Proc. Natl. Acad. Sci. USAVol. 90, pp. 4112-4116, May 1993Cell Biology

Inhibition of a reductive function of the plasma membrane bybacitracin and antibodies against protein disulfide-isomerase

(diphtheria toxin/thiol-disulfide interchange/surface-bound proteins/endocytosis)

RICHARD MANDEL, HUGUES J.-P. RYSER*, FAROOQ GHANI, MIN WU, AND DAVID PEAK

Department of Pathology, Boston University School of Medicine, Boston, MA 02118

Communicated by R. John Collier, February 8, 1993

ABSTRACT Evidence had been provided that a disulfide-linked [12511iodotyramine/poly(D-lysine) conjugate was reduc-tively cleaved when bound nonspecifically to the surface ofChinese hamster ovary (CHO) cells and that this cleavage wasabolished by membrane-impermeant sulfhydryl blockers. Thesame blockers were subsequently found to inhibit the cytotox-icity of diphtheria toxin, a disulfide-linked heterodimer thatbinds to a specific surface receptor and must undergo chainseparation to exert its cytotoxicity. This suggested that thedisulfides of both macromolecules might be cleaved by athioldisulfide interchange reaction, possibly mediated by pro-tein disulfide-isomerase (PDI, EC 5.3.4.1). We tested whetherinhibitors of PDI-in particular, bacitracin and anti-PDI an-tibodies-might mimic the two effects of sulfhydryl blockers.Both bacitracin and anti-PDI antibodies were effective ininhibiting both reductive processes. This strongly suggests thatthe disulfide cleavage in the two membrane-bound macromol-ecules is mediated by PDI and that this enzyme, besides itsknown retention in the endoplasmic reticulum, must also beexposed at the plasma membrane. This paper points to otherpotentially important disulfide reductions that might be cata-lyzed by surface-associated PDI. It thereby broadens theknown functions of an enzyme already known for its multi-functional properties.

While studying the reductive cleavage of a disulfide-linkedmembrane-bound conjugate, [1251]iodotyramine conjugatedwith poly(D-lysine) via a 3,3'-dithiobis(propionic acid)spacer, (125I-Tyn-SS-PDL), Feener et al. (1) found that anearly phase of cleavage occurring in the first minutes ofchasewas totally inhibited in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or p-chloromercuribenzenesul-fonic acid (pCMBS), two membrane-impermeant sulfhydrylblockers. This indicated that the cell surface had a previouslyunknown reductive function mediated by surface sulfhy-dryls. The same two inhibitors were found to prevent thecytotoxicity of membrane-bound diphtheria toxin (DT), sug-gesting that the surface-associated reductive mechanism alsoactivated DT, presumably by cleaving the toxin's interchaindisulfide (2). Such a cleavage must occur prior to the trans-location of DT's chain A across the membrane of acidifiedearly endosomes. These two findings spurred an effort toidentify the mechanism of this reaction and the criticalsulfhydryls that were blocked by DTNB and pCMBS. It waspostulated that, if the sulfhydryls belonged to a surfaceenzyme, protein disulfide-isomerase (PDI, EC 5.3.4.1)-alsoknown as glutathione-insulin transhydrogenase or glu-tathione:protein-disulfide oxydoreductase (EC 1.8.4.2)-would be a possible candidate (1). PDI is found most prom-inently in the lumen of the endoplasmic reticulum (ER) whereit is retained by the carboxyl-terminal Lys-Asp-Glu-Leu

(KDEL) sequence (3). It has also been reported to occur atthe surface of mammalian cells (4, 5). This paper presentsevidence that PDI is indeed responsible for cleaving thedisulfides of these membrane-bound macromolecules andshows that cleavage is inhibited by monoclonal anti-PDIantibodies and by bacitracin, an antibiotic known to inhibitboth the reductive (6) and the oxidative (7) functions of PDI.

