Early Events in Glycosylphosphatidylinositol Anchor Addition

SUBSTRATE PROTEINS ASSOCIATE WITH THE TRANSAMIDASE SUBUNIT Gpi8p*

Tracey D. Spurway, Jane A. Dalley, Stephen High, and Neil J. BulleidDagger

From the University of Manchester, School of Biological Sciences, 2.205 Stopford Building, Manchester M13 9PT, United Kingdom

Received for publication, November 7, 2000, and in revised form, February 22, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Glycosylphosphatidylinositol (GPI)1 is a complex glycolipid that is covalently attached to many eukaryotic cell surface proteins. GPI provides an alternative anchoring mechanism to a hydrophobic polypeptide transmembrane domain, enabling stable association of protein with the lipid bilayer. In mammalian cells, more than 100 polypeptides are anchored to the plasma membrane via a GPI moiety (1). These encompass a functionally diverse set of proteins including cell surface enzymes, receptors, adhesion molecules, and differentiation antigens, which have been implicated in a number of cellular functions such as immune recognition, complement regulation, and transmembrane signaling (1). In mammalian cells, the GPI anchor is not essential for survival at the cellular level and cell lines defective in various stages of GPI backbone assembly, or transfer of this glycolipid to the polypeptide chain have been established (2, 3). However, GPI anchoring is essential for embryogenesis (4) and skin development (5), and defective cell surface expression of GPI-linked proteins may lead to specific disease states (6, 7). Therefore, determining the molecular mechanisms involved in the production of GPI-anchored proteins will provide an insight into the biosynthesis of proteins that are central to both normal and abnormal cell physiology.

The addition of GPI anchors to proteins entering the secretory pathway occurs post-translationally. The GPI moiety is synthesized in the endoplasmic reticulum (ER), and the overall biosynthetic pathway for the production of the GPI precursor is now well understood (8). Pre-formed GPI is transferred en bloc to proteins destined to be GPI-anchored. These precursor proteins contain an ER-targeting N-terminal signal sequence and a C-terminal signal sequence for GPI anchoring (9, 10). This GPI signal sequence consists of a C-terminal hydrophobic domain, preceded by a short hydrophilic spacer linked to the GPI attachment (omega ) site (10). The removal of the C-terminal signal sequence and replacement with pre-formed GPI at the omega -site is catalyzed by a transamidase that resides within the ER membrane (11). The GPI transamidase is proposed to bind to the GPI signal sequence and attack the carbonyl group of the omega -site amino acid. Cleavage of the signal sequence results in formation of a carbonyl intermediate between precursor protein and enzyme. The amino group on the terminal ethanolamine of GPI then attacks this intermediate to produce GPI-anchored protein (12, 13). Although this proposed reaction mechanism for GPI addition is now widely accepted, isolation of an active enzyme capable of such anchor attachment has not been achieved to date.

Genetic approaches have identified two genes, GAA1 and GPI8, which are required for addition of GPI to proteins in Saccharomyces cerevisiae (14, 15). Homologues of both these genes have now been identified in humans (16, 17). Transamidation of GPI-anchored proteins is therefore thought to depend upon the gene products Gpi8p and Gaa1p, and it has been suggested that these two proteins may constitute a transamidase complex (18). In agreement with this finding, mammalian cell lines defective in either Gpi8p or Gaa1p do not attach GPI to proteins and accumulate complete GPI lipids as well as GPI precursor proteins (18, 19). Human Gpi8p is a 45-kDa type I ER membrane protein that belongs to a novel cysteine protease family and has been postulated to be the catalytic subunit of the transamidase (18, 20). Human Gaa1p, a 67-kDa ER protein, with several membrane-spanning domains forms a complex with Gpi8p (18) and therefore constitutes part of the transamidase. However, the role that Gaa1p plays in the transamidation reaction remains unknown. The exact function for both transamidase subunits and their functional interaction with each other and with the precursor protein and GPI substrates also remains to be elucidated.

Processing of nascent proteins to GPI-anchored forms has been extensively studied using cell-free systems. Model GPI-anchored proteins can be translated in vitro and then processed by rough microsomal membranes (RM) derived from mammalian cells. In early studies translated human placental alkaline phosphatase (PLAP) was processed by RM to a mature GPI-anchored form, which was identified by reaction to site-specific antibodies (21). However, PLAP was not ideal for studying N- and C-terminal signal sequence processing due to the increased size of the glycosylated protein. Therefore a truncated version of PLAP, prepromini-PLAP was constructed to act as a reporter protein in this cell free system (22). Nascent prepromini-PLAP translated in vitro by microsomal membranes was processed by cleavage of the N-and C-terminal signal sequences, with subsequent GPI addition, to GPI-anchored mini-PLAP (22). Studies using this microsomal cell-free assay system have demonstrated the strict amino acid requirements at or adjacent to the GPI attachment (omega ) site (22, 23) and have shown that soluble components of the ER lumen are needed for formation of GPI-mini-PLAP (24). These cell-free systems have also provided evidence that GPI anchoring proceeds via a transamidation mechanism, as small nucleophiles such as hydrazine can replace the GPI anchor (13, 25), and that the process is energy-independent (12). However, these in vitro studies have not directly identified molecular components of the processing machinery and are not appropriate for a study of whether protein folding has a role on the overall process of GPI anchor addition.

