Protein-disulfide Isomerase-mediated Reduction of the A Subunit of Cholera Toxin in a Human Intestinal Cell Line*

(Received for publication, June 11, 1996, and in revised form, October 18, 1996)

Palmer A. Orlandi Dagger

From the Membrane Biochemistry Section, Laboratory of Molecular and Cellular Neurobiology, NINDS, National Institutes of Health, Bethesda, Maryland 20892-4440

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

A key step in the action of cholera toxin (CT) is the reduction of its A subunit to the A1 peptide. The latter is an ADP-ribosyltransferase, which activates the alpha -subunit of the stimulatory G protein of adenylyl cyclase. In this study, the enzymatic reduction of membrane-bound CT in CaCo-2 human intestinal epithelial cells was characterized. Whereas diphtheria toxin was found to be reduced by a cell surface population of protein-disulfide isomerase (PDI) and its cytotoxicity was inhibited by p-chloromercuribenzenesulfonic acid, bacitracin, or anti-PDI antibodies, these inhibitors had no effect on CT reduction or activity in intact cells. In contrast, the reduction of CT in vitro by either postnuclear supernatants (PNS) or microsomal membranes in the presence of Triton X-100 was significantly inhibited by p-chloromercuribenzenesulfonic acid and bacitracin. Anti-PDI monoclonal antibodies likewise inhibited the in vitro reduction of CT and also were effective in depleting reductase activity from PNS. Since inhibition and depletion were not observed in the absence of detergent, these results suggested that the reductase activity was a soluble component localized to the lumen of microsomal vesicles and correlated with the presence of protein-disulfide isomerase. This was further confirmed by showing a corresponding depletion of reductase activity and PDI in alkali-treated microsomes. This activity was restored when purified bovine PDI was added back to alkali-treated microsomes in a redox buffer that reflected conditions found in the lumen of the endoplasmic reticulum (ER). When the CT-related reductase activity was assayed in subcellular fractions of PNS-derived membranes isolated on a 9-30% Iodixanol gradient, the activity, as measured by CT-A1 peptide formation localized to those fractions containing PDI. Likewise CT-A1 peptide formed in intact cells co-localized to those membrane fractions containing the majority of cellular PDI. Furthermore, the banding density corresponded to a region of the gradient containing ER-derived membranes. These results indicated that CT was a substrate for PDI-catalyzed reduction in intact cells and supported the hypothesis that CT reduction and activation occurs in the ER.


INTRODUCTION

Cholera toxin (CT)1 consists of a pentameric B subunit that binds to specific cell surface receptors identified as ganglioside GM1 and an A subunit that activates adenylyl cyclase (see Ref. 1 for review). The A subunit consists of A1 and A2 peptides linked by a disulfide bond. The primary function of the A2 peptide is to noncovalently attach the A subunit to the B pentamer (2-4). Since its C terminus extends through and beyond the pore of the B pentamer (2) and contains the endoplasmic reticulum (ER) retention signal, KDEL, the A2 peptide may also function as a signal for the intracellular transport of the toxin. The A1 peptide is an ADP-ribosyltransferase that catalyzes the covalent modification of the alpha -subunit of the stimulatory G protein (Gs) and results in the persistent activation of adenylyl cyclase (for review, see Ref. 5). In target cells, the cytotoxic effect of CT is dependent on the reduction of CT-A to release the A1 peptide (6, 7). There is a distinct lag period between toxin binding and the activation of adenylyl cyclase, during which the toxin must be internalized and processed. At the end of this lag period, small amounts of A1 peptide appear in the cells in parallel with the activation of the cyclase. The mechanism of reduction and the intracellular location at which it occurs have yet to be identified.

Bound holotoxin is thought to undergo endocytosis through noncoated invaginations of the plasma membrane and appear first in noncoated vesicles and then in a tubulovesicular compartment (8). There is no indication that CT readily dissociates during this transit. Some insight into the intracellular pathway required for toxin activation may be provided by the recently described effects of brefeldin A (BFA) on CT action. The ability of BFA to disrupt the morphology of the Golgi apparatus and interfere with vesicular routing is well documented (9). In the human intestinal epithelial cell line, CaCo-2, as well as two different neurotumor cell lines, BFA completely inhibits the action of CT (10). Specifically, BFA prevents the reduction of CT to CT-A1 without any observable effects on toxin internalization. Thus, an intact, functional Golgi apparatus is essential for the intracellular activation of CT. Similar results were later reported by investigators using other cell lines (11-13). Several possible explanations for the profound effects of BFA on CT action were proposed (10): a direct inhibition of an unidentified reductase; the inability of internalized CT to be transported via a retrograde pathway to the site of reduction; or the relocation of the reductase to a new cellular compartment not accessible to the internalized toxin molecule. The identification and characterization of such a reductase may provide additional information about the intracellular processes involved in CT activation and those sites through which the toxin must pass.

Of the enzymes that catalyze thiol-disulfide interchange reactions, protein-disulfide isomerase (PDI; EC 5.3.4.1) is the best known. Found predominantly as a soluble protein within the lumen of the ER, the chief function of PDI is considered to be in the proper folding and processing of membrane and secretory proteins (14). PDI is also thought to act as a molecular chaparone, since it is able to interact and bind to a broad variety of peptides with no discernible requirement for sequence composition (15). It has also been identified as a thyroid hormone-binding protein and as the beta -subunit of prolylhydroxylase, although its capacity in these regards is not fully understood (15, 16). This 57-kDa enzyme is present in extremely high concentrations (for review, see Ref. 15), and its distribution within the ER is thought to be maintained in part by its carboxyl-terminal KDEL sequence (14), which prevents it from being secreted. Its high level of expression may influence its presence throughout the secretory pathway and the plasma membrane. PDI has been described in association with the Golgi apparatus (17), the trans-Golgi network (18), and the plasma membrane of mammalian cells (19, 20).

