Mechanism of action of the monosialoganglioside GM1 as a modulator ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Val. 268. No. 2, Issue of January 15,pp. 1368-1375,1993 Printed in U.S.A.

Mechanism of Action of the Monosialoganglioside GMl as a Modulator of CD4 Expression EVIDENCE THAT GMl-CD4 INTERACTIONTRIGGERS INTERNALIZATION AND DEGRADATION*

DISSOCIATION OF p56Ick FROM CD4,AND

CD4

(Received for publication, June 19, 1992)

Daniela SaggioroS, Claudio SorioB, Francesca Calderazzo, Lanfranco Callegaroq, Marina Panozzo, Giorgio Bertong, and Luigi Chieco-Bianchi From the Institute of Oncology, Interuniversity Center for Cancer Research, Istituto Scientific0 Tumori-Genova, Section of Biotechnology, University of Padova and the $Institute ofGeneral Pathology, University of Verona,and IlFidia Research Laboratories, A b a m Terme, Padova, Italy

Analyzing the mechanisms underlying the capability of the monosialogangliosideGMl to induce CD4 modulation we observed that GMl has a dual effect on the CD4 molecule.GM1 treatment of the lymphoma cell line MOLT-3 and CD4-transfected HeLa cells for times shorter than 30 min prevented binding of monoclonal antibodies (mAbs) recognizing epitopes located within the firstNHz-terminal domains of CD4, but not of the OKT4 mAb,which binds to the region of CD4 proximal to the transmembrane domain. However, no binding of the OKT4 mAb was observed after a few hours of treatment with GMl in both MOLT-3 cells and HeLa cells transfected with an intact CD4 molecule,but not in HeLa cells transfected with a CD4 moleculelacking the bulk of the cytoplasmic domain, suggesting that modulation of CD4 by GMldepends on the integrity of the cytoplasmic domain. GMl treatment blocked binding of several mAbs which recognize epitopes located within the first two NH2-terminal domains of CD4 and did not induce CD4 down-modulation if MOLT-3 cells were preincubated withthe OKT4A or the OKT4 mAbs. Immunoprecipitation studies with [35S]methionine-labeled MOLT-3 cells showed that GM1-induced CD4 down-modulation was accompanied by CD4 degradation,andthiswas preceded by dissociation of p56ICkfrom CD4. GM1-induced CD4down-modulation, dissociation of p56Ickfrom CD4, and CD4 degradation were unaffected by staurosporine, which, on the contrary, blocked these events inresponse to phorbol 12myristate 13-acetate. These observations demonstrate that the firstaction ofGMl is to mask epitopes located within the first two NH,-terminal domains; then, GMI triggers protein kinase C-independent signals which cause p56ICkdissociation from CD4 and the deliveryof the molecule to an intracellular compartment whereit is eventuallydegraded.

The CD4 antigen is an integral membrane glycoprotein

* This work was supported by grants from Minister0 Sanita, Progetto AIDS, Grants 7204-22 and 628.005 and from Associazione Italiana Ricerca sul Cancro totheInstitute ofOncologyof the University of Padova and the Institute of General Pathology of the University of Verona. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $ T o whom correspondence should be addressed Institute of Oncology, Via Gattamelata, 64, 35128 Padova, Italy. Tel.: 049-807-1859 or 1863; Fax: 049-807-2854.