In this report we describe a convenient assay for GPI anchoring based on a cell-free system using digitonin-permeabilized cells. These semipermeabilized (SP) cells are capable of replacing microsomal membranes as a source of intact ER in a conventional in vitro translation system (26). PLAP was used as a model protein for the biosynthesis, processing, and folding of GPI-anchored proteins in these experiments. The results indicate that the initial stages of GPI anchor addition to PLAP can be reconstituted in vitro using the SP cell system and approximate events seen in vivo. An objective of this work is to determine the role that protein folding has on GPI addition to PLAP. The data presented show that folding of PLAP is not influenced by the addition of a GPI anchor and that GPI anchor addition does not require protein folding, demonstrating that these two processes can occur independently of each other. Use of a mutant cell line defective in GPI-protein biosynthesis, combined with this flexible in vitro translation system, has provided evidence of an association between protein substrate and the transamidase subunit hGpi8p. The significance of these findings with regard to possible functions of the hGpi8p subunit of the transamidase is discussed.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Plasmid Construction-- Plasmids containing human cDNA coding mini-PLAP (pGEM-4Z/miniPLAP) and full-length PLAP (pGEM-4Z/PLAP) were kindly provided by Dr. M. E. Medof (Case Western Reserve University, Cleveland, OH). PLAP was subcloned as an EcoRI-HindIII fragment into pBluescript SK- (Stratagene, Cambridge, United Kingdom (UK)) to generate pBS/PLAP. Introduction of the mutation D484P into pBS/PLAP was achieved by site-directed mutagenesis using a Quickchange mutagenesis kit (Stratagene) with the following primers: 5'-GCCCCCCGCCGGTACCACCCCCGCCGCGCACCCG-3' and 5'-CGGGTGCGCGGCGGGGGTGGTACCGGCGGGGGGC-3', creating the plasmid pBS/PLAP-D484P.

Transcription in Vitro-- Transcription reactions were carried out as described by Gurevich et al. (27). Recombinant plasmids were linearized with HindIII; pBS/PLAP and pBS/PLAP-D484P were transcribed using T3 RNA polymerase (Promega, Southampton, UK), and pGEM4Z/miniPLAP was transcribed with T7 RNA polymerase (Promega).

Preparation of Semipermeabilized Cells-- The human lymphoblastoid cell line K562 was obtained from the European collection of animal cell cultures. The class K mutant and class IA mutant K562 cell lines were a gift from Dr. M. E. Medof. Cell lines were cultured in RPMI 1640 media supplemented with 10% fetal calf serum. Semipermeabilized (SP) cells were prepared by treatment with digitonin, as described previously (26).

Translation in Vitro-- RNA was translated using a rabbit reticulocyte lysate (FlexiLysate, Promega). The translation reaction (25 µl) contained 17.5 µl of reticulocyte lysate, 40 µM methionine-free amino acid mixture, 45 mM KCl, 15 µCi of L-[35S]methionine (PerkinElmer Life Sciences, Houndslow, UK), 1 µl of transcribed mRNA, and appropriate SP cell preparation (~2 × 105 cells). Hydrazine (10 mM) or DTT (5-10 mM) were included in translations where indicated. Translations were incubated at 30 °C for 60-90 min and were terminated by centrifugation (12,000 × g for 3 min at 4 °C) to isolate SP cells. Translation products were then processed for further analysis as detailed below.

Endoglycosidase H Treatment-- After translation, isolated SP cells were solubilized in dissolution buffer (50 mM Tris-HCl, pH 8.0, 1% (w/v) SDS) for 5 min at 95 °C. Insoluble material was removed by centrifugation at 13,000 × g for 10 min. An equal volume of 150 mM sodium citrate buffer, pH 5.5, containing PMSF (0.5 mM) was added to the soluble fraction. The sample was divided into two identical aliquots, which were incubated for 12 h at 37 °C with either 1 unit of endoglycosidase H (Roche Molecular Biochemicals, Lewes, UK) or buffer alone. Treated and untreated reactions were then subjected to analysis by SDS-PAGE.

Proteinase Protection Assays-- After translation, isolated membrane fractions were resuspended in KHM buffer (20 mM HEPES, pH 7.2, 110 mM potassium acetate, 2 mM magnesium acetate) supplemented with 10 mM CaCl2 and were treated with proteinase K (Roche Molecular Biochemicals) at a concentration of 250 µg/ml for 30 min at 0 °C, either in the presence or absence of 1% (v/v) Triton X-100. Following digestion, proteinase K was inhibited by the addition of PMSF to a final concentration of 1 mM, and samples were then analyzed by SDS-PAGE.

Triton X-114 Extraction-- After translation SP cells were isolated and resuspended in 500 µl of TBS (10 mM Tris-HCl buffer, pH 7.4, 150 mM NaCl) containing 2% (v/v) Triton X-114. Resuspended cells were sonicated for 3.5 min to solubilize proteins and incubated on ice for 15 min. Samples were centrifuged for 10 min at 10,000 × g and the supernatant incubated at 37 °C until turbid. The aqueous and detergent phases were then separated by centrifugation at 10,000 × g for 10 min. The aqueous phase was re-extracted three times with TBS containing 2% (v/v) Triton X-114, and all aqueous phases were combined. The detergent phase was re-extracted three times with TBS, and all detergent phases were combined. The radiolabeled material in the detergent and aqueous phases was then analyzed by SDS-PAGE.