The plasma membrane-associated population of PDI has recently been shown to facilitate the reduction and activation of diphtheria toxin (DT), another ADP-ribosylating toxin. As with CT, the activation of DT requires the cleavage of an interchain disulfide bond to exert its cytotoxic effects. Treatment of intact Chinese hamster ovary (CHO) cells with anti-PDI monoclonal antibodies, membrane-impermeant sulfhydryl blockers, or bacitracin prior to toxin exposure inhibits DT cytotoxicity (21). In contrast, neither the cellular reductase responsible for the activation of CT nor the site at which the reduction occurs in target cells has been fully established. While it had been demonstrated that purified bovine PDI increased the rate of CT activation in solution in the presence of dithiothreitol or glutathione (22), there has been no direct evidence to suggest that this enzyme is responsible for the cellular activation of CT. This study examines the characteristics of membrane-bound CT reduction and the cellular site of toxin reduction and explores the potential role of the intracellular population of PDI in this process.


EXPERIMENTAL PROCEDURES

Materials

CT, CT-A, and DT were obtained from List Biological Laboratories (Campbell, CA). Sigma was the source of 3-isobutyl-1-methylxanthine, para-chloromercuribenzenesulfonic acid (p-CMBS), N-ethylmaleimide (NEM), and monoclonal antibodies against beta -COP and the 58-kDa Golgi protein. Anti-KDEL antibodies were purchased from Affinity BioReagents, Inc. (Golden, CO). Bacitracin and forskolin were purchased from Calbiochem; Immobilon-P membranes were from Millipore; protein A-Sepharose and the Mab Trap kit were from Pharmacia Biotech Inc.; and purified bovine liver PDI was from Panvera (Takara Biomedicals). Na125I, 125I-protein A (9.5 µCi/µg), and [3,4,5-3H]leucine (152.2 Ci/mmol) were purchased from DuPont NEN. Iodixanol (OptiprepTM) was purchased from Life Technologies, Inc.

Cells and Cell Culture

CaCo-2 cells were obtained from the American Type Culture Collection (Rockville, MD) and maintained in Eagle's minimal essential medium (EMEM) supplemented with nonessential amino acids, sodium pyruvate, 2 mM glutamine, and 20% NuSerum IV (Collaborative Biomedical, Bedford, MA) as described previously (7). For assaying cAMP accumulation and DT cytotoxicity, cells were grown in 24 × 16-mm clusters; for assaying the formation of CT-A1, they were grown in 6 × 35-mm clusters; and for preparing cellular fractions, they were grown in 75- or 175-cm2 flasks.

Toxin Cytotoxicity Assays

Cells were washed once in serum-free EMEM buffered with 25 mM HEPES (EMEM/HEPES) and preincubated in the same medium containing 0.01% bovine serum albumin and p-CMBS or bacitracin where indicated for 60 min at 37 °C. Cells treated with anti-PDI monoclonal antibody RL90 were preincubated for 60 min at 4 °C. The exposure of cells to these inhibitors or antibodies was continuous throughout the experiment. The cells then were exposed to 1 nM CT or 1.7 nM DT for 2 h at 37 °C. For those cells treated with CT, 1 mM 3-isobutyl-1-methylxanthine was included in the culture medium throughout the experiment. Following exposure to CT, cells were assayed for cAMP by radioimmune assay as described previously (23). To control for the effects of p-CMBS or bacitracin on adenylyl cyclase activity, some of the cells treated with the inhibitors were exposed to 100 µM forskolin instead of CT for 30 min at 37 °C and assayed for cAMP. Those cells exposed to DT were washed with serum-free EMEM/HEPES, pulsed an additional 1 h at 37 °C with 1 µCi of [3H]leucine, and assayed for trichloroacetic acid-insoluble radioactivity as described previously (24).

Preparation of PNS and Membrane Fractions

CaCo-2 cells were grown to confluence and maintained in culture for at least 14 days before harvesting. For labeling with 125I-CT, cells were washed once with serum-free EMEM/HEPES and chilled. Cells then were labeled with 125I-CT (106 cpm/ml; ~0.3 nM) in the same medium containing 0.01% bovine serum albumin for 30 min either (a) at 4 °C, washed, and harvested immediately (t = 0) or (b) at 15 °C, washed, and incubated for 60 min at 37 °C (t = 60) prior to harvesting. When harvested, cells were rinsed once with ice-cold PBS, scraped in PBS, and pelleted by low speed centrifugation. Those cells labeled with 125I-CT were harvested in the presence of 1 mM NEM to prevent any further reduction of CT (6). The cell pellets were suspended in 50 mM Tris-HCl, pH 7.4, containing 250 mM sucrose (Tris/sucrose buffer) and homogenized on ice with 10-20 strokes of a Dounce homogenizer. The homogenate was spun at 3,000 × g to pellet unbroken cells, cell debris, and nuclei. The PNS was removed, and a portion of it was centrifuged at 100,000 × g for 1 h at 4 °C. The resulting microsomal pellet was suspended in Tris/sucrose buffer, and both the PNS and microsomal membranes (~1 mg/ml) were stored in 1-ml portions at -80 °C.

In Vitro Reductase Assay

To assay the enzymatic reduction of CT, PNS or microsomal membranes (1-10 µg of protein) were incubated with 125I-labeled CT in 0.2 ml (106 cpm, ~1-2 nM) for 30 min at 4 °C and centrifuged to pellet the resulting CT-bound membranes. The latter were then suspended in 50 µl of 50 mM sodium acetate buffer, pH 5.5, and 1% Triton X-100 unless otherwise stated. Samples were incubated for 1 h at 37 °C and rapidly neutralized by diluting with 450 µl of ice-cold PBS, pH 7.4, containing 1% Triton X-100, 1 mM NEM, and 20% ethylene glycol. Reductase assays in the presence of PDI inhibitors or anti-PDI antibodies were conducted in a similar manner. When the effects of sulfhydryl blockers were examined on intact microsomal vesicles, membranes were incubated with the indicated inhibitor in Tris/sucrose buffer for 30 min at 37 °C, pelleted, washed in the same buffer, and assayed for reductase activity as described above with the exception that 125I-CT was added directly to the solubilized membranes. In some experiments, PNS were incubated overnight at 4 °C in PBS, 1% Triton X-100 with and without 500 µg/ml of anti-PDI antibodies. After the samples were absorbed with protein A-Sepharose for 1 h at 4 °C, they were centrifuged to pellet the beads, and the resulting supernatants were assayed for reductase activity. The resulting protein A-Sepharose beads were also analyzed by SDS-PAGE and Western blotting to verify the presence or absence of PDI. The amount of CT-A1 generated during reductase assays was determined by SDS-PAGE. Each sample (~25,000 cpm) was mixed with an equal volume of 0.2% SDS sample buffer, heated for 10 min at 37 °C, and separated on a 15% gel. The gels were then fixed, dried, and exposed to Kodak XAR-5 film to visualize the labeled products by autoradiography.