expressed on the surface of T cells restricted to the class I1 major histocompatibility complex (MHC),’ and cells of the monocyte/macrophage lineage (for review, see Refs. 1 and 2). By binding to class I1 MHC antigens expressed by antigenpresenting cells, CD4 provides essential accessory signals for T helper cell stimulation through the T cell receptor-CD3 complex (see Ref. 3); furthermore, CD4 serves as a receptor for the human immunodeficiency virus (HIV) (4). Different experimental approaches have addressed CD4 expression regulation. The constitutive internalization ofCD4by T cells was shown to be very low (5); however, following treatment with phorbol esters (6-9) or ligation of the T cell receptorCD3 complex by specificantigens (10) or antibodies (11, 12), CD4 surface expression decreased substantially. Down-modulation of surface CD4 is thought to reflect its internalization in an intracellular compartment.Evidence has been presented that thecytoplasmic tail of CD4is essential for its endocytosis (8,9,13,14),and residues of the cytoplasmic domain, essential for phorbol 12-myristate 13-acetate (PMA)-induced endocytosis, were recently identified (9). In the lastfew years, it has been appreciated that sialyated glycosphingolipids (gangliosides) also reduceCD4 surface expression on T cells from different species (15); both monosialo- (GMl) and disialoganglioside,as well as some ganglioside derivatives (16), appear to be effective. It was also demonstratedthat ganglioside-induced CD4 down-modulation is accompanied by inhibition of HIV infectivity (17-19). It is definitively intriguing that these simple constituents of eukaryotic cell membrane cause so selective a modulation of CD4 expression on T cells, as revealed by the absence of any effect on several other surface markers’ (15). The mechanism of action of gangliosides as modulators of CD4 expression has remained elusive. Although it was suggested that gangliosides induce CD4 internalizationin an endocytic compartment (20), it is not clear how they exert their effect. This studyaddressed the mechanisms of ganglioside action and demonstrates that the monosialoganglioside GM, masks epitopes located within or around its first and second NH,-terminal domain. We also show that GM1 causes the dissociation of ~ 5 6 ’ ‘tyrosine ~ kinase and rapid CD4 delivery to a compartment where it undergoes degradation. These GM1effects were clearly independent of a possible The abbreviations used are: MHC, major histocompatibility complex; HIV, human immunodeficiency virus; PMA, phorbol 12-myristate 13-acetate; GMl, IPNeuAc-GgOse4Cer;mAb; monoclonal antibody; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate. *D. Saggioro, C. Sorio, F. Calderazzo, L. Callegaro, M. Panozzo, G. Berton, and L. Chieco-Bianchi, unpublished observation.

1368

Mechanism of Action of

GM1 as Modulator

of CD4 Expression

1369

protein kinase C activation, since staurosporine, a protein OKT4 or B9.12.1 (IgG2a anti-class I MHC, kindly provided by Dr. kinase C inhibitor, blocked PMA, but neither GMl-induced R. Accolla, Institute of Immunological Sciences, Verona, Italy) mAbs fluids. After 2 h of incubation the lysates were centrifuged at down-modulation of CD4 expression, nor dissociation of ~ 5 6 ' ' ~ascitic 12,000 X g for 10 min and the supernatants transferred to tubes from CD4, and CD4 degradation. On the basisof these find- containing 5 pl of 10% S. aureus in lysis buffer. The incubation was ings, a mechanism of CD4 down-modulationmay be envisaged prolonged for afurther 45 min, and the immunocomplexes were based on ligation, and possible induction of conformational collected by centrifugation for 1 min at 12,000 X g. The pellets were changes, of CD4 and independentof phosphorylation of crit- then washed twice in RIPA buffer (25 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% sodium dodecyl ical intracytoplasmic residues. MATERIALSANDMETHODS