MATERIALS AND METHODSMaterials. Bacitracin, DTNB, and pCMBS were purchased

from Sigma. DT was from List Biological Laboratories,Campbell, CA. Thioredoxin and monobromotrimethyl-ammoniobimane (Thiolyte MQ) were from Calbiochem. Ly-phophilized ascites fluid containing anti-PDI monoclonalantibodies RL77, RL90, and HP13 (8) and the parent hybrid-oma cells were a gift from Charlotte S. Kaetzel, CaseWestern Reserve University School of Medicine. For someexperiments the antibody-containing ascites fluid or condi-tioned medium was purified by protein G affinity chroma-tography (Mab Trap G kit from Pharmacia LKB). 125I-Tyn-SS-PDL conjugate was prepared as described (1). The ex-periments were carried out on Chinese hamster ovary (CHO)cells.

Isolation ofPDI and Assay ofPDI and Thioredoxin. PDI wasisolated from calf liver as described by Hillson et al. (9) andwas 90-95% pure by electrophoresis. It was assayed asdescribed by Carmichael et al. (10), except that the glu-tathione concentration was reduced to 250 AM. The amountand radioactivity of 125I-insulin were 50 ,ug and 105 cpm persample. Under these conditions, PDI had a specific activityof 5200 units/mg in the standard 5-min assay. In the assaysof Fig. 1 and Table 1, the reaction time was extended to 30min and the reaction was stopped with 10 mM N-ethylma-leimide. The PDI concentration was 1.2 ,ug/ml. PDI activitywas calculated as the difference between acid-soluble radio-activity generated in 30-min incubations at 37°C in thepresence or absence of PDI. Controls were run to determineacid-soluble radioactive contamination of the 125I-insulinpreparations (usually 2-6%). Assays were carried out inpresence and absence of aprotinin to monitor possible pro-teolytic cleavage of 125I-insulin. They showed that insulinproteolysis was not a factor under our assay conditions. Inthe assays of Table 1, anti-PDI antibodies were preincubatedwith the enzyme for 30 min at 37°C, and glutathione wasraised from 250 ,uM to 500 ,uM. In vitro cleavage of 125I-Tyn-SS-PDL was measured during a 30-min incubation at 37°C,with the conjugate instead of 1251-insulin as substrate in the

Abbreviations: PDI, protein disulfide-isomerase; DT, diphtheriatoxin; 125I-Tyn-SS-PDL, [1251]iodotyramine conjugated with poly(D-lysine) via a 3,3'-dithiobis(propionic acid) spacer; DTNB, 5,5'-dithiobis(2-nitrobenzoic acid); pCMBS, p-chloromercuribenzene-sulfonic acid; T3BP, 3,3',5-triiodo-L-thyronine-binding protein; ER,endoplasmic reticulum.*To whom reprint requests should be addressed.

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PDI assay and with the concentration of glutathione de-creased from 250 to 100 ,uM. The activity of thioredoxin wasdetermined in the turbidimetric assay described by Holmgren(11).

Reductive Cleavage of Membrane-Bound Conjugate. Inexperiments testing the effect of bacitracin, the procedurewas essentially that described (1), except that bacitracin wasused instead of DTNB or pCMBS and was present duringboth the 60-min labeling period at 0°C and the subsequentincubation at 37°C. As described (1), the incubation mediumcontained bovine serum albumin at 4 mg/ml. Acid-solublecell-bound radioactivity was generated during both the la-beling period and the subsequent incubation. Both were

entered separately in Fig. 2. In experiments testing the effectof anti-PDI antibodies, the described procedure was modifiedas follows. Cells grown in 24-well plates were rinsed withice-cold medium and preincubated for 30 min at 0°C inserum-free Eagle's medium containing anti-PDI antibodies or

irrelevant IgG and 25 mM Hepes buffer (pH 7.4), in a totalvolume of 0.3 ml. 125I-Tyn-SS-PDL (1 ,g/ml, 1.8 x 105 cpmper well) was then added to each well to label the surface ofthe cells for 30 min at 0°C. The cells were washed twice andreincubated in prewarmed medium (37°C) containing anti-bodies or irrelevant IgG for a 30-min incubation. A furthermodification was the omission of bovine serum albumin fromthe incubation medium.