Subcellular Fractionation and Gradient Centrifugation

Subcellular fractionation was always performed on freshly prepared PNS or microsomal membranes from either unlabeled cells or those labeled with 125I-CT. PNS or microsomal preparations were adjusted to 35% Iodixanol, and 3 ml was layered under a linear 10-ml gradient of 9-30% Iodixanol in Tris/sucrose buffer. Samples were centrifuged in an SW 28 rotor at 52,000 × g for 90 min at 4 °C using a Beckman L8-70M ultracentrifuge. Fractions (~0.5 ml) were collected from the top of the gradient using a density gradient fractionator (ISCO). A 15-µl portion of each gradient fraction was used to determine its refractive index (eta ) as measured on an Abbe MARK II digital refractometer. From the refractive indices of standard Iodixanol solutions in Tris/sucrose buffer and their corresponding densities (rho ), the following equation was calculated to determine the densities of individual gradient fractions and is in agreement with that reported by Graham et al. (25): rho  = 3.4911eta  - 3.6664.

Gradient fractions from cells labeled with 125I-CT for 60 min at 37 °C were analyzed for CT-A1 peptide formation by immunoprecipitation of the individual fractions. Every two fractions along the gradient were pooled, treated with 1% Triton X-100, and immunoprecipitated with anti-CT-A1 antiserum overnight at 4 °C. Samples were analyzed by SDS-PAGE and autoradiography as described. Fractions from unlabeled cells were similarly pooled, precipitated with 10% trichloroacetic acid and 100 µg of bovine serum albumin as carrier, washed twice with 95% ice-cold ethanol, and solubilized with SDS-sample buffer. Samples were separated by SDS-PAGE, electroblotted onto Immobilon-P, and probed with anti-PDI antibodies. For the analysis of CT-related reductase activity in isolated gradient fractions, membranes from individual gradient fractions were diluted 4-fold with Tris/sucrose buffer and the membranes pelleted by a brief centrifugation in a TLA-100.2 fixed angle rotor at 200,000 × g for 15 min at 4 °C using a Beckman benchtop TL-100 ultracentrifuge. Isolated membrane fractions were then solubilized in 90 µl of 50 mM sodium acetate buffer, pH 5.5, containing 1% Triton X-100, and each was separated into two equivalent portions. One portion was subsequently treated with 1 mM NEM for 30 min at 37 °C. All sample volumes were then adjusted to 50 µl with the addition of 125I-CT and buffer, incubated for 60 min at 37 °C, and analyzed for the formation of CT-A1 as described above. A portion (25 µl) of these reaction mixtures was simultaneously probed for PDI by Western blotting as described above.

Reduction of CT by Purified Bovine PDI

Reduction of membrane-bound CT was performed using alkali-treated membranes from PNS. Alkali-treated membranes were prepared as described (26). Briefly, 100 µg of microsomal membranes were pelleted, suspended in 0.1 ml of 100 mM Na2CO3, pH 11.5, and incubated at 4 °C for 30 min. Control membranes were treated in a similar manner except that Tris/sucrose buffer was used throughout. Membranes were then pelleted, rinsed, and suspended in 0.1 ml of Tris/sucrose buffer containing 10 nM 125I-CT (107 cpm/pmol). Toxin was allowed to bind at 4 °C for 60 min, and the membranes were pelleted. Assays for the reduction of membrane-bound CT were then performed either in 50 mM sodium acetate, pH 5.5, or 50 mM sodium phosphate buffer, pH 7.0, in the presence of purified bovine PDI, GSH, and GSSG at concentrations described in Fig. 6. No detergent was included in assays involving alkali-treated membranes. Samples were incubated at 37 °C for 30 min, neutralized, and prepared for SDS-PAGE.


Fig. 6. Reduction of CT by purified bovine PDI. Alkali-treated membranes were prepared as described under "Experimental Procedures" to obtain membranes devoid of PDI. The Western blot above A represents 10 µl of the 50-µl reaction mixture in the absence or presence of increasing concentrations of exogenously added PDI. PDI was detected with RL77 anti-PDI antibodies to demonstrate the effectiveness of alkali treatment on the removal of PDI. A, the concentration-dependent reduction of CT by purified bovine PDI. 125I-CT was bound to membranes, pelleted, resuspended in either 50 mM sodium acetate, pH 5.5 (bullet , open circle ), or 50 mM sodium phosphate buffer, pH 7.0 (black-square, square ), in the presence (bullet , black-square) or absence (open circle , square ) of 1 mM GSH in a final volume of 50 µl. Samples were incubated at 37 °C for 30 min and neutralized, and the formation of CT-A1 peptide was assessed as before. B, effects of altering the GSH/GSSG ratio on the PDI-catalyzed reduction of CT. 125I-CT-bound membranes were resuspended in 50 mM sodium phosphate buffer, pH 7.0, containing 56 µM PDI (3 mg/ml), 1 mM GSH, and increasing concentrations of GSSG. The formation of CT-A1 peptide was assayed as above.
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Other Methods

Monoclonal antibodies RL77, RL90, and HP13 (27) were purified from either ascites (RL77, HP13) or conditioned culture supernatants (HP13) by protein G affinity chromatography. The generation of CT-A1 by intact cells was determined as described previously (6). CT was iodinated by a chloramine-T procedure (28) and routinely had a specific activity of ~15-20 µCi/µg. Protein was determined by the method of Lowry et al. (29), using bovine serum albumin as the standard. SDS-PAGE was performed according to Laemmli (30). Proteins separated by SDS-PAGE were electrophoretically transferred to Immobilon-P membranes (31). PDI was detected by immunoblotting with a purified IgG fraction (25 µg/ml) from RL77 ascites and 125I-labeled protein A or by chemiluminescence (Boehringer Mannheim). Densitometry of autoradiographs was performed using a Lacie Silverscanner II and quantified using NIH Image software, version 1.55.