Cell Lines and Treatment of Cells with MonosialogangliosideMOLT-3 lymphoma cells, kindly provided by Dr. R. Gallo (NIH), were cultured inRPMI 1640 medium (Flow Laboratories, Irvine, Scotland) supplemented with 10% fetal calf serum (Flow) and 2% Lglutamine. HeLa cells transfected with a CD4-expressing construct (HeLa CD4')' were a gift of Dr. G. Lederkremer (University of Tel Aviv, Israel), whereas HeLa cells transfected with a deleted CD4 gene lacking the sequence coding for the cytoplasmic domain (8, HeLa CD4 cyt-) were kindly provided by Dr. P. Clapham (Institute for Cancer Research, Chester Beatty Laboratory, London, United Kingdom). HeLa cells were grown in Dulbecco's modified Eagle's medium (Flow) supplemented with 10% fetal calf serum and 2% L-glutamine; the cells were detached from the culture plates by incubation for 10 min at 37 "C in phosphate-buffered saline (PBS) containing 5 mM EDTA. Monosialoganglioside ( G M l ) was kindly provided by FIDIA Research Laboratories (Abano Terme, Padua, Italy). MOLT-3 and HeLa cells were washed with PBS and incubated at a concentration ~ and time of incubation varied of 1 X 10' cells/ml in PBS; G Mdosage in different experiments and are specified under "Results." After incubation with G M , , the cells were washed twice with PBS and then analyzed for CD4 expression or ~ 5 6 ' kinase '~ activity, as detailed in the following sections. Antibodies-Fluorescein isothiocyanate (F1TC)-conjugated antiT4 (DAKO T4) and phycoerythrin-conjugated Dako T4 were purchased from Dakopatts (Glostrup, Denmark); unconjugated OKT4 and OKT4A, B, D, E, F were kindly provided by Dr. G. De Chirico (Ortho Pharmaceutical Division, Raritan, NJ). As second antibody for indirect immunofluorescence staining, FITC-conjugated rabbit anti-mouse immunoglobulins (Dakopatts) were used. Immunofluorescence-Surface expression of CD4was detected either by direct or indirect immunofluorescence assays. For direct immunofluorescence, the cells were stained for 30 min at 4 "C with the DAKO T4 mAb. For indirect immunofluorescence, the cells were incubated for 30 min at 4 'C with unconjugated anti-CD4 mAbs, washed with PBS, and incubated under the same conditions with FITC-conjugated rabbit anti-mouse Ig. Dual fluorescence was performed by staining the cells with OKT4 mAb plus FITC-conjugated rabbit anti-mouseIg and thenwith phycoerythrin-conjugated DAKO T4. Cells were analyzed for fluorescence intensity after excitation with a 448-nm argon ion laser, using an Epics C or Epics Elite flow cytometer (Coulter Electronics, Hialeah, FL). Vital cells were gated on the basis of forward angle light scatter and 90" light scatter parameters. 5,000 cells were counted for every histogram to evaluate the percentage of CD4-positive cells. Cell Labeling-Labeling of MOLT-3 cells was performed in methionine-free RPMI 1640 medium supplemented with 5% dialyzed fetal calf serum. After 1 h of preincubation, 50-100 pCi of [35S]methionine (cell labeling grade, Amersham International plc, Amersham, U. K.; 10 mCi/ml), were added to 1 ml of medium containing 10 X 10' cells and the incubation prolonged for further 6 h. The cells were then washed twice with PBS, resuspended at 1 X 106/mlin complete RPMI 1640 without serum, and dispensed in 24-well plates. Cells were incubated for various time periods (indicated under "Results") in the absence or presence of G M I . At the end of the incubation cells were collected and washed once with ice-cold P B S 1 X 10' cells were then lysed in 250 p1 of 25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 100 p M sodium orthovanadate, 1 mM EDTA, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 p M leupeptin, 10 p M pepstatin, 1 mM dithiothreitol (lysis buffer). The lysates were kept for 10 min at 4 "C in a rotating wheel and then centrifuged at 12,000 X g for 10 min; the supernatants were rapidly frozen in liquid nitrogen and stored at -70 "C until use. Immunoprecipitation-Lysates were thawed and placed in an Eppendorf tube containing 75 pl of pelleted 10% Staphylococcus aureus (Calbiochem) in lysis buffer. After 30 min of incubation in a rotating wheel, the lysates were centrifuged for 5 min at 12,000 X g, and the supernatants were transferred to Eppendorf tubes containing 2 p1 of

sulfate (SDS), 1 mM dithiothreitol, 100 p~ sodium orthovanadate), and once with Opper 1 (25 mM Tris, pH 7.5, 1 M NaC1,0.1% Nonidet P-40), Opper 2 (25 mM Tris, pH 7.5,200 mM NaC1,l mM EDTA, 1% Nonidet P-40, 0.1% SDS), and Opper 3 (25 mM Tris, pH 7.5, 0.1% Nonidet P-40). The pellets were resuspended in 25 p1 of SDS-polyacrylamide gel electrophoresis sample buffer, incubated at 95 'C for 3 min, and the supernatantswere subjected to a 10% SDS-polyacrylamide gel electrophoresis. Fluorography was performed according to Laskey and Mills (21) using XAR film (Eastman Kodak) at -70 "C. In Vitro Kinase Assays-Ten X IO6cells/ml were lysedas described above. Supernatants were incubated with 2 pl of OKT4 or B9.12.1 mAbs ascitic fluids or 2 pl of anti-p56Ickserum (kindly provided by Immunocomplexes were Dr. S. Fisher, INSERM,Paris,France). collected as described, washed three times in RIPA buffer, once in TBS (25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM dithiothreitol, 100 p~ sodium orthovanadate), and finally suspended in 20 pl of kinase buffer (22) with or without 5 pg of acid-denaturated enolase. Samples were incubated for 5 min at 4 "C and then for 10 min at 30 "C to favor initially ~ 5 6 ' 'autophosphorylation ~ and then phosphorylation of the exogenous substrate e n ~ l a s eThe . ~ reaction was stopped, and the samples were analyzed as described previously (22). RESULTS

