Glycosylphosphatidylinositol Moiety Research Articles

In vivo incorporation of an azide-labeled sugar analog to detect mammalian glycosylphosphatidylinositol molecules isolated from the cell surface

N-Acetylgalactosamine (GalNAc) linked to the first mannose of glycosylphosphatidylinositol (GPI) core has been previously reported to be heterogeneously present on some mammalian GPI-anchored proteins. Here we present a method for profiling GalNAc-containing GPI-anchored proteins in mammalian cells by metabolic labeling with tetraacetylated N-azidoacetylgalactosamine (GalNAz) followed by biotinylation of the incorporated sugar analog. We have labeled both endogenous and recombinant GPI-anchored proteins with GalNAz, and demonstrated that the azide-activated sugar gets incorporated into the GPI glycan, likely as an unsubstituted side branch of the core structure. GalNAz was detected only on GPI molecules attached to proteins, and not on GPI precursors, indicating that GalNAc modification takes place after the GPI anchor is transferred to protein. We have highlighted the utility of this cell labeling approach by demonstrating the ability to examine specific GalNAc-containing GPI-anchored proteins isolated non-destructively from separate membrane domains (apical and basolateral) in polarized epithelial cells. This study represents the first demonstration of site-specific in vivo labeling of a GPI moiety with a synthetic sugar analog.

Characterization of conformation-dependent prion protein epitopes.

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP(C)), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27-30), a protease-resistant counterpart of the pathogenic scrapie form (PrP(Sc)) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126-218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27-30) both renature to a common structure that reconstitutes the globular domain.

Phagocyte Responses to Protozoan Infection and How Toxoplasma gondii Meets the Challenge

Phagocyte Responses to Protozoan Infection and How Toxoplasma gondii Meets the Challenge

Efficient Glycosylphosphatidylinositol (GPI) Modification of Membrane Proteins Requires a C-terminal Anchoring Signal of Marginal Hydrophobicity

Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is "marginal": less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating that the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 (ω−2, where ω is the GPI moiety at E160), P159 (ω−1), and M162 (ω+2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide–anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.

The Receptor for Urokinase Regulates TLR2 Mediated Inflammatory Responses in Neutrophils

The urokinase-type plasminogen activator receptor (uPAR), a glycosylphosphatidylinositol (GPI) anchored membrane protein, regulates urokinase (uPA) protease activity, chemotaxis, cell-cell interactions, and phagocytosis of apoptotic cells. uPAR expression is increased in cytokine or bacteria activated cell populations, including macrophages and monocytes. However, it is unclear if uPAR has direct involvement in the response of inflammatory cells, such as neutrophils and macrophages, to Toll like receptor (TLR) stimulation. In this study, we found that uPAR is required for optimal neutrophil activation after TLR2, but not TLR4 stimulation. We found that the expression of TNF-α and IL-6 induced by TLR2 engagement in uPAR-/- neutrophils was less than that in uPAR+/+ (WT) neutrophils. Pretreatment of neutrophils with PI-PLC, which cleaves GPI moieties, significantly decreased TLR2 induced expression of TNF-α in WT neutrophils, but demonstrated only marginal effects on TNF-α expression in PAM treated uPAR-/- neutrophils. IκB-α degradation and NF-κB activation were not different in uPAR-/- or WT neutrophils after TLR2 stimulation. However, uPAR is required for optimal p38 MAPK activation after TLR2 engagement. Consistent with the in vitro findings that uPAR modulates TLR2 engagement induced neutrophil activation, we found that pulmonary and systemic inflammation induced by TLR2, but not TLR4 stimulation is reduced in uPAR-/- mice compared to WT counterparts. Therefore, our data suggest that neutrophil associated uPAR could be a potential target for treating acute inflammation, sepsis, and organ injury related to severe bacterial and other microbial infections in which TLR2 engagement plays a major role.

