GPI-anchored Alkaline Phosphatase Research Articles

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.

A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles

Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl–phosphatidyl–inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders.

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.

IDENTIFICATION, ISOLATION AND CLONING OF cDNA ENCODING AMINOPEPTIDASE FROM THE MIDGUT OF THE EGYETIAN COTTON LEAF WORM THAT SERVES AS A RECEPTOR FOR CRY TOXIN

Bacillus thuringiensis (Bt) is an aerobic, spore-forming bacterium that produces crystalline inclusions during the sporulation phase of growth that are composed of proteins known as δ-endotoxins (Bravo et al., 2005). The δ-endotoxins comprise two multigenic families, cry and cyt. Cry proteins are specifically toxic to different insect orders, Lepidoptera, Coleoptera, Hymenoptera, Diptera or nematodes (Go´mez et al., 2007). The crystal inclusions ingested by susceptible larvae, dissolve in the alkaline environment of the gut, and the solubilized inactive protoxins are cleaved by midgut proteases yielding active monomeric 60–70 kDa protease-resistant toxin (Pardo-Lo´ pez et al., 2009; Bravo et al., 2005), then the Cry toxic fragment binds to specific receptors located in the microvilli of the apical membrane of midgut epithelial cells that leads to membrane insertion, pore formation, cell lysis and the eventual death of the host (Bravo et al., 2004 & 2005). For Cry1A toxins (lepidopteran specific toxins), at least four different binding-proteins have been described as receptors for the Cry toxins; a cadherin-like protein (CADR), a glycosylphos-phatidyl–inositol (GPI) - anchored amin-opeptidase-N (APN), a GPI-anchored alkaline phosphatase (ALP) and a 270 kDa glycoconjugate (Vadlamudi et al., 1995; Knight et al., 1994; Jurat Fuentes et al., 2004; Valaitis et al., 2001). APN is the most extensively studied Cry receptor identified and isolated from several lepidopteran insects. APN serves as a binding molecule for Cry1C in Manduca sexta (Luo et al., 1996) and Spodoptera litura (Agrawal et al., 2002), for Cry1Aa in Bombyx mori (Nakanishi et al., 2002; Jenkins and Dean 2001; Yaoi et al., 1999) and for Cry1Ac in M. sexta (Garczynski et al., 1995; Knight et al., 1994 & 1995; Sangadala et al., 1994), Lymantria dispar (Lee et al., 1996; Valaitis et al., 1995) and Heliothis virescens (Gill et al., 1995; Luo et al., 1997; Oltean et al., 1999) The characterization of receptors in the economically important pests by studying their binding to different Cry toxins will help in evaluating the molecular mechanism of action of Cry toxin and will lead to a better understanding of the mechanism of development of resistance to these proteins in insects. The Egyptian cotton leaf worm (Spodoptera littoralis Boisduval) is an important pest in Mediterranean and Asian countries; it populates fields in relatively close locations (Hamal et al., 1991) and is considered a totally polyphagous pest (Lutfallah et al., 1993) affecting various economically important crops in Egypt such as cotton, maize, rice, soybeans and vegetables (Salama and Matter, 1991). To identify the precise toxin binding targets in insects, midgut cell membranes have been electrophoretically separated and incubated with Bt cry toxins in Western blot analysis. We have identified the proteins involved in CryIAc toxicity to Spodoptera littoralis. A 109-kDa B. thuringiensis toxin-binding protein from the midgut brush-border membrane that binds the Cry1Ac toxin was determined. A cDNA clone encoding this protein was cloned, sequenced and characterized. The analysis of the predicted amino acids sequence reveals that the protein belongs to the aminopeptidase N family of proteins.

A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae

A 65 kDa GPI (glycosylphosphatidyl-inositol)-anchored ALP (alkaline phosphatase) was characterized as a functional receptor of the Bacillus thuringiensis subsp. israelensis Cry11Aa toxin in Aedes aegypti midgut cells. Two (a 100 kDa and a 65 kDa) GPI-anchored proteins that bound Cry11Aa toxin were preferentially extracted after treatment of BBMV (brush boder membrane vesicles) from Ae. aegypti midgut epithelia with phospholipase C. The 65 kDa protein was further purified by toxin affinity chromatography. The 65 kDa protein showed ALP activity. The peptide-displaying phages (P1.BBMV and P8.BBMV) that bound to the 65 kDa GPI-ALP (GPI-anchored ALP) and competed with the Cry11Aa toxin to bind to BBMV were isolated by selecting BBMV-binding peptide-phages by biopanning. GPI-ALP was shown to be preferentially distributed in Ae. aegypti in the posterior part of the midgut and in the caeca, by using P1.BBMV binding to fixed midgut tissue sections to determine the location of GPI-ALP. Cry11Aa binds to the same regions of the midgut and competed with P1.BBMV and P8.BBMV to bind to BBMV. The importance of this interaction was demonstrated by the in vivo attenuation of Cry11Aa toxicity in the presence of these phages. Our results shows that GPI-ALP is an important receptor molecule involved in Cry11Aa interaction with midgut cells and toxicity to Ae. aegypti larvae.