Inhibition of Protein Synthesis by DT. The cytotoxicity ofDT was assessed by measuring its inhibitory effect on theamino acid incorporation into cellular proteins. The proce-dure was essentially that described (2), using bacitracin or

anti-PDI antibodies as inhibitors instead of DTNB andpCMBS. In experiments testing the effect of anti-PDI anti-bodies, cells were preexposed to medium containing anti-bodies as described for experiments measuring conjugatecleavage, except that the antibodies were given as aliquots ofreconstituted solutions of lyophilized ascites fluid containingprotein at 10 mg/ml. Controls contained the same proteinamount as a mixture of irrelevant IgG and bovine serumalbumin at the ratio found in ascites fluid (1:9). DT (50 ,ul) wasadded in each well to the antibody-containing medium, andthe culture plates were incubated for 2 hr at 37°C andprocessed as described (2). We had found in previous exper-iments that simple pretreatment of cells with DTNB for 1 hrat 0°C did not inhibit the cytotoxicity ofa 2-hr exposure to DTat 37°C and that pretreatment with pCMBS led to only partialinhibition. Anti-PDI antibodies were therefore present bothduring preincubation at 0°C and during incubation at 37°Cwhen either conjugate cleavage or DT cytotoxicity was

measured.

RESULTS

PDI was tested in the insulin-reduction assay in the presenceof bacitracin or anti-PDI antibodies. Bacitracin at 3 mMcaused a 95% inhibition of PDI activity (Fig. 1). In the sameassay, three membrane-impermeant sulfhydryl blockerscaused 100% inhibition at a concentration of 1.0 mM and hadsimilar dose-inhibition curves that were steeper than that ofbacitracin (Fig. 1). The activity of the enzyme thioredoxin,which like PDI catalyzes disulfide reductions, was not inhib-ited by bacitracin. On the contrary, 3 and 6 mM bacitracinenhanced insulin cleavage by 12% and 5%, respectively (datanot shown).

Affinity-purified monoclonal antibodies against rat (RL77)and human (HP13) PDI (8) were tested for their inhibitoryeffect in the same PDI assay. Both antibodies caused dose-

dependent inhibitions of PDI (Table 1). The observation thatRL77 caused only 49% inhibition ofPDI activity is consistentwith the finding of Kaetzel et al. (8) and with their suggestion

be

c0

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0.01 0.1 1.0 10

Concentration (mM)

FIG. 1. Inhibition of PDI activity by bacitracin and membrane-impermeant sulfhydryl reagents. The activity of PDI isolated fromcalf liver was determined in the presence of bacitracin (e) or one ofthree membrane-impermeant sulfhydryl blockers: DTNB (0),pCMBS (n), and monobromotrimethylammoniobimane (A).

that these antibodies bind at or near only one ofthe two activesites of PDI.

Effect of Bacitracin on Reductive Functions. In preliminaryexperiments testing whether PDI could cleave the disulfide ofour model conjugate, 1251-Tyn-SS-PDL was used as substratein the way 125I-insulin is used in the classic PDI assay. In a30-min incubation at 37°C, PDI cleaved 25% of the addedconjugate, a value that compares favorably with the cleavageof 1251-insulin (29%) by the same amount of PDI.