Characterization of the Dimerization Interface of Membrane Type 4 (MT4)-Matrix Metalloproteinase

MT4-MMP (MMP17) belongs to a unique subset of membrane type-matrix metalloproteinases that are anchored to the cell surface via a glycosylphosphatidylinositol moiety. However, little is known about its biochemical properties. Here, we report that MT4-MMP is displayed on the cell surface as a mixed population of monomeric, dimeric, and oligomeric forms. Sucrose gradient fractionation demonstrated that these forms of MT4-MMP are all present in lipid rafts. Mutational and computational analyses revealed that Cys(564), which is present within the stem region, mediates MT4-MMP homodimerization by forming a disulfide bond. Substitution of Cys(564) results in a more rapid MT4-MMP turnover, when compared with the wild-type enzyme, consistent with a role for dimerization in protein stability. Expression of MT4-MMP in Madin-Darby canine kidney cells enhanced cell migration and invasion of Matrigel, a process that requires catalytic activity. However, a serine substitution at Cys(564) did not reduce MT4-MMP-stimulated cell invasion of Matrigel suggesting that homodimerization is not required for this process. Deglycosylation studies showed that MT4-MMP is modified by N-glycosylation. Moreover, inhibition of N-glycosylation by tunicamycin diminished the extent of MT4-MMP dimerization suggesting that N-glycans may confer stability to the dimeric form. Taken together, the data presented here provide a new insight into the characteristics of MT4-MMP and highlight the common and distinct properties of the glycosylphosphatidylinositol-anchored membrane type-matrix metalloproteinases.

Differential Clustering of Membrane Anchors Observed by FCCS in Live Cells

Proper lateral organization of protein signaling elements in the cell membrane is crucial for proper cell behavior. Several important signaling proteins, such as some members of the Src family of kinases, are anchored to the membrane by covalent acyl, alkyl, and glycosylphosphatidylinositol (GPI) moieties. It has been proposed that these membrane anchors help to laterally sort proteins into signaling nanoclusters of unknown sizes. We set out to understand the role of these membrane anchors in the lateral sorting of proteins in the plasma membrane of a live cell. In order to observe this, we genetically encode lipidation motifs from a number of different cell signaling proteins fused to either a monomeric red or green fluorescent protein in living cells. Using Fluorescence Cross-Correlation Spectroscopy (FCCS) we are able to quantify the amount of dynamic colocalization of red and green fluorescent proteins anchored in the cell membrane. By only illuminating an area of the membrane with the cross-section of a focused laser beam, FCCS allows us to observe dynamic colocalization on the nanometer length scale, and unlike Forster Resonance Energy Transfer (FRET), FCCS can detect positive colocalization regardless of orientation and at lengths beyond the Forster radius of FRET pairs. We seek to understand if our fluorescent membrane anchors alone can dynamically colocalize in domains and if these domains can discriminate between different anchors. We have performed pairwise comparison of green-labeled and red-labeled membrane anchors in live cells and have seen different levels of cross-correlation for different anchor pairs.

Endogenous Proteolytic Cleavage of Disease-associated Prion Protein to Produce C2 Fragments Is Strongly Cell- and Tissue-dependent

The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection.

Cloning and functional characterization of chicken stem cell antigen 2

Stem cell antigen 2 (SCA2) is a Ly6 family member whose function is largely unknown. To characterize biological properties and tissue distribution of chicken SCA2, SCA2 was expressed in E. coli, purified, and a polyclonal antibody developed. Utilizing the polyclonal antibody, SCA2 is a 13 kDa cell surface protein anchored by a glycosyl-phosphatidylinositol (GPI) moiety. SCA2 is expressed in connective tissues of thymus and bursa based on immunohistochemistry, immunoprecipitation, and western blots. In bursal follicles, SCA2 is specifically expressed on the cortical-medullary epithelial cells (CMEC) surrounded by MHC class II presenting cells. Expression profiles of bursal cells induced by contact with SCA2-expressing cells shows down-regulation of numerous genes including CD79B, B cell linker ( BLNK), spleen tyrosine kinase ( SYK), and gamma 2-phospholipase C ( PLCG2) that are involved in the B cell receptor (BCR) and immune response signaling pathways. These results suggest chicken SCA2 plays a role in regulating B lymphocytes.

GPI Glycan Remodeling by PGAP5 Regulates Transport of GPI-Anchored Proteins from the ER to the Golgi

Many eukaryotic proteins are attached to the cell surface via glycosylphosphatidylinositol (GPI) anchors. How GPI-anchored proteins (GPI-APs) are trafficked from the endoplasmic reticulum (ER) to the cell surface is poorly understood, but the GPI moiety has been postulated to function as a signal for sorting and transport. Here, we established mutant cells that were selectively defective in transport of GPI-APs from the ER to the Golgi. We identified a responsible gene, designated PGAP5 (post-GPI-attachment to proteins 5). PGAP5 belongs to a dimetal-containing phosphoesterase family and catalyzed the remodeling of the glycan moiety on GPI-APs. PGAP5 catalytic activity is a prerequisite for the efficient exit of GPI-APs from the ER. Our data demonstrate that GPI glycan acts as an ER-exit signal and suggest that glycan remodeling mediated by PGAP5 regulates GPI-AP transport in the early secretory pathway.

