Mechanism for release of alkaline phosphatase caused by glycosylphosphatidylinositol deficiency in patients with hyperphosphatasia mental retardation syndrome

Hyperphosphatasia mental retardation syndrome (HPMR), an autosomal recessive disease characterized by mental retardation and elevated serum alkaline phosphatase (ALP) levels, is caused by mutations in the coding region of the phosphatidylinositol glycan anchor biosynthesis, class V (PIGV) gene, the product of which is a mannosyltransferase essential for glycosylphosphatidylinositol (GPI) biosynthesis. Mutations found in four families caused amino acid substitutions A341E, A341V, Q256K, and H385P, which drastically decreased expression of the PIGV protein. Hyperphosphatasia resulted from secretion of ALP, a GPI-anchored protein normally expressed on the cell surface, into serum due to PIGV deficiency. In contrast, a previously reported PIGM deficiency, in which there is a defect in the transfer of the first mannose, does not result in hyperphosphatasia. To provide insights into the mechanism of ALP secretion in HPMR patients, we took advantage of CHO cell mutants that are defective in various steps of GPI biosynthesis. Secretion of ALP requires GPI transamidase, which in normal cells, cleaves the C-terminal GPI attachment signal peptide and replaces it with GPI. The GPI-anchored protein was secreted substantially into medium from PIGV-, PIGB-, and PIGF-deficient CHO cells, in which incomplete GPI bearing mannose was accumulated. In contrast, ALP was degraded in PIGL-, DPM2-, or PIGX-deficient CHO cells, in which incomplete shorter GPIs that lacked mannose were accumulated. Our results suggest that GPI transamidase recognizes incomplete GPI bearing mannose and cleaves a hydrophobic signal peptide, resulting in secretion of soluble ALP. These results explain the molecular mechanism of hyperphosphatasia in HPMR.     

Biochemical background of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by paroxysms of intravascular hemolysis. A considerable part of erythrocytes in patient blood is susceptible to autologous complement activation because of the deficiency of CD59, which is a glycosylphosphatidylinositol (GPI)-anchored protein and inhibits the formation of the membrane attack complex (MAC) of complement. The deficiency of CD59 is derived from the inability of GPI-anchor synthesis. Although more than 10 proteins are involved in the GPI-anchor synthesis, the mutation of only one protein, PIG-A, causes the defect in about 200 patients with PNH who have been analyzed. The reason why only PIG-A causes the deficiency of GPI anchor is due to the location of its gene on X chromosome. The clonal stem cell mutated with PIG-A gene in the bone marrow loses the capability of the synthesis of GPI-anchor. The mutation of PIG-A gene alone, however, seems to be insufficient to account for the survival of the PIG-A-deficient cells in the bone marrow. Thus, a fraction of the mutant stem cells probably gain a survival advantage by some additional changes, either additional mutations or changes in immunological circumstances. The release of the surviving cells into blood stream results in a clinical syndrome with PNH.     

Glucose phosphate isomerase deficiency: enzymatic and familial characterization of Arg346His mutation

Homozygous glucose phosphate isomerase (GPI) deficiency is one of the most important genetic disorders responsible for chronic non-spherocytic hemolytic anemia (CNSHA), a red blood cell autosomal recessive genetic disorder which causes severe metabolic alterations. In this work, we studied a patient with CNSHA due to an 82% loss of GPI activity resulting from the homozygous missense replacement in cDNA position 1040G>A, which leads to substitution of the protein residue A346H mutation. The enzyme is present in a dimeric form necessary for normal activity; the A346H mutation causes a loss of GPI capability to dimerize, which renders the enzyme more susceptible to thermolability and produces significant changes in erythrocyte metabolism.     

GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors. The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene. The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B. YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans. Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay. Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1- 6(acyl) Inositol-P-lipid. The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins. This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells. Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.      

Isolation of Novel Animal Cell Lines Defective in Glycerolipid Biosynthesis Reveals Mutations in Glucose-6-phosphate Isomerase

Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 → Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.     

Caffeine sensitivity of the yeast Saccharomyces cerevisiae MCD4 mutant is related to alteration of calcium homeostasis and degradation of misfolded proteins

The MCD4 gene of Saccharomyces cerevisiae encodes a protein involved in glycosylphosphatidylinositol (GPI) biosynthesis. However, some mutations in the MCD4 gene have pleiotropic effects that may not be related to the defects in GPI anchor synthesis. The ssu21 mutation in the MCD4 gene studied here causes sensitivity to caffeine. The screen for multicopy suppressors of caffeine sensitivity caused by the ssu21 mutation revealed genes involved in aminoglycerophospholipid metabolism, unfolded protein response, and protein degradation. The suppressor effect of all isolated genes was enhanced by an increase in concentration of external calcium, which is consistent with the ability of caffeine to block calcium entry into the yeast cell. Obtained results indicate that caffeine sensitivity caused by the ssu21 mutation could be due to cytoplasmic accumulation of misfolded proteins destined for degradation.     

Clinical symptoms and biochemical properties of three new glucosephosphate isomerase variants

Glucosephosphate isomerase deficiency as the cause of macrocytic congenital nonspherocytic hemolytic anemia is described in three unrelated families. The biochemical properties of the variant glucosephosphate isomerases indicate that the patients have new variants, designated as GPI Kiel, GPI Hamburg, and GPI Homburg. The severity of the clinical symptoms depended on the amount of residual GPI activity and the biochemical properties of the variant enzyme. Thus the patient with GPI Kiel (34% residual activity) whose variant GPI was slightly unstable showed a mild chronic hemolytic anemia. The patient with GPI Homburg (7% residual activity) whose variant enzyme was stable and had a reduced specific activity, suffered from severe congenital hemolytic anemia and neuromuscular symptoms. Due to the special properties of GPI Homburg, we assume that both the hematological and neuromuscular symptoms of the patient with GPI Homburg are caused by his GPI deficiency. The twins with GPI Hamburg (27% residual activity) had a distinctly unstable variant enzyme and had suffered from hemolytic crises since birth. Only GPI Homburg showed an altered electrophoretic mobility and an increased affinity for fructose-6-phosphate. The other two variants had normal values.     