Measurement of the Anchorage Force between GPI-Anchored Alkaline Phosphatase and Supported Membranes by AFM Force Spectroscopy

Mammalian alkaline phosphatases (AP) are glycosylphosphatidylinositol (GPI) anchored proteins that are localized on the outer layer of the plasma membrane. The GPI anchors are covalently attached to the C-termini of proteins and consist of a glycan chain bonded to phosphatidylinositol with two acyl chains anchored into the membrane bilayer. Force spectroscopy, based on atomic force microscope (AFM) technology, was used to determine the adhesion of alkaline phosphatase in the absence and presence of anchors. The GPI anchors increase markedly the adhesion frequency (i.e., the protein affinity for the membrane). An adhesion force of 350 +/- 200 pN is measured between GPI-anchored AP (AP(GPI)) and supported phospholipid bilayers of dipalmitoylphosphatidylcholine (DPPC) presenting structural defects (holes). In the absence of defects, the adhesion force (103 +/- 17 pN) and the adhesion frequency are reduced. These results indicate that AP(GPI) poorly spontaneously insert into membranes in vivo and open new perspectives for the characterization of the interactions between GPI proteins and membranes.

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems.

Insertion of GPI-anchored alkaline phosphatase into supported membranes: a combined AFM and fluorescence microscopy study.

A new method based on combined atomic force microscopy (AFM) and fluorescence microscopy observations, is proposed to visualize the insertion of glycosylphosphatidyl inositol (GPI) anchored alkaline phosphatase from buffer solutions into supported phospholipid bilayers. The technique involves the use of 27 nm diameter fluorescent latex beads covalently coupled to the amine groups of proteins. Fluorescence microscopy allows the estimation of the relative protein coverage into the membrane and also introduces a height amplification for the detection of protein/bead complexes with the AFM. The coupling of the beads with the amine groups is not specific; this new and simple approach opens up new ways to investigate proteins into supported membrane systems.

AFM Detection of GPI Protein Insertion into DOPC/DPPC Model Membranes

Membrane microdomains are involved in numerous cellular processes such as membrane transport and signal transduction. Among these functionally microdomains, rafts are enriched in sphingolipids and cholesterol and their existence is actually mainly explained by a fluid-ordered lipid lateral phase separation. They also concentrate proteins like these with a C-terminal glycosylphosphatidylinositol (GPI) modification that serves as an anchor to the plasma membrane. In order to better understand how GPI proteins partition into microdomains, the insertion of the GPI-anchored alkaline phosphatase (AP), added exogenously into supported phospholipid bilayers, was studied in real-time using atomic force microscopy (AFM). Fluid-gel (lipid ordered) phase-separated bilayers were made by fusion of dioleoylphosphatidylcholine/dipalmitoylphosphatidylcholine (DOPC/DPPC, 1:1) vesicles. Time-dependent insertion of AP through its GPI anchor was observed at the periphery of the gel-phase domains. This demonstrates that GPI proteins can spontaneously localize in lipid ordered phases.

Spontaneous insertion and partitioning of alkaline phosphatase into model lipid rafts.

Several cell surface eukaryotic proteins have a glycosylphosphatidylinositol (GPI) modification at the C-terminal end that serves as an anchor to the plasma membrane and could be responsible for the presence of GPI proteins in rafts, a type of functionally important membrane microdomain enriched in sphingolipids and cholesterol. In order to understand better how GPI proteins partition into rafts, the insertion of the GPI-anchored alkaline phosphatase (AP) was studied in real-time using atomic force microscopy. Supported phospholipid bilayers made of a mixture of sphingomyelin-dioleoylphosphatidylcholine containing cholesterol (Chl+) or not (Chl-) were used to mimic the fluid-ordered lipid phase separation in biological membranes. Spontaneous insertion of AP through its GPI anchor was observed inside both Chl+ and Chl- lipid ordered domains, but AP insertion was markedly increased by the presence of cholesterol.