GMl Modulation of CD4 Antigen Expression on MOLT-3 Lymphoma Cells and CD4-transfected HeLa Cells-Studies demonstrating that GM1 caused a selective down-modulation of CD4 expression did not clarify whether this action was a consequence of direct interactionof GM1 with CD4. To address this issue we investigated the effect of GM1 on the bindingof mAbs that react with different epitopes of the extracellular domain of CD4, i.e. DAKO T4 and OKT4mAb which recognize epitopes proximal to the two extremes of the extracellular portion of CD4. Following incubation with 100 pg/ml GM1 for 30 min at 37 "C, the epitopelocalized at the NH2-terminal domain, and recognized by DAKO T4 mAb, was no longer detectable on MOLT-3 cells, as well as on HeLa CD4' or HeLa CD4 cytcells; on the contrary, OKT4 mAb binding was unaffected (Fig. 1). This finding showed that GM1 acts by masking a region of CD4 close totheNH2-terminaldomainthatis recognized by DAKO T4 and analogous (Leu3a and OKT4A, not shown) mAbs. However, MOLT-3 and HeLa CD4' cells were not stained by OKT4 mAb if incubation with GM1 was prolonged up t o 4 h; significantly, HeLa CD4 cyt- cells were still bound by the OKT4 mAb even after this time. These observations suggest that following prolonged incubation with GMI, a true CD4 internalization occurs in cells that express an intactCD4 molecule but not incells expressing a molecule lacking the bulk of the cytoplasmic tail. The masking effect of GM, on DAKO T4 binding was, as described previously (15-17), dose-dependent. 30 pg/ml inhibited DAKO T4 binding t o MOLT-3 and CD4-transfected HeLa cells by 70-80% (not shown). Since the masking effects of GMl were shown to be selectivealso at higher concentrations (15-17), we performed the investigations described below at the maximally inhibitory concentrationsof 100 pg/ml. The kinetics of GM1 masking of the DAKO T4 epitope in MOLT-3 cells is shown in Fig. 2. Inhibition of DAKO T4 binding was evident already after 5 min of incubation with G M at ~ 100 pg/ml; it increased prolonging the time of incubation and was complete at 30 min (Fig.2A). As expected, S. Courtneidge, personal communication.

Mechanism of Action of GM,as Modulator of CD4 Expression

1370

MOLT-3 cells were still bound by the OKT4 mAb up to 30 min of treatment with GM1 (Fig. 2B). In another experiment, MOLT-3 cells were treated for 30 min at 37 “C with 100 pg/ml GM1 and thenmonitored by dual fluorescence using the OKT4 mAb plus FITC-conjugated anti-mouse Ig and phycoerythrin-labeled DAKO T4 mAb. Untreated cells (Fig. 3A) were double-positive for both mAbs, D M 0 T4

OKT4

100 90 80

70

MOLT-3

60

50 40

30 20 10 0

3

W V W

g 0

a

.+

0

0 L

0 bp

1 DO 90

80

70 HcLo CD4+

60 50 40

30 20 10 0 100

HeLa CD4 cyl40

30 20 10

0 CONTROL CUI 100 ug/rnl 30 rnin m CUI 100 ug/rnl 4 h

FIG.1. Differential effect of GMl on Dako T4 andOKT4 mAb binding. MOLT-3, HeLa CD4+ and HeLa CD4 cyt- cells were incubated at 37 “C for 30 min or 4 h with 100 pg/ml GM,.CD4 expression was monitored using the DAKO T4 or the OKT4 mAb, as described under “Materials and Methods.”