Phospholipids influence the aggregation of recombinant ovine prions: From rapid extensive aggregation to amyloidogenic conversion

The transformation of prion protein (PrP) into its insoluble amyloid form correlates with neurodegeneration in transmissible spongiform encephalopathies. PrP is connected to the neuronal membrane by a covalently-linked glycosylphosphatidylinositol (GPI) anchor. The current study demonstrates that phosphatidylinositol and phosphatidylethanolamine in low concentrations (0.5–50 μM) stimulate rapid unlimited aggregation of PrP. At a higher concentration (500 μM), lipid particles prevent the formation of large PrP aggregates and induce an increase in the β-sheet structure content of PrP protein. Thus, the liberation of PrP from the membrane and its direct interaction with its own GPI moiety, as well as with membrane lipids, can promote the formation of aggregated structures of PrP. The phospholipids studied are also able to upregulate the aggregation of oligomeric PrP forms (12-mers and 36-mers), the neurotoxicity of which has been reported recently. Low phosphatidylinositol concentrations induce these oligomers to form aggregates of smaller size when compared with aggregates formed directly from monomers. The inhibition of extensive aggregation observed at a high concentration of phosphatidylinositol (500 μM) results in both the formation of amyloids from PrP monomers and the interaction of protein molecules with lipid micelles. Thus, phospholipids are not only involved in the aggregation of prion monomers and their amyloidogenic conversion, but also regulate the aggregative status of prion oligomers already formed. Consequently, depending on their micellar status, phospholipids can either promote amyloidogenic conversion and conserve neurotoxic oligomeric forms (lipid micelles) or mediate the formation of large-size amorphous aggregates (non-micellar phospholipids).

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Identification and Role of the Homodimerization Interface of the Glycosylphosphatidylinositol-anchored Membrane Type 6 Matrix Metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.

Characteristics of Laboratory Screening Suggest That Paroxysmal Nocturnal Hemoglobinuria (PNH) Is Perceived Primarily as a Bone Marrow Failure Syndrome Rather Than as a Hemolytic Anemia