PLAP Assay-- Following translation for 1 h, isolated cells were solubilized in KHM buffer (60 µl per 25-µl translation) containing 0.05% (v/v) Triton X-100 for 30 min at 0 °C. Insoluble material was removed by centrifugation at 13,000 g for 10 min. Enzymatic activity of solubilized, translated PLAP (15-µl aliquots) was measured using a commercial kit (Great Escape SEAP, CLONTECH, Basingstoke, UK), as described in manufacturer's instructions for a 96-well format. PLAP activity was measured by dephosphorylation of the chemiluminescent substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'chloro)-tricyclo[3.3.1.1]decan-4-yl)phenyl phosphate (CSPD), with concomitant release of light, which was detected by a luminometer. PLAP activity was measured relative to a standard of the secreted form of PLAP and is expressed as relative light units emitted by the sample. The equivalent amount of N-terminally processed PLAP formed in each assay was estimated by immunoprecipitating an aliquot (10 µl) of Triton X-100-solubilized PLAP with amino-Ab. Following immunoprecipitation, PLAP was removed from protein A-Sepharose by addition of 50 mM Tris, pH 8, 1% (w/v) SDS with boiling for 3 min. Radioactivity incorporated into processed PLAP was then estimated by liquid scintillation counting. The specific activity of PLAP is expressed as relative light units emitted/amount radioactivity incorporated into N-terminally processed forms of PLAP. Background values obtained when translation was carried out in the absence of added RNA were subtracted from both the activity and radioactivity measurements. The results are the mean values from three separate experiments ± standard error.

Cross-linking-- Following in vitro translation, membrane fractions were resuspended in KHM buffer containing Me2SO (solvent control) or 100 µM bismaleimidohexane (BMH) (Pierce and Warriner, Warrington, UK), and incubated at room temperature for 10 min. Cross-linking was then quenched by the addition of 10 mM DTT, and the samples were left on ice for 10 min. Samples were then immunoprecipitated, under either native or denaturing conditions as detailed below.

Immunoprecipitation-- Polyclonal rabbit antibodies were commercially produced (SigmaGenosys, Cambridge, UK) against synthetic peptides conjugated to keyhole limpet hemocyanin. Three site-specific antibodies (Abs) to human PLAP-513 (endo-antibody, amino-antibody, and exo-antibody) were generated to specific amino acid sequences in the PLAP molecule, as described previously (21). Antibodies were also raised against components of the human transamidase, via peptides in the amino terminus of the mature form of hGpi8p (SHIEDQAEQFFRSGHTNNW) and to a peptide sequence representing residues 74-89 in hGaa1p (ARDFAAHRKKSGALP). Antibody to calreticulin was obtained from Cambridge Biosciences (Cambridge, UK) and to ERp57 from Dr. T. Wileman (IAH, Pirbright, UK).

For immunoprecipitations under native conditions, samples were solubilized in 1 ml of immunoprecipitation buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% (v/v) Triton X-100, 0.02% (w/v) azide) and incubated with 50 µl of protein A-Sepharose (10% (w/v) (Zymed Laboratories Inc., San Francisco, CA) for 30 min at 4 °C. Samples were centrifuged to remove protein A binding contaminants and precleared supernatants incubated overnight with 50 µl of protein A-Sepharose and 1-10 µl of relevant antisera. In the case of denaturing immunoprecipitations, samples were denatured for 5 min at 95 °C in the presence of 1% (w/v) SDS and then diluted with 1 ml of immunoprecipitation buffer to give a final concentration SDS < 0.05% (w/v). Samples were then precleared and incubated with antibody as described above. In each case, protein A-Sepharose-bound material was isolated by centrifugation at 13,000 × g for 1 min, washed three times with immunoprecipitation buffer, and then subjected to analysis by SDS-PAGE, isoelectric focusing (IEF), or radioactivity incorporated into protein measured by scintillation counting.

Electrophoresis-- Samples for IEF were immunoprecipitated under native conditions and material immobilized on protein A-Sepharose solubilized in IEF solubilization buffer containing 8 M urea, 0.5% (v/v) Triton X-100, 0.01 g/ml Ampholine, pH 3.5-9.5 (Amersham Pharmacia Biotech, Lower Chalfont, UK), 20 mM DTT, for 1 h at room temperature. Protein A-Sepharose was then removed by centrifugation (13,000 × g, 1 min) and supernatants were loaded onto an Immobiline DryPlate pH 4-7 isoelectric focusing gel (Amersham Pharmacia Biotech), that had been preswollen overnight in IEF solubilization buffer as described in manufacturer's instructions. After focusing gels were stained and fixed in 0.1 (w/v) CuSO4, 0.05% (w/v) R-250 Coomassie Blue, 17% acetic acid, 23% ethanol, destained in 30% (v/v) ethanol, 7% acetic acid, dried, and then visualized by either autoradiography or imaged using a FujiBas 2000 phosphorimager.

Samples for SDS-PAGE were resuspended in SDS-PAGE sample buffer (0.0625 M Tris-HCl, pH 6.8, containing 2% (w/v) SDS, 10% (v/v) glycerol, bromphenol blue) plus 50 mM DTT and were boiled for 5 min. Proteins were subjected to SDS-PAGE through either 12.5% (mini-PLAP) or 10% gels (PLAP and derivatives). Gels were fixed in 10% (v/v) methanol and 10% (v/v) acetic acid, dried, and then visualized using autoradiography or a phosphorimager as described above.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Reconstitution of GPI Anchor Addition in SP Cells-- We have previously shown that SP cells can efficiently translate, glycosylate, and assemble a variety of secretory and membrane-bound proteins (26). This approach has the advantage over using microsomal membranes in that the reactions occur within a morphologically intact ER and are generally more efficient (26). To determine if this system could be applied to study the translocation and subsequent processing of GPI-anchored proteins, we utilized the well characterized GPI anchor substrate, which is a truncated version of human placental alkaline phosphatase (prepromini-PLAP). The various different processed forms of this protein can be readily resolved on SDS-PAGE and have been previously characterized in translations with microsomal membranes (22). When mRNA coding for prepromini-PLAP was translated in the absence of SP K562 cells, a single translation product of ~28 kDa was generated (Fig. 1A, lane 1). In addition to prepromini-PLAP, two further translation products were generated in the presence of SP K562 cells (Fig. 1A, lane 2). The three translation products showed essentially the same migration pattern to those derived from identical translations in the presence of RM (22). The identity of the translation product synthesized in the absence of SP cells was confirmed as prepromini-PLAP by the sensitivity of this product to proteinase K digestion in the presence of SP cells (Fig. 1A, lane 3). The 27- and 25-kDa species synthesized in the presence of SP cells were shown to be resistant to proteolysis (Fig. 1A, lane 3), indicating that these polypeptides were translocated across the ER membrane. Addition of detergent prior to protease digestion demonstrated the susceptibility of the translocated portions of the protein to digestion after membrane solubilization (Fig. 1A, lane 4).