whereas GMl-treated cells (Fig. 3B) were stained only by OKT4 mAb. These findings further support that one of the effects of GM1‘ on CD4 antigen is to mask epitopes localized at itsNHz-terminal domain. To define more precisely the epitopes of CD4 masked by GM1,we used mAbs directed against distinct epitopes of the molecule (Figs. 4 and 5). Following incubation of MOLT-3 cells for 30 min at 37 “C with 100 pg/ml GMl, binding of mAbs mapping either within or around the first NHz-terminal domain (OKT4A, D, E, F) or below the second NH2-terminal domain (OKT4B) did not occur; again, the cells were OKT4positive. GMl also inhibited the binding (not shown) of SIM.4 mAb, which cross-reacts with the Leu3a/OKT4A epitope*; SIM.2 mAb, which recognizes a distinct epitope but blocks HIV-induced syncytium formation4; and B66.6,mAb which should bind to themore distal portionof the CD4 extracellular domain (28). These results demonstrate that GMl masks a large part of the extracellular portion of CD4 and leaves the domain that is closer to the plasma membrane, where the epitope recognized by OKT4 mAb is located, free to interact with OKT4 mAb. GMlModulation of the CD4 Antigen Occurs through Mechanisms Independent of Protein Kinase C Activation-In view of the above findings and the fact that phorbol esters induce internalization of CD4 in an endocytic compartment (9), we investigated whether the effects of a prolonged treatment with GMl were possiblycaused by protein kinase C activation. As shown in Fig. 6, PMA induced down-modulation of CD4 on MOLT-3 cells, and staurosporine, a widely used inhibitor of protein kinase C activity, totally blocked its effects. In contrast, the capability ofGM1 to modulate CD4 expression on MOLT-3 cells was not influenced by staurosporine, strongly suggesting that its effect is independent of protein kinase C activity. Other evidence that GMl effects are independent of protein kinase C activation will be described in the following section (see Fig. 10). GMl Effect on CD4 Antigen Expression on MOLT-3 Cells Pretreated with Anti-CD4 rnAbs-As the above findings distinctly implied that GMl acts, at least in part, by interacting with the CD4 antigen, we reasoned that preincubation with anti-CD4 mAbs should prevent modulation of CD4 antigen expression. Indeed, preincubation of MOLT-3 cells with OKT4A mAb prevented GM1-inducedCD4 modulation after both 30 min and 4 h ofGMl treatment as revealed by detection of preE. K. Hildreth, personal communication.

OKTL

OAK0 14

FIG.2. Kinetics of DAKO T4 epitope masking by G M ~MOLT-3 . cells were incubated at 37 “C for the times indicated with 100 pg/mi Gm. CD4 expression was monitored using DAKO T4 or OKT4 mAb, as described under “Materials and Methods.”

.I 1001 FLUORESCENCE

INTENSITY

1 100

(log)

10

Mechanism of Action of GMlas Modulator of CD4 Expression

1371

FIG. 3. Selective masking of the DAKO T4 epitope by GM,. MOLT-3 cells were incubated at 37 "C for 30 min without (panel A ) or with100 pg/ml GMI (panel B ) . At the end of the incubation, cells were stained with OKT4 mAb plus Ig FITC-conjugatedrabbitanti-mouse and then counterstained with phycoerythrin-conjugated DAKO T4 mAb. Cells wereanalyzed by cytofluorometryfor green (x axis) and red ( y axis) fluorescence. .l