Cleavage of surface-bound '251-Tyn-SS-PDL during a 30-min incubation at 37°C is shown by the lower curve of Fig. 2.Bacitracin inhibited cleavage in a dose-dependent fashion.Between 0.3 and 3 mM, the inhibition increased from 40% to76%, an increment comparable to the one seen in the inhi-bition of PDI activity in vitro (60% to 95%; Fig. 1). Surpris-ingly, we found that acid-soluble cell-bound radioactivity wasgenerated during the 60-min labeling at 0°C and that thiscleavage was also decreased in a dose-dependent fashion bybacitracin present during labeling. This bacitracin-inducedinhibition of cleavage is represented by the upper curve ofFig. 2. It indicates that some surface-associated cleavageoccurs at a temperature known to arrest endocytosis and thusthat bacitracin can exert an inhibitory effect at the cellsurface. It is difficult to conceive how these bacitracin effectscould be nonspecific artifacts. Exposure of CHO cells to 3mM bacitracin for 2 hr at 37°C did not cause cell damage inthe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium(MTT) cytotoxicity assay (12). Moreover, a bacitracin leak-age of intracellular PDI into the medium would, if anything,increase the reductive cleavage of membrane-bound 125I-Tyn-SS-PDL. The bacitracin-induced inhibitions seen in Fig.

Table 1. Inhibition of PDI activity by monoclonal anti-PDIantibodies RL77 and HP13 in the insulin-reduction assay

% decrease in PDI activityConc.,,Lg/ml RL77 HP13

10 9.7 ± 6.8 (5) 14 ± 6.5 (3)20 10 ± 13 (4) 23 ± 9.7 (2)50 32 ± 11 (10) 30 ± 11 (3)100 41 ± 11 (2)200 49 (1)

PDI activity measured in the presence of irrelevant IgG was usedas control. Antibodies (8) were partially purified from ascites fluid orconditioned medium; concentrations refer to the protein content ofthese preparations. Numbers of experiments are given in parenthe-ses.

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FIG. 2. Bacitracin-induced inhibition of disulfide cleavage ofmembrane bound 125I-Tyn-SS-PDL. Cleavage was measured asrelease of acid-soluble 1251-Tyn-SH and expressed as percent ofcontrols. Lower curve shows cleavage measured during 30 min ofincubation at 37°C. The absolute value for the control (100% forlower curve) was 2.9 ± 1.3% of the total cell-bound radioactivity, afigure consistent with published values (1). This control underesti-mates the real cleavage since, as we found recently, some 1251-Tyn-SH released from 125I-Tyn-SS-PDL binds to bovine serumalbumin present in the medium and becomes acid-insoluble. Uppercurve shows cleavage occurring during the labeling period at 0OC.The acid-soluble membrane-bound radioactivity in the absence ofbacitracin (100lo for the upper curve) was 3.6 ± 1.4% of the totalcell-bound radioactivity. For both curves, the bacitracin-induceddecreases in acid-soluble radioactivity were calculated separately foreach experiment (n = 4), averaged, and expressed as percent of thecorresponding control.

2 must therefore be due to a specific inhibition of surface-associated PDI.

In experiments measuring the cytotoxicity of DT, bacitra-cin inhibited cytotoxicity in a sharply dose-dependent fashion(Fig. 3). Bacitracin alone (3 mM) caused a slight inhibition ofprotein biosynthesis (91 ± 5.9% of controls, n = 21), whichwas taken into account in the calculations. The magnitude ofprotection obtained with a given dose of bacitracin wasinfluenced by the initial level ofDT cytotoxicity, which in thethree experiments of Fig. 3 averaged 36% of controls. In alarger series of experiments, protection was found to be

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FIG. 3. Inhibition of DT cytotoxicity by bacitracin. CHO cellsseeded (6 x 105 per well) and grown for 45 hr were rinsed twice withserum-free medium with or without bacitracin, exposed for 2 hr at37°C to DT (average, 75 ng/ml) in serum-free medium with or withoutbacitracin, and processed for measurement of amino acid incorpo-ration into cellular protein as described (2). The inhibition of proteinsynthesis by DT alone varied between 24% and 55% of controls, witha mean of 36% (n = 3). Because inhibitions of DT cytotoxicity are

influenced by the level of initial cytotoxicity (see text), they were

calculated for each experiment and averaged (± SD).