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Fig. 1.   Co-translational processing of mini-PLAP in the presence of SP K562 cells. Panel A, mini-PLAP mRNA was translated using rabbit reticulocyte lysate in the presence (lanes 2-4) or absence (lane 1) of SP K562 cells for 1 h at 30 °C. Isolated cells were treated with proteinase K (250 µg/ml) (lanes 3 and 4) with addition of 1% (w/v) Triton X-100 as indicated (lane 4). After 30 min at 0 °C, the protease was inhibited by addition of PMSF (1 mM). Labeled translation products were analyzed by SDS-PAGE and autoradiography. Panel B, mini-PLAP mRNA was translated in vitro for 1 h at 30 °C in a rabbit reticulocyte lysate system supplemented with SP cells prepared from K562 cells (lanes 1-3). Processed forms of mini-PLAP were immunoprecipitated with three site-directed antibodies raised to specific peptides in PLAP: endo-Ab (lane 1), amino-Ab (lane 2), and exo-Ab (lane 3). The samples were analyzed by SDS-PAGE and autoradiography. Identities of processed mini-PLAP forms in panels A and B are shown according to Kodukula et al. (22).

To confirm the identity of the translation products, we generated three peptide-specific antibodies termed amino-, endo-, and exo-antibodies that have been used previously (22) to monitor the various stages of mini-PLAP processing. Amino-Ab was raised to a peptide sequence corresponding to the N terminus of the mature protein and will only react with polypeptides lacking the N-terminal signal sequence i.e. promini-PLAP and mini-PLAP. Endo-Ab was raised to a peptide sequence corresponding to the C terminus of the mature protein and reacts with all forms of the protein, whereas exo-Ab was raised to a peptide sequence corresponding the C-terminal signal for GPI addition and therefore only reacts with pre- and promini-PLAP. When the translation products synthesized in the presence of SP K562 cells were immunoprecipitated with endo-Ab, all three products were immunoprecipitated (Fig. 1B, lane 1). With amino-Ab the 27- and 25-kDa products, but not the 28-kDa product, were immunoprecipitated, confirming that the 28-kDa product is prepromini-PLAP (Fig. 1B, lane 2). With exo-Ab only the 28- and 27-kDa products were immunoprecipitated, confirming the identity of the 25-kDa product as mini-PLAP (Fig. 1B, lane 3). These results are identical to those obtained with RM (22), and demonstrate that the SP cells reconstitute GPI signal processing.

Full-length PLAP Is GPI-anchored in SP Cells-- One of the objectives of this work was to determine the influence of protein folding on the addition of GPI anchors to proteins and to also investigate whether GPI addition was required for efficient protein folding. It has been suggested previously that molecular chaperones within the ER influence the processing and addition of GPI anchors to proteins (24). This conclusion was based on studies showing that removal of soluble proteins from microsomal membranes prevents GPI addition. These studies were carried out with the model substrate prepromini-PLAP, an artificial molecule that does not fold correctly and forms a prolonged interaction with the molecular chaperone BiP (11). We therefore investigated the influence of protein folding on GPI addition using authentic full-length prepro-PLAP. PLAP is not ideal for studying N- and C-terminal signal processing, as the prepro-protein becomes glycosylated on translocation into the ER, making it difficult to analyze processed products differing by a few kilodaltons in size by SDS-PAGE. Hence, before assessing the influence of protein folding on GPI addition, we needed to prove that PLAP was processed to GPI-anchored form in our SP cell system.

When PLAP mRNA was translated in the absence of SP K562 cells, a single labeled product of approximately Mr 58,000, corresponding to prepro-PLAP was formed (Fig. 2, lane 3). In translations supplemented with SP cells, a second larger form of PLAP with an approximate Mr of 63,000 was seen (Fig. 2, lane 4), which was protected from proteolysis by proteinase K (Fig. 2, lane 5). This larger form was shown to correspond to a glycosylated product, as it was sensitive to digestion with endoglycosidase H (Fig. 2, lane 2). These results confirm previous work carried out with RM (21) and indicate that the full-length product was translocated across the ER membrane and glycosylated following translation in the presence of SP cells.


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Fig. 2.   Translocation of PLAP into the ER of SP K562 cells. PLAP mRNA was translated using rabbit reticulocyte lysate in the presence (lanes 1, 2, and 4-6) or absence (lane 3) of SP K562 cells for 1 h at 30 °C. Isolated cells were incubated with endoglycosidase H (1 unit) at 37 °C for 12 h (lane 2) or treated with proteinase K (250 µg/ml) (lanes 5 and 6) with addition of 1% Triton X-100 as indicated (lane 6). After 30 min at 0 °C, the protease was inhibited by addition of PMSF (1 mM). Labeled translation products were then analyzed by SDS-PAGE and autoradiography.