RESULTS

Cell Surface Sulfhydryl Groups Are Not Required for CT Reduction and Action

When CaCo-2 cells containing bound 125I-CT were incubated at 37 °C, the cells generated small amounts of CT-A1 after a lag of at least 15 min (Fig. 1A). Similar lags have been described previously (6) and are consistent with the time-dependent internalization of membrane-bound toxin that precedes the activation of adenylyl cyclase and subsequent accumulation of cAMP by these and other cells (6, 7, 10, 32). The delay in CT-A1 peptide formation reflects the possibility that endocytosed CT proceeds via a defined intracellular pathway that results in its reduction and subsequent activation of adenylyl cyclase. This is in direct contrast to the described mechanism for DT activation in which reduction of the toxin occurs at or near the plasma membrane (21, 33). This disulfide-exchange reaction is catalyzed via a cell surface pool of PDI. To compare the activation of both CT and DT in intact cells, the cytotoxic effects of both toxins on CaCo-2 cells were examined in the presence of several membrane-impermeant sulfhydryl blockers. Cells were pretreated with p-CMBS, bacitracin, or a combination of the two reagents prior to the addition of either toxin. Bacitracin has been shown to be an effective inhibitor of PDI activity (34, 35). As shown in Table I, both pCMBS and bacitracin inhibited the cytotoxic effects of DT at concentrations reported to inhibit PDI activity in vitro (21) and block DT cytotoxicity in CHO cells (33). In contrast, neither reagent, either alone or in concert, affected the ability of the cells to generate significant levels of CT-A1 peptide from bound CT (Fig. 1B). This was also reflected in the lack of any observable effects on CT activity as measured by the toxin-mediated increase in intracellular cAMP (data not shown). NEM, which is membrane-permeant, however, completely inhibited the formation of CT-A1 peptide.


Fig. 1. Reduction of CT to CT-A1 by intact CaCo-2 cells. A, CaCo-2 cells were incubated with 125I-CT for 30 min at 4 °C, washed, and incubated in warm medium at 37 °C for the indicated times. Cells were analyzed for the formation of CT-A1 by SDS-PAGE, autoradiography, and densitometry as described under "Experimental Procedures." B, cells were incubated in the absence (lanes 1 and 2) and presence of 10 µM NEM (lane 3), 10 µM p-CMBS (lane 4); 10 mM bacitracin (lane 5), or 10 µM p-CMBS and 10 mM bacitracin (lane 6) for 30 min at 37 °C. Cells then were chilled, labeled with 125I-CT for 30 min at 4 °C, washed, and incubated in warm medium at 37 °C for 60 min in the presence of the indicated sulfhydryl blockers. Some cells (lane 1) were maintained at 4 °C. Cells were then analyzed for CT-A1 formation as above.
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Table I.

Effect of membrane-impermeant inhibitors on the activity of DT in CaCo-2 cells

CaCo-2 cells were incubated with the indicated inhibitors for 60 min at 37 °C, exposed for 2 h to 1.7 nM DT, and assayed for protein synthesis as described under "Experimental Procedures." Values represent means ± S.D. from one of three experiments.
Inhibitor Toxin Incorporationa Inhibition of protein synthesis DT toxicity

dpm/well % of control %
None  - 39,600  ± 3770
+ 24,300  ± 1360 38.6  ± 4.3 100
10 µM p-CMBS  - 38,800  ± 5060
+ 27,900  ± 1070 28.1  ± 3.8 72.8  ± 12.7
10 mM bacitracin  - 38,800  ± 620
+ 31,500  ± 1750 18.6  ± 1.1 48.2  ± 2.3
10 mM bacitracin, 10 µM p-CMBS  - 36,300  ± 2460
+ 30,900  ± 1160 15.0  ± 1.2 38.9  ± 5.3

a  Values for the incorporation of [3H]leucine into cell protein have been corrected for the effects of p-CMBS and bacitracin on the total uptake of [3H]leucine into CaCo-2 cells in the absence of toxin. Levels of [3H]leucine uptake in the presence of p-CMBS and bacitracin were 94 and 73%, respectively, of untreated control cells.

To further study the involvement of cell surface-associated PDI in the activation of both CT and DT, CaCo-2 cells were incubated in the presence of anti-PDI monoclonal antibody RL90 prior to the addition of either toxin. Affinity-purified anti-PDI antibodies at a concentration of 0.5 mg/ml inhibited DT cytotoxicity by 36.9% as compared with 3.8% in the presence of an irrelevant mouse IgG control (Table II). This is consistent with results of a previous study on DT activity in the presence of anti-PDI antibodies (21). RL90 antibodies, however, had no effect on the activity of CT under the same conditions (Table III). Comparable levels of cAMP were generated in control and antibody-treated cells. The results (Fig. 1 and Tables I, II, III) indicated that CT, unlike DT, was not reduced and activated by the cell surface-associated population of PDI (or another reductase) although this did not exclude the possibility that the reduction of CT occurred at a later stage in its intracellular transit through the tubulovesicular network (8) with the possible involvement of the intracellular pool of PDI. Indeed, the potent inhibitory effects of NEM on the activation of CT (Fig. 1B) may reflect not only the involvement of the intracellular pool of PDI but also the role of intracellular vesicular transport in general and the internalization of plasmalemmal-derived vesicles in particular (36-38).

Table II.

Effect of anti-PDI monoclonal antibody RL90 on the activity of DT in CaCo-2 cells

CaCo-2 cells were incubated with the indicated antibody for 1 h at 4 °C, exposed for 2 h at 37 °C to 1.7 nM DT, and assayed for protein synthesis as described under "Experimental Procedures."
DT Antibodya Protein synthesis DT toxicity Inhibition of DT toxicity

% of control % %
 -  - 100
+  - 68.3  ± 1.5 100  ± 2.2
+ IgG 69.5  ± 2.6 96.2  ± 3.5 3.8  ± 0.1
+ RL90 80  ± 7.5 63.1  ± 5.9 36.9  ± 2.6

a  Affinity-purified RL90 was used at a concentration of 500 µg/ml in a total volume of 0.25 ml. The same concentration of an affinity-purified nonimmune mouse IgG fraction was used as an irrelevant antibody control.

Table III.