greatest when DT alone reduced protein biosynthesis to25-50o of control values. A similar correlation was noted inthe protective effect ofDTNB and pCMBS (2). The postulatethat this inhibition of DT cytotoxicity is due to an inhibitionof PDI is supported by a comparison of the dose-effectrelationships of Fig. 1, Fig. 2 (lower curve), and Fig. 3, whichplace the IC50 of bacitracin around 0.25, 0.62, and 0.90 mMfor the inhibitions of PDI activity, cleavage of surface-bound125I-Tyn-SS-PDL, and DT cytotoxicity, respectively. Thisconcordance is quite remarkable when one considers thatPDI acts in these cases on three different substrates and insystems with different end points.As had been found with the nonpermeant sulfhydryl block-

ers (2), bacitracin did not reduce the cytotoxicity ofricin and,in fact, slightly increased it. As discussed elsewhere (2), thisobservation is consistent with the currently accepted viewthat reductive cleavage of ricin's interchain disulfide occursin the trans-Golgi network or the Golgi apparatus-i.e., at asite not accessible to membrane-impermeant PDI inhibitors.HeLa cells, the only cell type other than CHO tested in thisfashion, were also consistently protected by bacitracinagainst DT cytotoxicity.The marked differences in structural and biochemical

properties ofDTNB and bacitracin suggested that they mightinhibit PDI by different mechanisms. It was of interest,therefore, to test whether the two categories of inhibitorswould have additive effects. In two experiments carried outat different levels ofDT cytotoxicity, the average protectionafforded by 3 mM bacitracin or 1 mM DTNB given alone orin combination was 39 + 17%, 24 ± 17%, and 68 ± 14%,respectively. A similar additivity was observed with 3 mMbacitracin and 0.1 mM pCMBS. In absence ofDT, bacitracincombined with DTNB depressed protein biosynthesis by13%, an effect of no consequence since the agents giventogether significantly counteract the inhibition of proteinbiosynthesis caused by DT.

Effect of Anti-PDI Antibodies on Reductive Functions. Thetwo monoclonal anti-PDI antibodies tested for their effect onPDI activity in vitro (Table 1), as well as a second antibodyagainst rat PDI (RL90), were tested for their ability to inhibitthe cleavage of surface-bound 125I-Tyn-SS-PDL during a30-min incubation at 37°C. When matched against the effectof comparable amounts of irrelevant IgG, all three antibodieshad marked inhibitory effects. The affinity-purified antibod-ies RL77 and HP13 at concentrations of25 ug per well causedinhibitions of 43% and 51%, respectively. RL90 tested inreconstituted ascites fluid (0.6 mg of protein per well) caused62% inhibition.The same antibodies were tested for an inhibitory effect on

DT cytotoxicity in four experiments in which DT alonedepressed amino acid incorporation into proteins to 47% ofcontrol values. These experiments used reconstituted solu-tions of lyophilized antibody-containing ascites fluid with aprotein content of 10 mg/ml (0.84 mg per well). Comparedwith the effect of identical concentrations of irrelevant IgG,all three antibodies caused significant inhibition of DT cyto-toxicity (Table 2). As noted with bacitracin, the magnitude ofinhibition of DT cytotoxicity was influenced by the level ofinitial DT cytotoxicity. In three experiments in which RL77was tested at two or more concentrations, its effect wasclearly dose-related.