As we were unable to demonstrate GPI anchoring of PLAP by differences in mobility of translation products after SDS-PAGE, we used two alternative approaches to demonstrate that this modification had indeed occurred. First, we predicted that GPI-anchored and non-GPI-anchored proteins would have different isoelectric points and, therefore, show altered migration patterns on IEF gels. To test this prediction PLAP mRNA was translated in the presence of either SP parental or mutant K class K562 cells, and then products were analyzed by IEF (Fig. 3A). The mutant K class cell line carries a mutation in one of the transamidase subunits Gpi8p, thereby preventing GPI anchor addition from occurring (16). As can be seen, three radiolabeled translation products were formed in SP K562 cells (Fig. 3A, lane 1). To determine the identity of these translation products, the three site-directed antibodies were utilized as described earlier. In SP K562 cells, all forms of PLAP were immunoprecipitated with the endo-Ab and amino-Ab, indicating that the N-terminal signal sequence had been cleaved from these translation products (Fig. 3A, lanes 2 and 3). Exo-Ab did not immunoprecipitate either translation product (Fig. 3A, lane 4). It should be noted that the amount of the most acidic translation product formed is low in comparison to the more basic product, which could explain the lack of immunoprecipitation with exo-Ab. When the translation products generated by translation of PLAP RNA in the presence of SP K class cells were analyzed by IEF, the major product was the more acidic form of PLAP that was immunoprecipitated by amino-Ab and exo-Ab (Fig. 3A, lanes 5 and 6). Taken together these results suggest that the acidic form corresponds to pro-PLAP, whereas the major more basic form is C-terminally cleaved PLAP. The identity of the third most basic form is unknown but could represent C-terminally cleaved non-GPI-anchored PLAP (22) or different charge forms of PLAP.


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Fig. 3.   C-terminal processing of PLAP. Panel A, PLAP mRNA was translated for 1 h at 30 °C with rabbit reticulocyte lysate in the presence of SP K562 cells (lanes 1-4) or SP K class cells (lanes 5 and 6). An aliquot of each translation was removed and cells isolated by centrifugation. Remaining translation products were immunoprecipitated with endo-Ab (lane 2), amino-Ab (lanes 3 and 5), or exo-Ab (lanes 4 and 6). Isolated cells or immunoprecipitates were then resuspended in IEF solubilization buffer prior to focusing on an Immobiline DryPlate, pH 4-7. Differently charged forms of PLAP were identified using autoradiography and are indicated on the right. Panel B, PLAP mRNA was translated in vitro using rabbit reticulocyte lysate and either SP K562 cells (lanes 1 and 2) or SP K class cells (lanes 3 and 4) with (lanes 2 and 4) or without (lanes 1 and 3) 10 mM hydrazine for 90 min at 30 °C. Following translation, samples were immunoprecipitated with amino-Ab and samples were separated by SDS-PAGE and visualized by autoradiography. The putative PLAP-hydrazide product is indicated. Panel C, PLAP mRNA was translated using a rabbit reticulocyte lysate and either SP K562 cells (lanes 1 and 2) or SP K class cells (lanes 3 and 4) for 90 min at 30 °C. Translation products were partitioned into aqueous and detergent phases by Triton X-114 extraction as described under "Materials and Methods." Samples were separated by SDS-PAGE and visualized by autoradiography.

From the reactivity of site-specific antibodies with PLAP, it was evident that the C-terminal signal sequence was cleaved from this polypeptide during processing in SP K562 cells. However, it was not clear if this was accompanied by addition of a GPI anchor to the cleaved PLAP molecule. To determine whether the C-terminally processed PLAP was GPI-anchored, we used hydrazine as an alternative nucleophilic agent to GPI. Hydrazine should act as an effective acceptor for an enzyme-activated omega -carbonyl intermediate, and has been shown to promote formation of the hydrazide of free mini-PLAP in translations with RM (28). Mobility by SDS-PAGE of translation products synthesized in SP K class cells was unaffected by inclusion of hydrazine (Fig. 3B, lanes 3 and 4). This confirms previous studies, which showed that RM isolated from K class cell line efficiently produced the pro-form of mini-PLAP, but additional COOH processing of this product did not occur either in the presence or absence of hydrazine (19). In the present study, incubation of SP K562 cells with hydrazine resulted in production of a faster migrating product (Fig. 3B, compare lanes 1 and 2). In these experiments, the hydrazide form of free PLAP was the major translation product and may arise directly from either processed pro-PLAP or via cleavage of the GPI moiety from GPI-anchored PLAP. Cleavage of C-terminal signal sequence at the omega -site of pro-PLAP by the transamidase must therefore be accompanied by GPI anchor addition to the protein, as there was no evidence for the formation of free PLAP in translations with SP K562 cells (Fig. 3B, lane 1). From Fig. 3B it can be seen that on SDS-PAGE mature PLAP appeared to co-migrate with pro-PLAP (lane 1 compared with lane 3), and it is logical to suggest that the difference in size between mature PLAP and non-GPI-anchored, free PLAP (lane 1 versus lane 2) is due to GPI anchor attachment. Therefore, the additional translation product identified in K562 cells compared with K class cells by IEF must be due to GPI anchor addition to C-terminally cleaved PLAP. These results, combined with cross-reactivity of various forms of PLAP with site-specific antibodies, suggest that in SP K562 cells the majority of PLAP was fully processed and also GPI-anchored.

Further indirect confirmation that PLAP was GPI-anchored in SP K562 cells came from experiments with phospholipase D, which specifically cleaves the lipid moiety from GPI-anchored proteins. Incubation of putative GPI-anchored PLAP synthesized in SP K562 cells with phospholipase D produced a cleavage product that was not observed on incubation with non-GPI-anchored PLAP synthesized in K class cells (results not shown). Additionally, when subjected to phase partition between Triton X-114 and water, 40-50% of the translation product synthesized in K562 cells partitioned into the Triton X-114 phase (Fig. 3C, lane 1). This contrasts to only 10-15% of the translation product synthesized in K class cells partitioning into the detergent phase (Fig. 3C, lane 3). Therefore, taken together, these observations indicate that a large proportion of PLAP is GPI-anchored in SP K562 cells.