Effect of anti-PDI monoclonal antibody RL90 on the activity of CT in CaCo-2 cells

CaCo-2 cells were incubated with 500 µg/ml of either control mouse IgG or RL90 for 1 h at 4 °C, stimulated for 2 h at 37 °C with 0.03 nM CT, and assayed for cAMP accumulation as described under "Experimental Procedures."
CT Antibody cAMP accumulation Inhibition of CT

pmol/well %
 -  - 4.4  ± 1.5
+  - 71.9  ± 12 0
+ IgG 65.4  ± 4 9
+ RL90 72.3  ± 4 0

Cell-free Reduction of CT

To identify the cellular reductase(s) responsible for the formation of CT-A1 peptide, we next assayed for the reduction of CT in a cell-free system using either PNS or microsomal vesicles derived from PNS (Fig. 2). Because toxin subunit interactions are susceptible to pH (39, 40), the effects of pH on CT-A1 peptide formation were first examined. 125I-CT was incubated with CaCo-2-derived PNS at 4 °C, and the toxin-bound membranes were washed and pelleted by centrifugation. The membranes were then solubilized in buffers ranging from pH 7.5 to 3.0 containing 1% Triton X-100 and incubated at 37 °C for 60 min. Under these conditions, holotoxin was most efficiently reduced in a slightly acidic environment (Fig. 2A). Maximum reduction of CT to CT-A1 peptide occurred between pH 5.5 and 6.0. It was these parameters (50 mM sodium acetate, pH 5.5, containing 1% Triton X-100) that were used to further examine the reduction of membrane-bound CT in vitro. When the CT-related reductase activity in PNS was compared with that of the derived microsomal and cytosolic fractions, the majority of the activity was found in the microsomal fraction (Fig. 2B, lane 3). This distribution correlated with the distribution of PDI as assessed by immunoblotting of these same samples with anti-PDI antibodies (Fig. 2B, lanes 7-9).


Fig. 2. The reduction of membrane-bound CT in vitro. A, effects of pH on toxin reduction and analysis of CT-A1 peptide formation by SDS-PAGE. B, PNS, cytosolic, and microsomal fractions were prepared as described under "Experimental Procedures." Each fraction was incubated with 125I-CT and assayed for CT reduction. Lane 1, PNS; lane 2, cytosolic fraction; lane 3, microsomal fraction. The same fractions (lanes 7-9) also were analyzed for PDI by Western blotting with anti-PDI antibody RL77. Intact microsomes also were treated in the absence (lane 3) or presence (lanes 4-6) of 10 µM NEM, 10 µM p-CMBS, or 10 mM bacitracin (BAC) in Tris/sucrose buffer. The treated microsomes were then washed, solubilized in reaction buffer, incubated with 125I-CT, and assayed for CT-A1 peptide formation. C, PNS was treated for 30 min at 37 °C with 1% Triton X-100 in the absence (lanes 1 and 7) or presence (lanes 2-5) of the same concentrations of inhibitors as described in B with the exception of lanes 6 and 7. In lane 6, PNS was assayed in the presence of 10 µg/ml BFA, and in lane 7 PNS was prepared from cells treated with 10 µg/ml BFA for 1 h at 37 °C and assayed as above. Following the addition of 125I-CT, the samples were incubated for 60 min at 37 °C and assayed for the formation of A1 peptide.
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Since PDI is a soluble protein that resides in the lumen of the ER (15), we next determined whether the endogenous reductase activity had similar properties. Intact microsomal vesicles were pretreated with NEM, p-CMBS, and bacitracin for 30 min at 37 °C to examine their effects on the in vitro reduction of CT. Microsomal vesicles were then washed, solubilized in reaction buffer and 125I-CT, and incubated for 60 min at 37 °C. As was observed when intact cells were exposed to these inhibitors (Fig. 1B), neither of the membrane-impermeant sulfhydryl blockers inhibited CT reduction (Fig. 2B, lanes 5 and 6). Only NEM inhibited the formation of A1 peptide. In contrast, when membranes were first solubilized with reaction buffer containing 1% Triton X-100 and treated with these same inhibitors prior to the addition of 125I-CT, A1 was not formed (Fig. 2C). The inhibition of CT reduction in the presence of p-CMBS and bacitracin was concentration-dependent with IC50 values of 10 µM and 3 mM for p-CMBS and bacitracin, respectively (data not shown). These concentration ranges closely paralleled those shown to inhibit the PDI-dependent reduction of disulfide-containing macromolecules inserted into the plasma membrane of CHO cells (21, 41). We also tested the effects of BFA on CT reduction in vitro. No inhibition of CT reduction was detected when 1 µg/ml BFA was added directly to the assay (Fig. 2C, lane 6) or when PNS was prepared from cells treated with 10 µg/ml BFA for 60 min at 37 °C (Fig. 2C, lane 7). The results shown in Fig. 2 demonstrated that without Triton X-100 to solubilize the vesicles, the reductase was not accessible to membrane-impermeant inhibitors such as p-CMBS and bacitracin. It was found that in the absence of nonionic detergent only 19% of CT-A was reduced to the A1 peptide using untreated microsomes compared with 58% in the presence of 1% Triton X-100 (data not shown). These results were consistent with the properties of PDI, the vast majority of which is found as a soluble protein in the lumen of the ER (15).

PDI-Specific Monoclonal Antibodies Inhibit the in Vitro Reduction of CT

CaCo-2 PNS were incubated with anti-PDI monoclonal antibody RL77 or RL90 in the presence of 1% Triton X-100, and any PDI-antibody complexes were then absorbed with protein A-Sepharose. Both antibodies were raised against rat liver PDI and previously shown to cross-react with human PDI as well as inhibit its activity in vitro (27). When the absorbed extracts were assayed for CT reductase activity, it was found that RL77 and RL90 reduced the level of CT-A1 generated by 55 and 80%, respectively when compared with control extracts of PNS that were absorbed with protein A-Sepharose alone. Immunoblotting confirmed that PDI was depleted from the PNS extracts and recovered in the immunoabsorbates (data not shown). When added directly to the reductase assay, both antibodies inhibited the reduction of 125I-CT by PNS in a concentration-dependent manner (Fig. 3). A third anti-PDI monoclonal antibody, HP13, raised against human placenta PDI, gave similar results, whereas an irrelevant mouse IgG was ineffective.