Cytochrome c forms complexes and is partly reduced at interaction with GPI-anchored alkaline phosphatase

Cytochrome (cyt) c forms complexes, undergoes a conformational change and becomes partly reduced at interaction with membrane anchored alkaline phosphatase (AP), a glycoprotein which is released into the body fluid in forms differing in hydrophobicity. The proportion of products formed in the mixtures depends on pH, ionic strength, temperature and the buffer composition. The reaction terminates in an equilibrium between cyt c(FeII) and other cyt c conformers. Optimal conditions for the rate of the reaction are 100 mM glycine/NaOH, pH 9.7–9.9, at which 68–74% of cyt c is found in the reduced state. The interaction affects compactness of the haem cleft as shown by changes induced in CD spectra of the Soret region and changes in optical characteristics of phenylalanine, tyrosine and tryptophan residues. Differential scanning calorimetry of AP+cyt c mixtures revealed a creation of at least two types of complexes. A complex formed by non-coulombic binding prevails at substoichiometric AP/cyt c ratios, at higher ratios more electrostatic attraction is involved and at 1:1 molar ratio an apparent complexity of binding forces occurs. The rapid phase of the cyt c(FeII) formation depends on the presence of the hydrophobic alkylacylphosphoinositol (glycosylphosphatidylinositol) moiety, the protein part of the enzyme participates in an electrostatic and much slower phase of cyt c(FeII) creation. The results show that non-coulombic interaction may participate at interaction of cyt c with cellular proteins.

Differential patterns of expression of glycosylphosphatidylinositol-anchored carcinoembryonic antigen and alkaline phosphatase in various cancer cell lines.

The expression of glycosylphosphatidylinositol (GPI-anchored) carcinoembryonic antigen (CEA) and alkaline phosphatase (ALP) on the cell surface of various cancer cell lines and a lung diploid cell line (WI38) was investigated, with exposure of the cell lines to a cell differentiation agent (sodium butyrate) to induce cell differentiation and expression of the two tumor-associated antigens. In three colon (SW1222, SW1116, and HT-29) and stomach (MKN-45) cancer cell lines, all of which are double producers of CEA and ALP, the maximum expression of GPI-anchored CEA occurred with butyrate at a lower concentration than did that of GPI-anchored ALP. GPI-anchored ALP derived from colon (SW1222 and SW1116) and stomach (MKN-45 and MKN-1) cancer cell lines was heat-stable with and without exposure to butyrate, but GPI-anchored ALP derived from lung cancer cell lines (PC-6, PC13, PC-14, WI26VA4, and WI38VA13) showed a variety of heat stabilities, depending on cell line, butyrate exposure, and SV40 transformation.

Expression of intracellular and GPI-anchored forms of GPI-specific phospholipase D in COS-1 cells

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) is a secretory protein present in high amounts in mammalian body fluids. Its cDNA has been isolated and encodes a signal peptide of 23 amino acids and the mature protein of 816 amino acids. We generated cDNAs encoding a signal peptide-deficient and a GPI-anchored form of GPI-PLD and transiently transfected these constructs into COS-1 cells. The signal peptide-deficient form of GPI-PLD was expressed as a 90-kDa protein that was catalytically active and was localized intracellularly. Cells transfected with cDNA encoding the GPI-anchored form of GPI-PLD expressed a catalytically active enzyme of 100 kDa that could be labelled with [ 3H]ethanolamine demonstrating its modification by a GPI structure. Expression of the GPI-anchored form of GPI-PLD resulted in the release of endogenous GPI-anchored alkaline phosphatase from COS-1 cells, whereas expression of the intracellular form of GPI-PLD had no effect on membrane attachment of endogenous alkaline phosphatase. Similarly, in cells cotransfected with GPI-anchored placental alkaline phosphatase (PLAP) and the GPI-anchored form of GPI-PLD, PLAP was released into the cell culture supernatant while expression of the signal peptide-deficient form of GPI-PLD did not affect the amount of cell-associated PLAP.