OKTt

+

a - m o u s e lg FlTC

by the finding that binding of the FITC-labeled DAKO T4 mAb was inhibited; as shown in Figs. 1-3 this mAb is a very sensitive marker of the G M l masking effect on CD4 antigen. These observations suggest that G M l masks a quite discrete OKT4 D region of CD4 andthen induces conformationalchanges OKT4 E which favor displacement of antibodies from their binding OKT4 A site. DAKO-TI As the portion of CD4 masked by GMl is clearly distinct from the epitope recognized by OKT4 mAb (Figs. 1-3 and 51, we thought that preincubation of MOLT-3 cells with OKT4 OKT4 F would not inhibit CD4 modulation, revealed as the disappearance of the OKT4 epitope. Unexpectedly, however, preincubation with OKT4 blocked GMl capacity to induce the loss of the detectability of OKT4 epitope which occurs after 4 h of OKT4 B treatment. In this case as well, the fact that PMA was still able to induce CD4 modulation indicated that OKT4 binding does not hamper antigen internalization. Sincewe could not exclude that OKT4 binding blocked the interaction of G M , with the NH,-terminal part of CD4, we investigated G M 1 binding on the basis of its capacity to compete with DAKO T4 mAb. Even in OKT4-preincubated MOLT-3 cells, treatment withG M , prevented DAKO T4 binding, thus confirming that G,, had interacted with CD4 (Table 11). Induction of the CD4 Antigen Degradation and Dissociation of ~ 5 6 ' 'from ~ CD4 by GMl-To obtain direct proof that a prolonged treatment with GMl induces delivery of CD4 in an endocytic compartment and its eventual degradation we analyzed the pool of immunoprecipitable CD4 after G M l treatment using two approaches (Fig. 7 ) . CD4 was immunoprecipitated from lysates of [35S]methionine-labeled MOLT-3 cells after treatment with 100 pg/ml GMl for various time intervals. Immunoprecipitation of total cell lysates showed that CD4 was degraded about 35% in 30 COOH min, 60% in 1 h, and almost totally in 2-4 h (Fig. 7 A ) . CD4 FIG. 4. Schematic representation of the CD4 molecule and degradation was also evident as a decrease in p56lCkdetectathe putative regionsrecognized by the mAbs used in this study bility in anti-CD4 immunoprecipitates.Indeed, 30 minof GMl treatment causeda 65% decrease inautophosphorylated ( s e e Refs. 23-27). p56'", as revealed by in uitro kinase assaysperformed on antibound OKT4A mAb by FITC-conjugated anti-mouse Ig; sig- CD4 immunoprecipitates (Fig. 7 8 ) . After this time, modulanificantly, PMA-induced CD4 down-modulation was not af- tion of CD4 expression on MOLT-3cells was only partial (see of the CD4 molecule assayed fected, thus indicating that the binding of mAbs that react Figs. 1,2, and 5), and degradation with theNH2-terminaldomain of CD4 does nothamper inparallel experiments (Fig. 7 A ) was about 35%. In three antigen internalization (Table I). independent experiments, after treatment with 100jtg/ml G M l We then investigated whether other mAbs reacting with for 30 min CD4 was degraded by 37.6 f 7.0%, butthe epitopes close to, or distinct from, the OKT4A epitopewere autophosphorylated ~ 5 6 ' "in~ anti-CD4immunoprecipitates also able to preventG M 1 effects. We found that OKT4B and decreased by 69.3 & 6.1%. These observations indicated that E were less efficient than OKT4A in preventingGM, effects. dissociation of ~ 5 6 " ' from CD4 precedes internalization and degradation of the CD4 antigen; however, as shown in Fig. G M , treatment decreased the detection of OKT4B prebound to MOLT-3 cells by about 30% and that of OKT4E by about 7 B , after 1-2 h of G M l treatmentit was not possible to 70% (Table I). T h a t GM, stillinteractedwith CD4 after distinguish between the two. To individuate the time interval ~ is clearly distinct from CD4 inpreincubation with OKT4B or E was demonstrated indirectly in which ~ 5 6 ' 'dissociation

I 1

1372

[2:

W

m

E

1)

z

J J

W

0

FLUORESCENCE

INTENSITY

(log)

FIG. 5. Effect of GMl on the binding of mAbs recognizing different CD4 epitopes. MOLT-3 cells were incubated at 37 "C for 30 min with 100 pg/ml GMI.At the end of the incubation CD4 expression was monitored using the indicated mAb plus FITC-conjugated rabbit anti-mouse Ig. Empty area, GMl-treatedcells; dashed area, control cells.

