Defective lipid remodeling of GPI anchors in peroxisomal disorders, Zellweger syndrome, and rhizomelic chondrodysplasia punctata

Many cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The predominant form of mammalian GPI contains 1-alkyl-2-acyl phosphatidylinositol (PI), which is generated by lipid remodeling from diacyl PI. The conversion of diacyl PI to 1-alkyl-2-acyl PI occurs in the ER at the third intermediate in the GPI biosynthetic pathway. This lipid remodeling requires the alkyl-phospholipid biosynthetic pathway in peroxisome. Indeed, cells defective in dihydroxyacetone phosphate acyltransferase (DHAP-AT) or alkyl-DHAP synthase express only the diacyl form of GPI-anchored proteins. A defect in the alkyl-phospholipid biosynthetic pathway causes a peroxisomal disorder, rhizomelic chondrodysplasia punctata (RCDP), and defective biogenesis of peroxisomes causes Zellweger syndrome, both of which are lethal genetic diseases with multiple clinical phenotypes such as psychomotor defects, mental retardation, and skeletal abnormalities. Here, we report that GPI lipid remodeling is defective in cells from patients with Zellweger syndrome having mutations in the peroxisomal biogenesis factors PEX5, PEX16, and PEX19 and in cells from patients with RCDP types 1, 2, and 3 caused by mutations in PEX7, DHAP-AT, and alkyl-DHAP synthase, respectively. Absence of the 1-alkyl-2-acyl form of GPI-anchored proteins might account for some of the complex phenotypes of these two major peroxisomal disorders.     

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.     

DPM1, the catalytic subunit of dolichol-phosphate mannose synthase, is tethered to and stabilized on the endoplasmic reticulum membrane by DPM3

Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.     

Testicular Angiotensin-converting enzyme with different glycan modification: characterization on glycosylphosphatidylinositol-anchored protein releasing and dipeptidase activities

We have previously found that the angiotensin-converting enzyme (ACE) carries GPI-anchored protein releasing activity (GPIase) as well as dipeptidase activity. Testicular ACE (tACE), the male germinal specific isozyme, plays a crucial role in male fertilization. The amino-terminal region of this isozyme is different from that of somatic isozyme (sACE) and contains potential O-linked glycosylation sites. By multiple mutagenesis after an in silico prediction, amino acid residues acquiring O-glycans were assigned. Both GPIase and dipeptidase activities were compared between O-glycan null mutant and wild-type molecules, but no differences were found. Furthermore, the wild-type tACE was produced in two different cells (COS7 and CHO) and its activities compared. The GPIase activity, but not dipeptidase, was apparently higher for CHO-derived molecule than COS7. Sensitivity to neuraminidase and O-glycosidase digestions and the profile of glycosylation were quite different between these two molecules. Moreover, serial digestions with neuraminidase and O-glycosidase have no influence on GPIase activity of both molecules, suggesting that the sialylation and the presence of O-glycan has no influence on tACE enzyme activities, while the set of glycans modulate GPIase activity.     

PGAP2 is essential for correct processing and stable expression of GPI-anchored proteins

Biosynthesis of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in the ER has been extensively studied, whereas the molecular events during the transport of GPI-APs from the ER to the cell surface are poorly understood. Here, we established new mutant cell lines whose surface expressions of GPI-APs were greatly decreased despite normal biosynthesis of GPI-APs in the ER. We identified a gene responsible for this defect, designated PGAP2 (for Post-GPI-Attachment to Proteins 2), which encoded a Golgi/ER-resident membrane protein. The low surface expression of GPI-APs was due to their secretion into the culture medium. GPI-APs were modified/cleaved by two reaction steps in the mutant cells. First, the GPI anchor was converted to lyso-GPI before exiting the trans-Golgi network. Second, lyso-GPI-APs were cleaved by a phospholipase D after transport to the plasma membrane. Therefore, PGAP2 deficiency caused transport to the cell surface of lyso-GPI-APs that were sensitive to a phospholipase D. These results demonstrate that PGAP2 is involved in the processing of GPI-APs required for their stable expression at the cell surface.     