PIG-B,a membrane protein of the endoplasmic reticulum with a large lumenal domain,is involved in transferring the third mannose of the GPI anchor.

Many eukaryotic cell surface proteins are bound to the membrane via the glycosylphosphatidylinositol (GPI) anchor that is covalently linked to their carboxy-terminus. The GPI anchor precursor is synthesized in the endoplasmic reticulum (ER) and post-translationally linked to protein. We cloned a human gene termed PIG-B (phosphatidylinositol glycan of complementation class B) that is involved in transferring the third mannose. PIG-B encodes a 554 amino acid, ER transmembrane protein with an amino-terminal portion of approximately 60 amino acids on the cytoplasmic side and a large carboxy-terminal portion of 470 amino acids within the ER lumen. A mutant PIG-B lacking the cytoplasmic portion remains active, indicating that the functional site of PIG-B resides on the lumenal side of the ER membrane. The PIG-B gene was localized to chromosome 15 at q21-q22. This autosomal location would explain why PIG-B is not involved in the defective GPI anchor synthesis in paroxysmal nocturnal hemoglobinuria, which is always caused by a somatic mutation of the X-linked PIG-A gene.     

Inherited glycosylphosphatidyl inositol deficiency: A treatable CDG

Tethering to cell membrane through attachment to the complex glycolipid anchor glycosylphosphatidyl inositol (GPI) is a mode of protein expression highly conserved in eukaryotes. The evolutionary purpose of such an elaborate way of expressing proteins is not clear and neither is the functional role of GPI itself. GPI-anchored proteins (GPI-AP) serve a variety of functions that include adhesion, receptors, signal transduction and complement activation. GPI biosynthesis, a process that is accomplished in at least 9 steps and involves several proteins, some with enzymatic activity, would be expected to be a fertile ground for development of inherited, autosomal recessive disorders. However, until recently, paroxysmal nocturnal haemoglobinuria, a rare haematological disorder caused by somatic mutations in the X-linked PIGA gene, was the only genetic disorder affecting GPI biosynthesis. Here we review the clinical spectrum, biochemical defect and genetic pathogenesis of inherited GPI deficiency, the first described form of inherited, autosomal recessive disorder of GPI biosynthesis and outline the molecular basis of targeted therapy for this condition.     

Null Mutation in PGAP1 Impairing Gpi-Anchor Maturation in Patients with Intellectual Disability and Encephalopathy

Many eukaryotic cell-surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). There are at least 26 genes involved in biosynthesis and remodeling of GPI anchors. Hypomorphic coding mutations in seven of these genes have been reported to cause decreased expression of GPI anchored proteins (GPI-APs) on the cell surface and to cause autosomal-recessive forms of intellectual disability (ARID). We performed homozygosity mapping and exome sequencing in a family with encephalopathy and non-specific ARID and identified a homozygous 3 bp deletion (p.Leu197del) in the GPI remodeling gene PGAP1. PGAP1 was not described in association with a human phenotype before. PGAP1 is a deacylase that removes an acyl-chain from the inositol of GPI anchors in the endoplasmic reticulum immediately after attachment of GPI to proteins. In silico prediction and molecular modeling strongly suggested a pathogenic effect of the identified deletion. The expression levels of GPI-APs on B lymphoblastoid cells derived from an affected person were normal. However, when those cells were incubated with phosphatidylinositol-specific phospholipase C (PI-PLC), GPI-APs were cleaved and released from B lymphoblastoid cells from healthy individuals whereas GPI-APs on the cells from the affected person were totally resistant. Transfection with wild type PGAP1 cDNA restored the PI-PLC sensitivity. These results indicate that GPI-APs were expressed with abnormal GPI structure due to a null mutation in the remodeling gene PGAP1. Our results add PGAP1 to the growing list of GPI abnormalities and indicate that not only the cell surface expression levels of GPI-APs but also the fine structure of GPI-anchors is important for the normal neurological development.     

Paroxysmal nocturnal haemoglobinuria (PNH) is caused by somatic mutations in the PIG-A gene.

Paroxysmal nocturnal haemoglobinuria (PNH), an acquired clonal blood disorder, is caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins due to a defect in a specific step of GPI-anchor synthesis. The cDNA of the X-linked gene, PIG-A, which encodes a protein required for this step has recently been isolated. We have carried out a molecular and functional analysis of the PIG-A gene in four cell lines deficient in GPI-linked proteins, obtained by Epstein-Barr virus (EBV) transformation of affected B-lymphocytes from PNH patients. In all four cell lines transfection with PIG-A cDNA restored normal expression of GPI-linked proteins. In three of the four cell lines the primary lesion is a frameshift mutation. In two of these there is a reduction in the amount of full-length mRNA. The fourth cell line contains a missense mutation in PIG-A. In each case the mutation was present in the affected granulocytes from peripheral blood of the patients, but not in normal sister cell lines from the same patient. These data prove that PNH is caused in most patients by a single mutation in the PIG-A gene. The nature of the mutation can vary and most likely occurs on the active X-chromosome in an early haematopoietic stem cell.