PNH is a hematopoietic stem cell disorder in which the predominant clinical manifestations are hemolysis, bone marrow failure and thrombophilia. PNH arises as a result of somatic mutation of PIGA, an X-linked gene required for synthesis of the glycosyl phosphatidylinositol (GPI) moiety that anchors some proteins to the cell surface; and consequently, progeny of affected stem cells are deficient in all GPI-anchored proteins (GPI-APs). The hemolysis of PNH is the result of deficiency of CD55 and CD59, GPI-APs that normally inhibit complement activation on the red cell surface, but the relationship between GPI-AP deficiency and the bone marrow failure and thrombophilia of PNH are enigmatic. The peripheral blood of patients with PNH is a mosaic of normal and abnormal cells, and the degree of mosaicism varies greatly among patients. By using fluorescently labeled antibodies, GPI-AP deficient cells (GPI-AP−) can be distinguished form GPI-AP sufficient cells (GPI-AP+) cells by flow cytometric analysis, allowing quantitation of mosaicism. Flow cytometry has been used diagnostically for more than a decade, and technical modifications have improved resolution so that very small populations of GPI-AP− peripheral blood cells can be accurately detected. The purpose of these studies was to generate insights into how PNH is perceived in the community by analyzing the results of a commercially available screening assay using data from a national clinical diagnostic laboratory (ARUP Laboratories, Salt Lake City, UT). The flow cytometric method used in these studies is a modification of the high-resolution two-color assay of Sugimori and colleagues (Blood 2006, 107:1308–1314). Clients are given the choice of testing for PNH by analyzing peripheral blood RBCs or PMNs (or both). The acidified serum test (Ham's test) and the sucrose lysis test (sugar water test) are also available for screening for PNH. For flow cytometric analysis of RBCs, a value of ≥0.005% GPI-AP− cells is considered abnormal, while for PMNs ≥0.003% is abnormal. From January 1, 2008 to June 30, 2008, 1,113 RBC assays and 133 PMN assays were performed. An abnormally large population of GPI-AP− RBCs was identified in 55 cases (5%). The percentage of GPI-AP− RBC ranged from 0.009–69.603% with a median of 1.405%. Twenty-two cases (40%) had >5% GPI-AP− RBCs, while 18 cases (33%) had >10% GPI-AP− RBCs. Of the 133 PMN assays performed, 15 (11%) were abnormal. The range of GPI-AP− PMNs was 0.004–97.727% with a median of 18.327 %. Eight samples (53%) had >10% GPI-AP− PMNs. During the 1-year period from July 1, 2007-June 30, 2008 the acidified serum lysis test (Ham's test) was performed on 212 samples while the sucrose lysis test was performed on 148 samples. These studies suggest that screening for PNH is common (~43 RBC assays/week compared to 44 assays/week for flow cytometric screening of peripheral blood for lymphoproliferative disorders and leukemia), but the vast majority of samples tested show normal expression of GPI-APs. That so many of the test samples are negative, and that the median for abnormal RBC samples is ~1.5 % GPI-AP− cells, suggest that most of the screening is done because of the association of PNH with bone marrow failure syndromes rather than because of evidence of intravascular hemolysis. These studies underscore the need to understand the pathophysiological basis and clinical implications of small populations of GPI-AP deficient cells in patients with bone marrow failure syndromes. Nonetheless, 18 cases with >10% GPI-AP− RBCs were detected during the 6 months of observation, indicating that the prevalence of classic PNH in the US is substantial. That PNH clone size is best determined by analysis of GPI-AP expression on PMNs does not appear to be widely appreciated in the community as the PMN assays is requested 12% as often as the RBC assay. Flow cytometry has largely, but not completely, replaced Ham's test and the sucrose lysis test as screening assays for PNH.

Glycosylation Analysis of IgLON Family Proteins in Rat Brain by Liquid Chromatography and Multiple-Stage Mass Spectrometry

IgLON family proteins, including limbic-associated membrane protein (LAMP), opioid-binding cell adhesion molecule (OBCAM), neurotrimin, and Kilon, are immunoglobulin (Ig) superfamily cell adhesion molecules. These molecules are composed of three Ig domains and a glycosylphosphatidylinositol (GPI) anchor and contain six or seven potential N-glycosylation sites. Although their glycosylations are supposed to be associated with the development of the central nervous system like other Ig superfamily proteins, they are still unknown because of difficulty in isolating individual proteins with a high degree of homology in performing carbohydrate analysis. In this study, we conducted simultaneous site-specific glycosylation analysis of rat brain IgLON proteins by liquid chromatography and multiple-stage mass spectrometry (LC-MS ( n )). The rat brain GPI-linked proteins were enriched and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The four proteins were extracted from the gel, and subjected to LC-MS ( n ) after proteinase digestions. A set of glycopeptide MS data, including the mass spectrum, the mass spectrum in the selected ion monitoring mode, and the product ion spectra, was selected from all data based on carbohydrate-related ions in the MS/MS spectrum. The peptide portion and the carbohydrate structure were identified on the basis of peptide-related ion and carbohydrate-related ions, and the accurate mass. The site-specific glycosylations of four proteins were elucidated as follows. N-Glycans near the N-terminal were disialic acid-conjugated complex- and hybrid-type oligosaccharides. The first Ig domains were occupied by Man-5-9. Diverse oligosaccharides, including Lewis a/x-modified glycans, a brain-specific glycan known as BA-2, and Man-5, were found to be attached to the third Ig domain. Three common structures of glycans were found in the GPI moiety of LAMP, OBCAM, and neurotrimin.