Influence of GPI Addition on PLAP Folding-- As discussed above, PLAP is an excellent model for investigating the sequence of events leading to catalytic addition of the GPI anchor to correctly folded protein. Like many transmembrane proteins, the extracellular domain of PLAP may contain intrachain disulfide bonds, which contribute to the stabilization of proteins. The contribution of disulfide bonds to PLAP biogenesis and the role that PLAP conformational maturation plays on GPI anchor addition remain undocumented. There is a paucity of information regarding number and position of disulfide bonds and active site conformation within PLAP, as the crystal structure of the human protein remains to be solved. Of the 513 amino acids present in nascent PLAP, 6 are cysteine residues, several of which are in close proximity to the C-terminal signal sequence. These cysteine residues may participate in the formation of up to two intrachain disulfide bonds within pro-PLAP (one cysteine residue is contained within the N-terminal signal sequence). The C-terminal signal sequence in PLAP is 29 amino acids long, implying that translation of the polypeptide has to be complete in order to expose the omega -site to the transamidase for GPI attachment. Hence, PLAP folding, especially within the vicinity of the C-terminal signal sequence, and the assumption of the correct protein conformation could play a part in positioning pro-protein at the omega -site for optimal recognition by the transamidase. Therefore, the location and kinetics of disulfide bond formation may play an important role in GPI addition to PLAP.

The activity of GPI-anchored PLAP expressed on the surface of mammalian cells has been measured previously and used to indicate that the active site was correctly folded (29). To our knowledge the folding of PLAP within an in vitro translation system has not previously been determined due to the very low (pmol) amounts of protein synthesized. The availability of a chemiluminescent assay, designed to determine as little as 10-13 g of a secreted form of PLAP protein (see "Materials and Methods") has enabled us to study folding of this hydrolytic enzyme using the SP cell system. To examine the role of disulfide bond formation on the folding of PLAP synthesized in the SP cell system, we translated PLAP mRNA under reducing (in the presence of 5 mM DTT) and non-reducing conditions and measured the specific activity of products formed. The rabbit reticulocyte lysate used in these experiments contains no added DTT and supports the formation of native disulfide bonds in proteins translocated across the ER membrane (26). When PLAP was synthesized in the absence of added DTT, the translation product was enzymically active (9.24 ± 2.04 RLU/dpm), indicating that it had folded correctly. However, when the translation was carried out in the presence of added DTT, essentially no enzyme activity was detected (0.13 ± 0.05 RLU/dpm). The activity was normalized to take into account any variation in the amount of protein synthesized in the presence and absence of DTT. Small shifts in mobility of PLAP monomer on non-reducing compared with reducing SDS-PAGE were also observed when the translations were carried out in the absence of added DTT, indicating the formation of intrachain disulfide bonds (results not shown). Thus, it appears that, in our SP cell system, PLAP contains at least one intrachain disulfide bond that must be formed to provide enzymically active protein.

To determine whether folding of PLAP into an active conformation required GPI anchor addition, we measured the activity of PLAP synthesized and translocated into the ER of the K class cell line that does not catalyze GPI anchor addition (16). PLAP may be folded as it is translocated across the ER, or alternatively the polypeptide may only attain the correctly folded conformation after GPI addition and release from the transamidase. The translation product synthesized in the presence of K class cells was enzymically active (3.4 ± 0.23 RLU/dpm), indicating that absence of a GPI anchor does not lead to improper protein folding. We observed variability between individual experiments, making it difficult to draw comparisons between the efficiency of folding in K562 and K class cells; however, the enzyme synthesized in K562 cells was consistently more active than the enzyme synthesized in K class cells. This could reflect subtle differences in the conformation of the protein when the C-terminal signal sequence is still present. These data show that PLAP activity measured within the ER of SP cells was derived from both the pro- and the GPI-linked protein and that PLAP was able to fold into an enzymically active conformation even in the absence of GPI addition.

Influence of PLAP Folding on GPI Addition-- Having established that preventing disulfide bond formation in PLAP prevented protein folding, we then went on to investigate the effect of preventing polypeptide folding on GPI anchor addition. PLAP mRNA was translated in either SP K562 or K class cells in the presence or absence of reducing agent as described above. Formation of GPI-anchored PLAP and pro-PLAP translation products were then monitored by IEF. As expected, in the absence of added reducing agent during translation, SP K class cells synthesized essentially only pro-PLAP (Fig. 4A, lane 3), whereas K562 cells produced both pro- and GPI-anchored PLAP (Fig. 4A, lane 1). This pattern of PLAP products remained unaffected in either cell type by the inclusion of reducing agent during translation at concentrations that we had established prevent correct protein folding (Fig. 4A, lanes 2 and 4). We also assessed the ability of the transamidase to catalyze the formation of the hydrazide form of free PLAP by carrying out translation of PLAP mRNA in the presence of hydrazine. When translation was carried out in the presence of K562 SP cells, the hydrazide form of free PLAP was formed in the presence of added DTT (Fig. 4B, lane 2). Hence, the GPI anchor or hydrazine was added to PLAP, whether the protein was in either the folded or the unfolded state. These results also agree with the observation that promini-PLAP, an engineered and probably incorrectly folded protein, undergoes GPI addition in this SP cell system. Folding of the extracellular domain of PLAP may therefore be independent from events leading to GPI addition. The fact that the transamidase recognizes both folded and unfolded PLAP means that the only requirement for substrate recognition may be the conformation of the C-terminal signal sequence. This may be governed largely by its hydrophobicity, rather than by the conformation of the entire protein, as changes in overall hydrophobicity of this signal sequence have been shown to prevent protein GPI anchoring (30).


