mice. were the of mice. inoculation as T. T. M. T. cell of the mouse a for Scholar). were the of mice of the strains as in and of the were mice mice were a the the of PrPC to is in PGAP4-KO mice, mice were of the in for and the for of by were as T. T. T. A. of a for the of of prion Scholar). were in of and and the and the of to M. 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We and for and We for of and for and for a of this T. and T. and T. T. A. T. M. A. and T. and M. T. T. A. and T. T. T. and T. T. and by a for to T. an to T. and of to T. and a to T. a for to a for the for the of a to a the to and a the to

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