Calnexin mediates the maturation of GPI-anchors through ER retention

The protein folding and lipid moiety status of glycosylphosphatidylinositol-anchored proteins (GPI-APs) are monitored in the endoplasmic reticulum (ER), with calnexin playing dual roles in the maturation of GPI-APs. In the present study, we investigated the functions of calnexin in the quality control and lipid remodeling of GPI-APs in the ER. By directly binding the N-glycan on proteins, calnexin was observed to efficiently retain GPI-APs in the ER until they were correctly folded. In addition, sufficient ER retention time was crucial for GPI-inositol deacylation, which is mediated by post-GPI attachment protein 1 (PGAP1). Once the calnexin/calreticulin cycle was disrupted, misfolded and inositol-acylated GPI-APs could not be retained in the ER and were exposed on the plasma membrane. In calnexin/calreticulin-deficient cells, endogenous GPI-anchored alkaline phosphatase was expressed on the cell surface, but its activity was significantly decreased. ER stress induced surface expression of misfolded GPI-APs, but proper GPI-inositol deacylation occurred due to the extended time that they were retained in the ER. Our results indicate that calnexin-mediated ER quality control systems for GPI-APs are necessary for both protein folding and GPI-inositol deacylation.     

V2 vasopressin receptor (V2R) mutations in partial nephrogenic diabetes insipidus highlight protean agonism of V2R antagonists

Inactivating mutations of the V2 vasopressin receptor (V2R) cause cross-linked congenital nephrogenic diabetes insipidus (NDI), resulting in renal resistance to the antidiuretic hormone AVP. In two families showing partial NDI, characterized by an apparently normal response to diagnostic tests and an increase in the basal ADH levels suggesting AVP resistance, we have identified two V2R mutations, Ser-333del and Y128S. Both mutant V2Rs, when expressed in COS-7 cells, show partial defects in vasopressin-stimulated cAMP accumulation and intracellular localization. The inhibition of internalization does not rescue their localization. In contrast, the non-peptide V2R antagonists OPC41061 and OPC31260 partially rescue the membrane localization and basal function of these V2R mutants, whereas they inhibit the basal activity of the wild-type V2R. These results indicate that a partial loss of function of Ser-333del and Y128S mutant V2Rs results from defective membrane trafficking. These findings further indicate that V2R antagonists can act as protean agonists, serving as pharmacological chaperones for inactivating V2R mutants and also as inverse agonists of wild-type receptors. We speculate that this protean agonism could underlie the possible dual beneficial effects of the V2R antagonist: improvement of hyponatremia with heart failure or polycystic kidney disease and potential rescue of NDI.     

PIG-W is critical for inositol acylation but not for flipping of glycosylphosphatidylinositol-anchor

Many cell surface proteins are anchored to a membrane via a glycosylphosphatidylinositol (GPI), which is attached to the C termini in the endoplasmic reticulum. The inositol ring of phosphatidylinositol is acylated during biosynthesis of GPI. In mammalian cells, the acyl chain is added to glucosaminyl phosphatidylinositol at the third step in the GPI biosynthetic pathway and then is usually removed soon after the attachment of GPIs to proteins. The mechanisms and roles of the inositol acylation and deacylation have not been well clarified. Herein, we report derivation of human and Chinese hamster mutant cells defective in inositol acylation and the gene responsible, PIG-W. The surface expressions of GPI-anchored proteins on these mutant cells were greatly diminished, indicating the critical role of inositol acylation. PIG-W encodes a 504-amino acid protein expressed in the endoplasmic reticulum. PIG-W is most likely inositol acyltransferase itself because the tagged PIG-W affinity purified from transfected human cells had inositol acyltransferase activity and because both mutant cells were complemented with PIG-W homologs of Saccharomyces cerevisiae and Schizosaccharomyces pombe. The inositol acylation is not essential for the subsequent mannosylation, indicating that glucosaminyl phosphatidylinositol can flip from the cytoplasmic side to the luminal side of the endoplasmic reticulum.