Fig. 3. Inhibition of the CT reductase activity in CaCo-2 PNS by anti-PDI monoclonal antibodies. Reductase assays were conducted in the presence of increasing concentrations of affinity-purified anti-PDI monoclonal antibodies (RL77 (bullet ), RL90 (open circle ), and HP13 (square )) or an irrelevant mouse IgG (black-square) as described under "Experimental Procedures." The formation of CT-A1 was analyzed by SDS-PAGE and autoradiography and quantified by scanning densitometry.
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Subcellular Co-localization of PDI and A1 Peptide Formation

To identify the intracellular site(s) of CT activation and the reductase(s) involved, PNS-derived microsomes were analyzed by subcellular fractionation on 9-30% Iodixanol gradients (25). CaCo-2 monolayers were either labeled with 125I-CT for 30 min at 4 °C, washed, and homogenized immediately to prepare the PNS fraction (t = 0), or they were labeled for 30 min at 15 °C, washed, and incubated an additional 30 min (t = 30) or 60 min (t = 60) at 37 °C prior to PNS preparation. Microsomes were then prepared, and the membranes were separated on the Iodixanol gradient. The 125I-CT density profiles for the subcellular fractionation of t = 0, t = 30, and t = 60 microsomes are shown in Fig. 4A. Bound toxin is not internalized at or below 15 °C (32); therefore, the t = 0 profile represents the distribution of bound toxin to plasma membrane elements. A significant amount of toxin was found in light fractions near the top of the t = 0 gradient. The peak fraction in this region was found to be rich in caveolin and suggested that a portion of the plasma membrane-bound toxin localized in caveolae.2 This is consistent with the observations of Tran et al. (8), who showed that CT bound to smooth invaginations on the cell surface. These have since been identified as caveolae and shown to be enriched in ganglioside GM1 (42).


Fig. 4. Subcellular fractionation of CaCo-2 cells labeled with 125I-CT. Cells labeled with 125I-CT were incubated at 37 °C for 0 time (------), 30 min (···), or 60 min (- - -), and microsomes were prepared as described under "Experimental Procedures." A, subcellular distribution of membrane-bound CT. Microsomal membranes were centrifuged through a 9-30% linear Iodixanol gradient, and fractions were collected from the top of the gradient and assayed for radioactivity and refractive index. Densities were calculated as described under "Experimental Procedures." B, subcellular distribution of CT-A1 peptide generated in cells incubated with 125I-CT for 30 and 60 min at 37 °C. Every two fractions from the t = 30 and t = 60 profile shown in A were pooled, treated with 1% Triton X-100, and immunoprecipitated with anti-CT-A1 antiserum overnight at 4 °C. Samples were analyzed by SDS-PAGE, autoradiography, and densitometry. A1 peptide formation is expressed as the percentage of total distribution along the gradient. C, subcellular distribution of PDI. An unlabeled microsomal fraction was prepared and separated on a linear 9-30% linear Iodixanol gradient as in A. Fractions were pooled as in B and analyzed for PDI by Western blotting as described under "Experimental Procedures." PDI distribution was quantified by densitometry and is expressed as a percentage of the total PDI detected.
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As shown in Fig. 1, when cells were warmed to 37 °C for 30 and 60 min, the internalized toxin was slowly reduced to generate minute amounts of the A1 peptide. The 125I-CT density profile of microsomal membranes prepared from these cells showed a significant shift in the distribution of the toxin (Fig. 4A) with the t = 30 profile reflecting an intermediate profile between the t = 0 and t = 60 samples. When fractions along the t = 30 and t = 60 profiles were analyzed for CT-A1 peptide, the majority of A1 present in each was distributed within a density range of 1.122-1.127 g/ml (Fig. 4B). Western blot analysis for the distribution of PDI using unlabeled microsomal membranes from a similar preparation detected PDI within the same density range of 1.121-1.129 g/ml (Fig. 4C).

We next examined the in vitro reduction of CT using membranes isolated from the subcellular fractionation of PNS on Iodixanol gradients. For these studies, we used freshly prepared PNS without first spinning down the microsomal membranes to avoid any perturbation of the subcellular organelles. Gradient fractions also were probed for the presence of PDI. As shown in Fig. 5A, maximum reduction of CT occurred using membranes isolated in the density range of 1.108-1.115 g/ml. These fractions also contained the majority of PDI when immunoblots of these fractions were probed with antibodies against PDI (Fig. 5B). To distinguish these fractions from those of the Golgi, we probed the same fractions with monoclonal antibodies against beta -COP and the Golgi marker, g58. As shown in Fig. 5, the Golgi markers appeared at a lighter density (1.077-1.08 g/ml). In contrast, anti-KDEL antiserum (used to further establish ER-derived fractions) detected only those proteins that co-localized to fractions containing PDI. Taken together, the results shown in Figs. 4 and 5 indicated that the endogenous CT-related reductase and PDI co-localized in the same ER-derived subcellular fractions and that these same fractions contained the bulk of the A1 peptide generated by intact cells after 1 h.


Fig. 5. In vitro reduction of CT by PNS subcellular fractions. PNS was prepared and separated by centrifugation on a linear 9-30% Iodixanol gradient. Membranes were isolated from individual fractions by a brief centrifugation step and solubilized in 50 mM sodium acetate buffer, pH 5.5, containing 1% Triton X-100 as described under "Experimental Procedures." Each fraction was then assayed for either the reduction of 125I-CT to CT-A1 peptide (A) or the distribution of PDI as detected by Western blotting (B). The reduction of CT was quantified by scanning densitometry. Values are expressed as a percentage of total CT-A present in each sample and have been corrected for any nonspecific reduction that may have occurred. Brackets in A represent the locations of beta -COP (a), 58-kDa Golgi (b), both found only in fractions 3 and 4 along the Iodixanol gradient, and anti-KDEL proteins (c).
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In Vitro Reduction of Membrane-bound CT by Purified Bovine PDI