Bioinformatics and Functional Analysis of an Entamoeba histolytica Mannosyltransferase Necessary for Parasite Complement Resistance and Hepatical Infection

The glycosylphosphatidylinositol (GPI) moiety is one of the ways by which many cell surface proteins, such as Gal/GalNAc lectin and proteophosphoglycans (PPGs) attach to the surface of Entamoeba histolytica, the agent of human amoebiasis. It is believed that these GPI-anchored molecules are involved in parasite adhesion to cells, mucus and the extracellular matrix. We identified an E. histolytica homolog of PIG-M, which is a mannosyltransferase required for synthesis of GPI. The sequence and structural analysis led to the conclusion that EhPIG-M1 is composed of one signal peptide and 11 transmembrane domains with two large intra luminal loops, one of which contains the DXD motif, involved in the enzymatic catalysis and conserved in most glycosyltransferases. Expressing a fragment of the EhPIG-M1 encoding gene in antisense orientation generated parasite lines diminished in EhPIG-M1 levels; these lines displayed reduced GPI production, were highly sensitive to complement and were dramatically inhibited for amoebic abscess formation. The data suggest a role for GPI surface anchored molecules in the survival of E. histolytica during pathogenesis.

Mammalian GPI-anchored proteins require p24 proteins for their efficient transport from the ER to the plasma membrane

The GPI (glycosylphosphatidylinositol) moiety is attached to newly synthesized proteins in the lumen of the ER (endoplasmic reticulum). The modified proteins are then directed to the PM (plasma membrane). Less well understood is how nascent mammalian GPI-anchored proteins are targeted from the ER to the PM. In the present study, we investigated mechanisms underlying membrane trafficking of the GPI-anchored proteins, focusing on the early secretory pathway. We first established a cell line that stably expresses inducible temperature-sensitive GPI-fused proteins as a reporter and examined roles of transport-vesicle constituents called p24 proteins in the traffic of the GPI-anchored proteins. We selectively suppressed one of the p24 proteins, namely p23, employing RNAi (RNA interference) techniques. The suppression resulted in pronounced delays of PM expression of the GPI-fused reporter proteins. Furthermore, maturation of DAF (decay-accelerating factor), one of the GPI-anchored proteins in mammals, was slowed by the suppression of p23, indicating delayed trafficking of DAF from the ER to the Golgi. Trafficking of non-GPI-linked cargo proteins was barely affected by p23 knockdown. This is the first to demonstrate direct evidence for the transport of mammalian GPI-anchored proteins being mediated by p24 proteins.

Requirement of Glycosylphosphatidylinositol Anchor of Cripto-1 for trans Activity as a Nodal Co-receptor

Cripto-1 (CR-1) has an indispensable role as a Nodal co-receptor for patterning of body axis in embryonic development. CR-1 is reported to have a paracrine activity as a Nodal co-receptor, although CR-1 is primarily produced as a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Regulation of cis and trans function of CR-1 should be important to establish the precise body patterning. However, the mechanism by which GPI-anchored CR-1 can act in trans is not well known. Here we confirmed the paracrine activity of CR-1 by fluorescent cell-labeling and immunofluorescent staining. We generated COOH-terminal-truncated soluble forms of CR-1 based on the attachment site for the GPI moiety (omega-site), which we identified in the present study. GPI-anchored CR-1 has a significantly higher activity than COOH-terminal-truncated soluble forms to induce Nodal signal in trans as well as in cis. Moreover, transmembrane forms of CR-1 partially retained their ability to induce Nodal signaling only when type I receptor Activin-like kinase 4 was overexpressed. NTERA2/D1 cells, which express endogenous CR-1, lost the cell-surface expression of CR-1 after phosphatidylinositol-phospholipase C treatment and became refractory to stimulation of Nodal. These observations suggest that GPI attachment of CR-1 is required for the paracrine activity as a Nodal co-receptor.

Cryptosporidium parvum glycoprotein gp40 localizes to the sporozoite surface by association with gp15

Cryptosporidium spp. are waterborne apicomplexan parasites responsible for outbreaks of diarrheal disease worldwide. Antigens involved in zoite invasion into host cells have been the focus of many investigations as these may prove to be good vaccine candidates. gp40/15 is a zoite antigen synthesized as a precursor protein and proteolytically cleaved into the mature glycoproteins, gp40 and gp15. gp15 is anchored in the sporozoite membrane by a glycosylphosphatidyl inositol moiety, while gp40 is predicted to be soluble. However, gp40 bears epitopes that recognize a host cell receptor. If this interaction is important for zoite invasion, then gp40 must have some mechanism of associating with the parasite membrane. In these studies we demonstrate that gp40 and gp15 co-localize to the surface membrane of sporozoites and merozoites, and co-immunoprecipitate, suggesting that these antigens associate after proteolytic cleavage to generate a protein complex capable of linking zoite and host cell surfaces.