Evidence for a glycosylinositolphospholipid-anchored alkaline phosphatase in the aquatic plant Spirodela oligorrhiza

Glycosylphosphatidylinositol (GPI)-anchored proteins occur widely, perhaps universally, on the surface of animal cells, where they perform a variety of important functions. However, the existence of GPI-anchored proteins on plant cells has never been established. Evidence is presented in this communication for the occurrence of a 50 kDa GPI-anchored alkaline phosphatase (AP) induced in the duckweed Spirodela oligorrhiza by phosphate deprivation. Triton X-114 partitioning of the Spirodela proteins yielded two forms of AP activity. The detergent-associated form was labeled prominently by [ 3H]ethanolamine, [ 3H]myristic acid and [ 3H]palmitic acid. This amphiphilic form of AP, like authentic GPI-anchored AP from mammals, was clearly resolved from the remaining, water-soluble AP activity by two types of incompletely-denaturing polyacrylamide gel electrophoresis. Lipid covalently bound to the solvent-delipidated amphiphilic AP was resistant to cleavage by phosphatidylinositol-specific phospholipase C. Strong acid or alkaline hydrolysis of the 3H-fatty acid-labeled amphiphilic AP yielded radioactive fatty acids and a radioactive lipid tentatively identified as a long chain base. The more abundant water-soluble AP was also radioactive in plants incubated with [ 3H]ethanolamine and was labeled to a lesser extent by 3H-fatty acids. The water-soluble AP, unlike its amphiphilic counterpart, could be freed of all fatty acid radioactivity by mild alkaline hydrolysis, indicating the continued presence of an ester-linked fatty acid. All evidence supports the conclusion that Spirodela AP is synthesized as an amphiphilic protein with a ceramide-containing GPI anchor.

Involvement of detergent-insoluble complexes in the intracellular transport of intestinal brush border enzymes.

A number of transmembrane digestive enzymes of the porcine small intestinal brush border membrane were found to be partially Triton X-100-insoluble at 0 degree C and colocalized in gradient centrifugation experiments with the GPI-anchored alkaline phosphatase in low-density, detergent-insoluble complexes commonly known as glycolipid "rafts". Thus, aminopeptidase N (EC 3.4.11.2), aminopeptidase A (EC 3.4.11.7), dipeptidyl peptidase IV (EC 3.4.14.5), and sucrase-isomaltase (EC 3.2.1.48-10) were 34-48% detergent-insoluble. Maltase-glucoamylase (EC 3.2.1.20) was markedly less detergent-insoluble (20%), and lactase-phlorizin hydrolase (EC 3.2.1.23-62) was essentially fully soluble in detergent. In radioactively labeled, mucosal explants, the newly synthesized brush border enzymes began to associate with detergent-insoluble complexes while still in their transient, high mannose-glycosylated form, and their insolubility increased to that of the steady-state level soon after they achieved their mature, complex glycosylation, i.e., after passage through the Golgi complex. Detergent-insoluble complexes isolated by density gradient centrifugation were highly enriched in brush border enzymes, and the enrichment was apparent after only 1 h of labeling, where aminopeptidase N, sucrase-isomaltase, and alkaline phosphatase together comprised 25-30% of the total labeled, detergent-insoluble proteins, showing that sorting of newly made brush border membrane proteins into the glycolipid "rafts" does take place intracellularly. I therefore propose that, in the enterocyte, the brush border enzymes are targeted directly from the trans-Golgi network toward the apical cell surface.

Purification of Glycosylphosphatidylinositol-Anchored Proteins by Modified Triton X-114 Partitioning and Preparative Gel Electrophoresis

Triton X-114 phase partitioning has frequently been used to obtain preparations enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and other hydrophobic proteins from crude cellular homogenates. We have developed a new modification of this phase-partitioning technique which allows two distinct GPI-anchored proteins of Tetrahymena mimbres to be separated from other hydrophobic as well as hydrophilic proteins and recovered in ∼90% yield. The unique feature of the new method is a 24-h incubation of the first Triton X-114 extract at −20°C. This improves the partitioning of GPI-anchored proteins into the detergent phase while promoting the aggregation of other hydrophobic proteins. Individual GPI-anchored proteins in the detergent phase are then purified to near homogeneity by one-step preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By substituting Triton X-114/water mixtures for the conventional SDS buffer used to collect proteins eluting from the preparative gel, the purified GPI-anchored protein can be rapidly concentrated from relatively large volumes of eluate by phase partitioning at 32°C. The method is also effective in separating mammalian GPI-anchored alkaline phosphatase from other proteins. It is likely to be of general utility in characterizing the GPI anchor structures associated with nonabundant and abundant GPI-anchored proteins coexisting within the same cell type.