Purified bovine PDI was tested for its ability to reduce membrane-bound CT in reconstitution experiments using membranes devoid of PDI. To examine PDI reductase activity on CT reduction, assay parameters also were altered to more closely reflect the redox environment and pH that are thought to exist within the lumen of the ER. CaCo-2 membranes were first treated with 100 mM sodium carbonate for 30 min at 4 °C. Such treatment causes membrane vesicles to open, flatten, and release their lumenal contents (26). Alkali treatment of PNS-derived membranes effectively removed all endogenous PDI as judged by Western blot analysis and resulted in a corresponding lack of CT reduction (Fig. 6). 125I-CT was then bound to the alkali-treated membranes for 60 min at 4 °C and repelleted. The reduction of membrane-bound 125I-CT was then monitored as a function of exogenously added PDI and glutathione. Assays were performed at pH 5.5 and pH 7.0 for comparison with previous results. Glutathione (GSH, reduced form) concentrations within the ER have been estimated to range between 0.5 and 1 mM (43). Similarly, PDI is found in such high concentrations within the lumen of the ER (0.4% of liver protein and over 2% of liver microsomal protein) as to suggest a direct participation in the redox buffering capacity of the ER (44). When experiments were conducted at pH 7.0 to examine the concentration-dependent effects of GSH on the reduction of membrane-bound CT in the absence of PDI, very little CT-A1 peptide was formed (3-6% reduction was routinely observed at concentrations of GSH between 0.5 and 1.0 mM). As demonstrated by Moss et al. (22), for the PDI-catalyzed reduction of CT in solution, reduction of membrane-bound CT at a neutral pH in the presence of 1 mM GSH was dependent on the presence of PDI and increased significantly with increasing concentrations of PDI added (Fig. 6A). The formation of CT-A1 peptide was increased ~13-fold at pH 7.0 and 56 µM PDI (3 mg/ml). No observable PDI-dependent reduction of CT occurred at neutral pH values in the absence of any exogenously added thiol. In contrast, when assays were conducted at pH 5.5, the reduction was increased nearly 27-fold at pH 5.5 and 19 µM PDI (1 mg/ml). In general, whereas the presence of PDI was effective in substantially decreasing the concentration of GSH necessary to generate a comparable level of CT-A1 peptide (data not shown), only at pH 5.5 were small amounts of CT-A1 peptide still formed by PDI in the absence of any exogenous thiol. The lowering of the dependence for GSH on the PDI-catalyzed reduction of CT at pH 5.5 may explain our earlier observations of more efficient CT reduction with PNS under slightly acidic conditions. It has likewise been suggested that reduction of disulfide bonds by PDI may also be dependent on protein conformation and accessibility of the disulfide bond to the enzyme.2,3 A subtle change in the conformation of CT-A at pH 5.5 may provide better access to the disulfide bridge and allow PDI to act even in the absence of optimal thiol concentrations.

We next examined the concentration effects of various GSH/GSSG buffers on the modulation of PDI-dependent reduction. The lumen of the ER is highly oxidizing with a GSH/GSSG ratio on the order of 3:1 to 1:1, whereas the overall cellular environment is considerably more reducing (30:1 to 100:1) (42). When the GSH/GSSG ratios were altered to more closely parallel the conditions of the ER, the PDI-dependent reduction of CT was decreased as the redox environment became more oxidizing. However, even when GSH/GSSG ratios ranged between 1:1 and 3:1, PDI still enhanced the reduction of CT from ~8- to 14-fold above levels generated in the presence of thiol only (Fig. 6B).


DISCUSSION

The intoxication of cells by CT requires the formation of CT-A1 inside the cells. After a defined lag phase, small amounts of CT-A1 are generated by the cells that are sufficient to activate adenylyl cyclase by ADP-ribosylation of Gsalpha . Although it is well established that CT treated with millimolar concentrations of reducing agents can catalyze the ADP-ribosylation of Gsalpha and activation of adenylyl cyclase in membranes (45), up to now little has been known about the cellular reduction of CT. In the present study, it was shown by several different methods that in CaCo-2 cells and their subcellular fractions, the reduction of CT was catalyzed by a reductase with the properties of intracellular PDI. These included the characterization and localization of the CT reductase, the effects of PDI-specific monoclonal antibodies and inhibitors on this reductase, and the co-localization of CT-A1 formed by intact cells in a subcellular compartment containing PDI. Additionally, this compartment contained markers characteristic of the ER with minimal overlap of those characteristic of the Golgi apparatus.

As a soluble enzyme, PDI can readily be released from its lumenal domain by the addition of detergent to membrane preparations. We have shown that the reductase activity found in microsomal vesicles had only limited access to membrane-bound CT unless a nonionic detergent such as Triton X-100 was present in the assay mixture. Similarly, in the absence of detergent, intact vesicles shielded the lumenal-resident reductase from the inhibitory effects of p-CMBS or bacitracin. In contrast, most of the reductase activity was lost from microsomal membranes following alkali treatment, which causes the release of lumenal and peripheral proteins (25). This loss closely correlated with the loss of PDI content in those same treated vesicles. The ability of several anti-PDI monoclonal antibodies to deplete the CT reductase from CaCo-2 PNS by immunoabsorption as well as to inhibit the in vitro reduction of CT confirmed the identity of the reductase acting on CT in vitro as PDI. Further, where assayed under conditions designed to reflect the redox conditions present in the lumen of the ER, purified bovine PDI was able to reduce CT bound to membranes depleted of cellular reductase(s).

Studies on the reductive cleavage of membrane-bound disulfide-linked macromolecules have shown that reduction can occur at two distinct stages within the cell (41), both of which appear to involve the activity of PDI. When 125I-labeled, disulfide-containing conjugates are bound to intact CHO cells and then chased into the cell, reduction is observed both at early times (within the first 15 min without a lag period) and at later times (beginning at ~30 min into the chase). Only the early stage of reduction is inhibited by membrane-impermeant sulfhydryl blockers such as p-CMBS and the PDI-specific inhibitor, bacitracin. Hence, this phase of reduction occurs at the cell surface or during the earliest stages of endocytosis, presumably with the involvement of cell surface sulfhydryl groups. The latter stage of reduction appears to occur in an unidentified, nonlysosomal compartment.

The present study provided direct evidence that the reduction of CT by target cells occurred at an intracellular location rather than at or near the plasma membrane. This is in contrast to several earlier reports based on photolabeling techniques (46, 47), which presented a model for toxin activation at the plasma membrane. While a population of PDI exists on the cell surface of mammalian cells (19, 20), our results indicated that it was the intracellular pool of PDI that catalyzed CT reduction. Neither p-CMBS, bacitracin, nor anti-PDI monoclonal antibody RL90 exhibited any inhibitory effects on CT activity. CaCo-2 cells exposed to these agents were able to generate levels of CT-A1 similar to those found in untreated cells, and there were no appreciable differences in cAMP accumulation.