DISCUSSIONPrevious work showed that the membrane-impermeant sulf-hydryl blockers DTNB and pCMBS were capable of inhib-iting both the reductive cleavage of membrane-bound 1251-Tyn-SS-PDL (1) and the reductive activation of membrane-bound DT (2). This implied that both reductive processeswere mediated by a plasma membrane-associated mechanism

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Table 2. Effect of anti-PDI antibodies on DT cytotoxicityIncorporation, Inhibition of

DT Antibody* % control toxicity,t %- - 100+ - 47 ± 4.9 0+ IgG 51±6.1 7.1±3.7+ RL77 75 ± 15 54 ± 24+ RL90 94 ± 10 85 ± 12+ HP13 80 ± 20 64 ± 33

Cytotoxicity was measured as the decrease in incorporation of14C-labeled amino acids into cellular proteins.*The reconstituted ascites fluids containing RL77, RL90, and HP13(8), as well as the IgG control solution, contained protein at 10mg/ml (0.84 mg per well). The protein concentration was thereforesubstantially higher than in the experiments of Table 1, in whichpartially purified antibodies were used.tThe inhibition of amino acid incorporation by DT varied from 41%to 53% with a mean of 47 ± 4.9% (n = 4). Because inhibition ofcytotoxicity is influenced by the level of initial cytotoxicity (seetext), it was calculated for each experiment and averaged. Com-pared with 7.1% (IgG control), the inhibitions were statisticallysignificant (P < 0.05 for RL77, P = 0.001 for RL90, and P < 0.05for HP13).

which required the presence of free sulfhydryl groups at thecell surface. The most plausible mechanism was a thiol-disulfide interchange reaction between cell surface sulfhy-dryls and the disulfide bonds of the exogenous macromole-cules. Such reactions can be enzyme-driven or occur spon-taneously in the presence of an excess of sulfhydryls, acondition that was not fulfilled in the experimental system (2).Enzymes known to carry out thiol-disulfide interchangereactions in mammalian cells include PDI (3), thioredoxin(13), and glutaredoxin (14). We chose to investigate PDIbecause the enzyme had been detected at the surface ofmammalian cells (4, 5). The finding that both reductivefunctions are markedly inhibited by bacitracin and by anti-bodies at concentrations that inhibit PDI activity in vitroprovides strong evidence that the reductive cleavage of125I-Tyn-SS-PDL and the reductive activation ofDT are bothcatalyzed by PDI. A major participation of thioredoxin inthese reactions is unlikely since concentrations of bacitracinthat strongly inhibited the membrane-associated reductionsenhanced the activity of thioredoxin in vitro. There is noreport that glutaredoxin occurs at the surface of mammaliancells. Comparison of the published sequences shows that theenzyme has little homology with PDI, either overall or at theactive site, and is more closely related to thioltransferasethan to PDI (14). It is not likely, therefore, that glutaredoxinwould crossreact with anti-PDI antibodies or that it would beinhibited by bacitracin. With the assumption that PDI and125I-Tyn-SS-PDL engage in a thiol-disulfide interchange atthe cell surface, it should be possible to obtain conclusivebiochemical evidence for the participation of PDI in thisprocess by isolating either the 1251-Tyn-SH adduct or thePDL-SH adduct of PDI from CHO cells that were exposed to125I-Tyn-SS-PDL. Involvement of PDI in a thiol-disulfideinterchange implies a mechanism capable of regenerating theenzyme's oxidized thiols, possibly a cascade of interchangereactions. Other possible participants in such an oxidoreduc-tion sequence have not been identified.The similarity between the inhibitory effects of bacitracin

and sulfhydryl reagents suggests that the critical surfacesulfhydryls blocked by DTNB and pCMBS and required forthe thiol-disulfide interchange are the cysteine sulfhydrylspresent at the catalytic sites of PDI. Unlike DTNB, bacitracindoes not contain active groups that can block free sulfhydrylsand is likely, therefore, to act by a different, albeit unknown,mechanism. Consistent with this view, the slope of itsdose-inhibition curve differs from that of three sulfhydryl