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Fig. 4.   Effect of reducing agents on GPI anchor addition. Panel A, PLAP mRNA was translated in vitro using rabbit reticulocyte lysate and either SP K562 cells (lanes 1 and 2) or SP K class cells (lanes 3 and 4) with (lanes 2 and 4) or without (lanes 1 and 3) 10 mM DTT for 60 min at 30 °C. Following translation samples were immunoprecipitated with amino-Ab and were evaluated by isoelectric focusing and autoradiography. The different forms of PLAP are designated on the right. Panel B, PLAP mRNA was translated for 90 min at 30 °C using a rabbit reticulocyte lysate and either SP K562 cells (lanes 1 and 2) or SP K class cells (lanes 3 and 4) in the presence of 5 mM DTT (lanes 1-4) with the addition of 10 mM hydrazine (lanes 2 and 4). Translation products were separated by SDS-PAGE and visualized by autoradiography.

It remains to be determined whether the hydrophobic C-terminal signal sequence must traverse the ER membrane prior to recognition by the transamidase (30) or if the signal sequence triggers its own integration into the lipid bilayer, enabling subsequent presentation of the omega -site to the transamidase (31). It has been shown that, in comparison with the expression of correctly processed GPI-anchored PLAP on the cell surface, non-GPI-anchored PLAP proteins became localized intracellularly, probably in association with the ER membrane (32). The retention of improperly processed GPI-anchored proteins within the ER has been attributed to determinants contained within the hydrophobic C-terminal signal sequence (33, 34). Our results suggest that folding of unprocessed precursor proteins is unlikely to account for the retention of these polypeptides by quality control mechanisms within the ER. We can therefore conclude that GPI anchoring at least for PLAP is not conformation-specific, and that protein folding and GPI addition constitute two distinct processes that are independent of one another.

Interaction of PLAP with Transamidase Subunits-- Two gene products (Gpi8p and Gaa1p) have recently been identified by genetic analysis to be essential for GPI anchor addition in yeast (14, 15) and in mammalian cells (16, 18) and probably constitute subunits of the transamidase. To determine whether either of these subunits interacts with pro-PLAP synthesized in our SP cell system, we used the bifunctional cysteine-specific cross-linking reagent BMH to cross-link proteins that were in close proximity to the nascent polypeptide chain. As mentioned previously, PLAP contains several cysteine residues, two of which are in close proximity to the omega -site allowing cross-linking at or near to the C-terminal signal sequence. PLAP mRNA was translated either in the presence of SP K562, K class, or mutant IA K562 cells and then treated with BMH. Mutant IA cells do contain a functional transamidase but are not able to synthesize the GPI moiety destined to be attached to processed PLAP (3). PLAP-D484P mRNA, which contains an Asp right-arrow Pro mutation at the omega -site that prevents GPI anchor addition (10), was also translated in the presence of SP K562 cells and cross-linked with BMH as above. Cross-linking partners to N-terminally processed forms of PLAP were identified by immunoprecipitation with amino-Ab, and PLAP cross-links to subunits of the transamidase were identified by immunoprecipitation with antibodies raised to either the hGpi8p or hGaa1p.

Discrete cross-linking products were obtained after the addition of BMH to translated PLAP constructs in all conditions studied (Fig. 5, A-D, lane 2). Several of these remain unidentified, but at least three can be attributed to an association of the folding ectodomain with calreticulin and ERp57 (Fig. 5E). This observation is not unexpected, as PLAP is a glycoprotein and most glycoproteins interact with calnexin, calreticulin, and ERp57 at an early stage during their biosynthesis (35). The appearance of the same cross-link products after immunoprecipitation of either calreticulin or ERp57 is due to an interaction between these two proteins as described previously (35). Two cross-linking products to the Gpi8 subunit of the transamidase were identified with relative molecular masses of ~120 and 130 kDa when PLAP was translated in SP mutant IA cells (Fig. 5C, lane 3). The presence of two cross-linking products with differing mobility probably reflects cross-linking from different positions within the respective proteins, although they may represent an additional interaction with a third, as yet unidentified, protein. These results indicate that PLAP was in close proximity to the Gpi8p subunit of the transamidase. We assume that the processed form of PLAP associate either directly with this subunit of the transamidase or indirectly via other transamidase subunits/cofactors. An interaction with the Gpi8p transamidase subunit was also seen when omega -site mutant mRNA was translated in SP parental K562 cells (Fig. 5D, lane 3). This form of PLAP contains an uncleavable C-terminal signal sequence and is therefore not GPI-anchored. Therefore, in two independent experiments in which PLAP was unable to undergo GPI addition, either because of a defect in GPI biosynthesis (mutant IA cell line) or because the C-terminal signal sequence was not cleaved from the protein (omega -site mutant), cross-linked products to Gpi8p were observed. The specificity of these cross-linked products was underlined by the finding that radiolabeled PLAP did not form an interaction with this transamidase subunit in mutant K cells. This cell line contains a non-functional Gpi8p, which we can conclude is needed for interaction with proteins destined to be GPI-anchored. No cross-linked product to PLAP translated in SP parental K562 cells was observed (Fig. 5A, lane 3). The lack of cross-linked product with K562 cells is likely due to the fact that cross-linking was studied after 1 h of translation and the majority of PLAP was processed and GPI-anchored after this time with SP K562 cells.