In contrast, these same membrane-impermeant reagents were effective inhibitors of DT in CaCo-2 cells, in agreement with earlier work on CHO cells (21, 33). Their inhibition of early stage reduction is coincident with the time during which DT is endocytosed and translocated across the membranes of acidic endosomes and indicated that surface sulfhydryls contributed by the cell surface pool of PDI were factors in the reductive cleavage and activation of DT. While reduction of DT may not necessarily occur at the plasma membrane, it may be catalyzed by PDI, which is originally present on the plasma membrane and, through endocytosis, becomes oriented on the lumenal side of early endosomes. Although small amounts of the sulfhydryl blockers present in the medium may be taken up by the cell, it is highly unlikely that they could proceed further than the early stages of the endosomal system. That these agents had no observable effect on the action of CT suggested that an intracellular pool of PDI (presumably that distributed within the ER and early Golgi complex) mediated the thiol-disulfide interchange reaction. Also, the distinct lag period prior to the CT-dependent activation of adenylyl cyclase coincides with the time frame of the later phase of PDI-dependent reduction of disulfide-containing conjugates described by Feener et al. (41).

The subcellular fractionation studies not only support the role of PDI in the reduction of CT but also provided additional insight into the intracellular transport of the toxin. Thus, the endogenous CT reductase activity co-localized to the same subcellular fractions as PDI and most probably represented the ER compartment. A second ER marker, the KDEL receptor, was also present in these same fractions, and on Iodixanol density gradients both ER markers appeared to band in a density range of 1.10-1.13 g/ml. This is in close agreement with values reported by Graham et al. (25) for the ER marker enzyme NADPH-cytochrome c reductase. This location was also quite distinct from CT-bound plasma membrane elements (as determined by CT binding at 4 °C). Golgi-associated membranes have been reported to band at a density of 1.06-1.09 (25), where we found the Golgi marker, gp58. Subcellular fractionation of CT-labeled cells showed that CT-A and the formation of CT-A1 peptide were localized to the same intracellular compartment as PDI. That these elements were coincident suggested that the intracellular trafficking of CT prior to its activation involved transit of CT-A through the ER. In showing the relationship between the toxin and an ER-resident reductase such as PDI, we further illustrated the importance of those organelles associated with the secretory pathway in the activation of CT.

We have previously provided evidence that an intact Golgi is required for the reduction of CT and the formation of CT-A1 peptide (7). Cells treated with BFA are unable to reduce CT to form the A1 peptide. While the effects of BFA on the morphology of the ER and Golgi complex, the redistribution of endogenous enzymes, and the inhibition of vesicular transport have been well studied (9), it was not clear how these effects contributed to the inhibition of toxin reduction. Whereas CaCo-2 cells pretreated with as little as 0.1 µg/ml of BFA are unable to reduce CT to the A1 peptide and are completely resistant to the action of the toxin (7), BFA had no effect on the reduction of CT in vitro. Likewise, PNS prepared from cells treated with 10 µg/ml of BFA for 60 min was as effective as PNS from untreated cells in reducing CT in vitro. These findings eliminated the possibility that BFA directly inhibited the reductase involved in CT reduction in intact cells. More study will be necessary to understand the exact relationship between the effects of BFA on Golgi organization and vesicular transport and to understand its ability to block toxin reduction. It is interesting to note, however, that PDI expresses the ER retention sequence, KDEL, at its C terminus (14). Its distribution in the ER and early Golgi would make this enzyme susceptible to the actions of BFA. Redistribution of ER- and Golgi-resident enzymes could contribute to the inability of BFA-treated cells to reduce internalized CT. Although some PDI has been reported to be present in the Golgi apparatus (17) and the trans-Golgi network (18), we have not been able to detect any significant levels of PDI and CT-A1 peptide co-localizing in these lighter fractions derived from CaCo-2 cells. Thus, the more likely possibility is that BFA treatment blocks the retrograde translocation of CT to the ER, where it is reduced by PDI.

Finally, the results of our in vitro experiments with purified PDI reflected those obtained with PNS and indicated that the two activities were the same. Additionally, PDI remained an effective catalyst for toxin reduction even under such suboptimal conditions as lower pH and thiol concentration (as defined for the oxidizing activity of PDI). Even under the oxidizing environment associated with the lumen of the ER, PDI-dependent reduction of CT was still favorable.

Although we cannot determine with absolute certainty that CT reduction occurs in the ER, all of the data are consistent with such a mechanism. The results of a recent study by Majoul et al. (48) support the conclusions drawn from our study. As in this work, their investigation demonstrated that small amounts of CT-A can be detected within the ER after 30 min. These levels were found to increase with time and were accompanied by the formation of minute amounts of CT-A1 peptide. In this regard, the intracellular processing of CT shares some similarities with another ADP-ribosylating toxin, Pseudomonas exotoxin A (see Refs. 7, 49, and 50). Both toxins need to be reduced in order to be activated; both toxins have C termini that end with sequences recognized by the KDEL receptor; the action of both toxins is blocked by BFA treatment; and the active fragments of both toxins have to undergo translocation from an extracytosolic compartment into the cytosol in order to reach their targets. In the case of exotoxin A, it is translocated out the ER into the cytosol via the ER protein-exporting mechanism (50). Whether CT uses the same mechanism remains to be determined.


FOOTNOTES

*   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: Bldg. 49, Room 2A28, National Institutes of Health, Bethesda, MD 20892-4440. Tel: 301-496-1624; Fax: 301-496-8244.
1    The abbreviations used are: CT, cholera toxin; DT, diphtheria toxin; Gs, stimulatory G protein; p-CMBS, para-chloromercuribenzenesulfonic acid; EMEM, Eagle's minimal essential medium; PNS, postnuclear supernatant(s); PBS, calcium- and magnesium-free phosphate-buffered saline; PDI, protein-disulfide isomerase; BFA, brefeldin A; PAGE, polyacrylamide gel electrophoresis; NEM, N-ethylmaleimide; ER, endoplasmic reticulum; CHO, Chinese hamster ovary; GM1, II3 Neu5AcG9Ose4Cer.
2    P. A. Orlandi and P. H. Fishman, unpublished observations.
3    C. S. Kaetzel, personal communication.

Acknowledgments

I thank Dr. Peter H. Fishman for guidance and helpful suggestions. I also thank Drs. Charlotte S. Kaetzel (University of Kentucky College of Medicine) and H. J. P. Ryser (Boston University School of Medicine) for helpful discussions on PDI and appreciate Dr. Kaetzel's generosity in providing the anti-PDI monoclonal antibodies and parent hybridoma cells.


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