reagents (Fig. 1) and it had an additive effect in suppressingDT cytotoxicity when given together with sulfhydryl block-ers. Since 125I-Tyn-SS-PDL and DT interact with the plasmamembrane at different sites, it can be inferred that PDI ispresent both in domains that interact nonspecifically with thepositive charges of poly(D-lysine) and in the receptor areathat specifically binds the B chain of DT. The data of Fig. 2indicate that bacitracin can act before internalization of1251-Tyn-SS-PDL. There can be little doubt, therefore, thatbacitracin as well as the anti-PDI antibodies exert theirinhibitory effect while the exogenous macromolecules arestill in their original membrane-bound state. As discussedelsewhere (2), this does not exclude the possibility that thereductive activation of DT might occur in early endosomes,since even membrane-impermeant inhibitors will be trappedin the fluid volume of nascent endocytic vesicles.Monoclonal antibodies directed against rat or human PDI

inhibited the activity of calf liver PDI in vitro, as well as tworeductive functions at the surface of CHO cells. This cross-reactivity is consistent with the extensive homology of hu-man, bovine, and rat PDI (15). Crossreactivity betweenhuman, bovine, and rat PDI was noted with polyclonalantibodies against human and rat PDI (8), as well as with amonoclonal antibody to purified 3,3',5-triiodo-L-thyronine-binding protein (T3BP) (16), now known to be identical to PDI(17).The best-known function of PDI is to catalyze the oxi-

doreduction of disulfide bonds in the ER, which leads to thecorrect folding of newly synthesized proteins (3, 17), andstrong immunocytochemical staining for PDI is found in theER (3). However, PDI has been detected at the surface ofmammalian cells (4, 5). Yoshimori et al. (5) showed thatsurface-associated PDI still displayed its characteristicKDEL retention signal and suggested that PDI reached thesurface as the result of an "overflow" from the ER. PDI isa multifunctional enzyme identical to the P subunit of prolyl-4-hydroxylase and to T3BP, and showing extensive homologywith three other proteins (17). Triiodothyronine binding siteshad been demonstrated at the surface of mammalian cells(18-20), suggesting that T3BP was exposed on the plasmamembrane (21). Now that T3BP and PDI are known to beidentical, these prior data strengthen our evidence that PDIis indeed present at the cell surface, where it can exertreductive functions.The surface-associated reductive mechanism initially re-

vealed by the cleavage of 125I-Tyn-SS-PDL (1) may servefunctions other than the reductive activation of DT. Forinstance, a2-macroglobulin, a well-characterized macromo-lecular carrier that interacts with a specific surface receptor,has been shown to form disulfide bonds with platelet-derivedgrowth factor (22), interleukin 1,3 (23, 24), and the deglyco-sylated A chain of ricin and ricin immunotoxins (25). Areductive mechanism capable of cleaving the interchain di-sulfide of membrane-bound DT and the intrachain disulfide of125I-Tyn-SS-PDL might be expected to cleave the disulfidelinkage of these membrane-bound a2-macroglobulin conju-gates and release the carried functional proteins at the cellsurface or in nascent endosomes. It has been reported thatDTNB inhibits several physiologic effects of insulin in fatcells, presumably by inhibiting the formation of a functionallyrelevant disulfide bond between insulin and its receptor (26).This and the fact that insulin is a commonly used substrate forPDI (10) suggest that this enzyme might play a role in thesequence of events initiated by the hormone-receptor inter-action. The presence of disulfides in a number of polypeptideeffectors and surface receptors may receive added interest inview of our evidence for a membrane-associated PDI activ-ity. Other recent data have indicated that disulfides presentin viral envelope proteins may be cleaved upon virus-cellinteraction (27) and that inhibition of this reductive cleavage

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may interfere with virus infection (28). These new perspec-tives broaden the possible role of membrane-associated PDIand the spectrum of action of an enzyme already known forits multifunctional properties.

We thank Dr. Charlotte S. Kaetzel, Case Western Reserve Uni-versity School of Medicine, for her generous gift of anti-PDI mono-clonal antibodies and parent hybridoma cells. This work was sup-ported by National Institutes of Health Grant CA14551 from theNational Cancer Institute.

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