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Fig. 5.   Interaction of in vitro synthesized PLAP with transamidase components. mRNA encoding PLAP (panels A-C and E) and omega  site mutant of PLAP (panel D) were translated in a rabbit reticulocyte system for 60 min in the presence of either SP K562 cells (panels A, D, and E), SP K class cells (panel B), and SP mutant IA cells (panel C) for 60 min at 30 °C. After termination of translation, cells were washed and, where indicated, treated with 100 µM BMH (panels A-E, lanes 2-4). The samples were denatured with 1% SDS prior to immunoprecipitation with PLAP amino-Ab (lanes 1 and 2), anti-Gpi8p (lane 3), and anti-Gaa1p (lane 4) for panels A-D and with amino-Ab (lane 1 and 3) or anti-calreticulin (lane 2) or anti-ERp57 (lane 4) for panel E. All samples were analyzed by SDS-PAGE and autoradiography. The cross-link products immunoprecipitated with Gpi8p antibodies are indicated with a star and to calreticulin and ERp57 with a filled circle.

These results demonstrate that, where the transamidase is functional but PLAP does not undergo processing to GPI-PLAP, an adduct to Gpi8p was formed. This is probably due to accumulation of the pro-form of the protein interacting directly with the Gpi8p transamidase subunit. The omega -site mutant contained a C-terminal signal sequence that was not processed in SP K562 cells (results not shown), and so probably remained in a "dead-end" complex with the transamidase. In the mutant IA cell line, GPI is not available to react with the carbonyl-enzyme intermediate. Therefore, a pro-PLAP-Gpi8p complex must have been formed, which was not released or was very slowly released from the transamidase, in comparison to the situation in K562 cells. From these results we can infer that nucleophilic attack by GPI is required for release of the pro-PLAP intermediate from the Gpi8p subunit of the transamidase. These data also demonstrate that, in the mutant IA cells, pro-PLAP interacts with Gpi8p in the absence of GPI. Therefore, direct or indirect recognition of pro-PLAP by Gpi8p occurs independently of GPI binding to the transamidase and may occur in either the presence or absence of GPI. It has been shown previously that, when prepromini-PLAP was translated in the presence of RMs derived from GPI-deficient cells, far smaller amounts of both GPI-linked and free mini-PLAP were synthesized compared with translation products synthesized in wild-type RMs (25). These workers suggested that GPI might act as an allosteric effector of the transamidase such that an activated complex between the pro-form of the protein and the transamidase only occurs in the presence of GPI. However, the fact that hydrazide-mini-PLAP was generated in microsomes derived from a GPI-deficient cell line provided evidence that a carbonyl intermediate forms in the absence of GPI (12). The identification of a pro-PLAP-Gpi8p transamidase intermediate formed in the absence of GPI in the present study is entirely consistent with the action of a transamidase.

Gpi8p has sequence homology to cysteine proteases and has been suggested to be the catalytic component that cleaves the GPI attachment signal peptide (20). The GPI signal sequence may be directly recognized by Gpi8p or may be presented to this putative catalytic subunit of the transamidase by another interacting subunit of the enzyme. It has been suggested that Gaa1p subunit of the transamidase is involved in the recognition of the protein substrate (14, 18), although the functions of Gaa1p cannot be predicted from the primary amino acid sequence of this protein. In the present study, we did not identify any cross-linking products that immunoprecipitated with antiserum recognizing the Gaa1p subunit of the transamidase (Fig. 5, A-D, lane 4). The antiserum used in these studies was able to react with Gaa1p on Western blots and by immunoprecipitation (results not shown). The failure to detect a cross-linking product to Gaa1p may be due to the fact that cysteine residues within PLAP and Gaa1p were not in close enough proximity for chemical linkage (BMH has a spacer arm of 16.1 Å). Therefore the absence of PLAP-Gaa1p intermediates was not necessarily due to the lack of molecular interactions between these two proteins. Alternatively, it may be that pro-PLAP may be transferred directly to the Gpi8p subunit of the transamidase. The Gaa1p subunit of the transamidase may bind GPI, recruiting this glycolipid to the pro-PLAP-Gpi8p complex and have a correspondingly brief interaction with protein destined to be GPI-anchored which cannot be detected using this approach. The fact that we were able to observe cross-links of PLAP with Gpi8p, but not with Gaa1p, may mean that the Gpi8p subunit of the transamidase has a very slow reaction rate and may catalyze the rate-limiting step of the transamidation reaction.

Quality control of post-translational modification is an important facet of protein biogenesis. GPI quality control is probably mediated by polypeptide retention in the ER followed by degradation via the cytoplasmic proteasome (33). Misfolding of PLAP monomers within the ER may not play an important role in this process, as both GPI-anchored and non-GPI-anchored PLAP were folded in the SP cell system. Alternatively, the direct interaction of precursor proteins with the transamidase demonstrated here may explain how precursor GPI-anchored proteins are retained in the ER. Modification of proteins by GPI addition would then release the protein from the transamidase and allow subsequent transport through the secretory pathway.

    ACKNOWLEDGEMENT

We thank Dr. M. E. Medof for providing PLAP clones and the K and IA class cell lines.

    FOOTNOTES

* This work was supported by Medical Research Council Grants G9722981 and G9722026 and by a grant from the Royal Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 44-61-275-5103; Fax: 44-61-275-5082; E-mail: neil.bulleid@man.ac.uk.

Published, JBC Papers in Press, February 23, 2001, DOI 10.1074/jbc.M010128200

    ABBREVIATIONS

The abbreviations used are: GPI, glycosylphosphatidylinositol; BMH, bismaleimidohexane; CSPD, 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'chloro)-tricyclo[3.3.1.1]decan]-4-yl)phenyl phosphate; DTT, dithiothreitol; ER, endoplasmic reticulum; IEF, isoelectric focusing; PAGE, polyacrylamide electrophoresis; PLAP, human placental alkaline phosphatase; RM, rough microsomal membrane; SP, semipermeabilized; PMSF, phenylmethylsulfonyl fluoride; TBS, Tris-buffered saline; RLU, relative light unit(s); Ab, antibody.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
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