-

CD4 by the OKT4 mAb (not shown). Fig. 9 shows that GMl treatment does not affect expression 90" W or activityof ~ 5 6 ' 'but ~ only the detectabilityof its activity in u 80" anti-CD4 immunoprecipitates. In fact, after1 or 4 h of treatw 2 70" ment with GMl, kinase activity inanti-p561ckimmunoprecipit ln 60" tates was unchanged, but in anti-CD4 immunoprecipitates it 50.was strongly reduced. 40While performing experiments of immunoprecipitation of 30the [35S]methionine-labeled CD4 we also investigated whether G 0 20" CD4 was possibly shed in the supernatantas reported previ8 10" ously (29). No [35S]methionine-labeled CD4 was detected in nthe supernatant of GMl-treated MOLT3 cells (not shown), Control GM1 100 ug/rnl PMA 100 ng/rnl FIG. 6. Effect of staurosporine on GMl capacity to induce thus indicating that CD4 shedding does not account for the CD4 modulation. MOLT-3 cells were incubated at 37 "C for 4 h GM1-induced CD4 modulation at the GM1 concentrations we with 100 pg/mI Gul or 100 ng/ml PMA in the presence (black columns) used. or the absence (white columns) of 50 nM staurosporine. A t the end of We also studied whether GM1-induced degradation of the the incubation CD4 expressionwas monitored using DAKOT4 mAb. CD4 molecule was because of a possible activation of protein kinase C. The data obtained by immunoprecipitating CD4 from [35S]methionine-labeled MOLT-3 cells confirmed the ternalization and degradation, we used the information obtained in the experiment reported in Fig. 2 which showed that results reported inFig. 6, ie. that inhibitorsof protein kinase GM1 masking of CD4 occurs in a few minutes. We found that C activity did not affect modulationof CD4 by GMl but only dissociation of ~ 5 6 from " ~ CD4 following G M treatment ~ is by PMA (data not shown). The GM1-induced dissociation of very rapid; in fact a clear dissociation is observed after 5-10 p56ICkfrom CD4 was also unaffected by staurosporine (Fig. min of treatment, a time at which CD4 expression,as revealed 10). As shown in Fig. 10, both GM1 and PMA induced dissoby OKT4 binding, is unchanged (Fig. 8). To exclude that ciation of p56Ickfrom CD4. As shown in previous studies (30), residual GM1 might have affected immunoprecipitation of the in PMA-treated cells the residual ~ 5 6 ' 'associated ~ with CD4 CD4 antigen from lysates of GMl-treated cells, we also per- migrated also as a form of higher molecular weight becauseof formed immunoprecipitation experiments by adding a n excess increased phosphorylation on serine and threonine residues of GM1 to lysatesof untreated cells; addition of GMl up to 100 (see Ref. 31). The form of p56ICkwith a retarded gel mobility pg/ml to cell lysates did not inhibit immunoprecipitation of was not observed after treatment with G M ~Moreover, . stau-

Y J

100"

T

Mechanism of Action of Gnnl as Modulator of CD4 Expression

1373

TABLEI Effect of preincubation with anti-CD4 mAbs on G,, activity MOLT-3 cells (1 X 106/ml) were preincubated with the OKT4A, OKT4B, and OKT4E mAbs and then treated with GM1 (100 pg/ml) or PMA (100 ng/ml) at 37 “C.At the end of treatments, the cells were stained either with FITC-conjugated anti-mouse Ig orDAKO T4 mAb. Each step was followed by four washes in PBS to remove the excess mAb or GM1. The percent of CD4-positive cells is reported asthe mean f S.D.of three or four experiments. mAb

None

one

Primary

OKT4A OKT4A OKT4A OKT4A

OKT4B OKT4B OKT4B OKT4B

one

one

OKT4E OKT4E OKT4E OKT4E

one

Treatment

None GMI(100cLg/ml) GMI(100 ag/ml) GMI(100 pglml) PMA ng/ml) (100

OKT4B None GMI (100 d m l ) GMI(100rg/ml) G M (100 ~ rglrnl)

OKT4E None GMI(100 rg/ml) GMI(100 d m l ) GMI(100 d m31.8 l)

Time

Secondary mAb

4h

OKT4A + a-mouse Ig-FITC + a-mouse Ig-FITC a-Mouse Ig-FITC a-Mouse Ig-FITC Ig-FITC a-Mouse Ig-FITC a-Mouse

OKT4B min 30 Ig-FITC min 30

Ig-FITC + a-mouse + a-mouse Ig-FITC a-Mouse a-Mouse Ig-FITC

min OKT4A 30 30 min

4h

30 min 11.8 OKT4E min 30 Ig-FITC 30 min

T4 DAKO Ig-FITC + a-mouse + a-mouse Ig-FITC a-Mouse a-Mouse Ig-FITC

T4 DAKO 30 min

CD4’ cells 56

97.0 & 1.3 5.4 f 6.2 98.9 f 1.6 94.9 f 7.4 86.3 f 2.4 13.1 f 17.2 96.0 f 2.7 0.5 f 0.1 96.8 64.2 f 6.8 98.9 f 3.3 96.9 & 2.4 0.3 f 0.1 90.1 27.1 f 4.3 98.9 f 0.7

TABLEI1 Effect of preincubation withOKT4 mAb on GM1 activity MOLT-3 cells (1 X 106/ml)were preincubated with the OKT4 mAb and then treated with GMl (100 pg/ml) or PMA (100 ng/ml) at 37 “C. At the end of treatments (30 min or 4 h), the cells were stained either with FITC-conjugated anti-mouseIg or DAKO T4 mAb. Each step was followed by four washes in PBS to remove the excess mAbs or G M ~The . percent of CD4 positive cells is reported asthe mean rt S.D. of three or four experiments. mAb

Primary

Treatment

Time

30 min 4h

30 min 4h 4h DAKO T4 min30

mAb

Secondary

OKT4 + a-mouse Ig-FITC OKT4 + a-mouse Ig-FITC OKT4 + a-mouse Ig-FITC a-Mouse Ig-FITC @-Mouse Ig-FITC a-Mouse Ig-FITC a-Mouse Ig-FITC DAKO T4

CD4+ cells

56

97.9 & 1.2 83.6 f 9.8 11.9 f 3.0 98.3 f 0.2 97.7 _t 0.9 88.5 & 7.6 8.7 98.2 rt 0.2 6.1 rt 5.5

rosporineinhibitedsubstantiallythe dissociation of ~ 5 6 ’ “ ~CD4 antigendetection by several non-cross-reactingantifrom CD4 induced by PMA but had no effect on the G M 1 - CD4 mAbs. Their conclusion that G M l simply modulates CD4 expression was also supported by the use of experimental induced dissociation. conditions, i.e. time of incubation with G,, of at least 1 h, DISCUSSION which cause internalization of CD4. Our study shows that G,, is indeed able t o mask epitopes Our findings demonstrate thatG M l has a dual effect on the CD4 antigen. G M 1 masksepitopes recognized by different that are recognized by different mAbs and scattered along a mAbs and located in the portion of the molecule including large portion of the CD4 antigen which includes the first and 30 min of G M 1 second NH2-terminal domains. In fact, within the first and the second NH2-terminaldomainsandalso triggers the generation of protein kinase C-independent sig- treatment, the binding of OKT4 mAb, which recognizes an epitope located proximally to the transmembrane region of nals which cause dissociation of p56lCkfrom CD4 and the CD4, was only partially inhibited, whereas mAbs that react delivery of CD4 to a compartment where itiseventually with the distal portion of the CD4 extracellular region were degraded. These results reconcile previous observations that inhibited by 90% or more. G M I inhibited the bindingof mAbs described G M l as a “modulator” of the CD4 antigen expression reacting with the “class I1 MHC” binding portion of CD4, but suggested different mechanisms of action. such as OKT4B, and those reacting with the “HIV-1 gp120” Offner et al. (15) first demonstrated that G M l selectively binding portion, such asOKT4A, D, E, F, B66.6, Leu3a (25). modulates CD4 expression on T cells from different species Several other observations support theconclusion that G M 1 (15).They also obtained strong evidence that modulation of masks the more distal portion of the extracellular region of CD4 antigen expression was a consequence of masking by CD4. G M l of discrete CD4 epitopes; in fact, preincubation of cells G M l effect was also evident in HeLa cells transfected with with mAbs reacting with the NH2-terminal portion of CD4 a CD4 molecule lacking the bulkof the cytoplasmic domain. prevented G M 1 effects (15).However, they concluded that G M l It was shown that this domain is essential for PMA-triggered masking of different epitopes was unlikely as G M l inhibited CD4 internalization (8, 9, 13), and later studies identified

Mechanism of Action of

1374

GMI

as Modulator of CD4 Expression

100

A

anti-lck

OKT4

8.9.12.1

80

f, 3

40

m 0

G

-

GM-1 Hwrs

+

M

-

I

+

+

-

+

+

-

+

+

-

Hours4 1 4 4 1 4 4 1 4 FIG.9. GMl decreases the amount of p56”’ associated with CD4 but not total ~ 5 6 ’ “activity. ‘ MOLT-3 cells were incubated ~ the times in the absence or the presence of 100 pg/ml G M for indicated. Cell lysates were immunoprecipitated with B9.12.1 (anticlass I MHC antigen), OKT4 (anti-CD4)mAbs, or rabbit anti-p56Ick serum, and in vitro kinase assays were performed as described under “Materials andMethods.” Immunoprecipitates obtained with control rabbit serum did not show the presence of any kinase activity (not shown). A representative of three experiments is shown.

+ - + - +

1 0.5 1 2 2 4 4

@L ” \

no*r

GM-1

-

t

+

+

+

Hours

4

0.5

1

2

4

FIG.I. GMl induces degradation of CD4 and dissociation of p56”‘ from CD4. Panel A, MOLT-3 cells were labeled with [3sS] methionine and chased for the times indicated, in the absence or the . were immunoprecipitated with presence of 100 pg/ml G M ~Lysates anti-CD4 (OKT4) or anti-class I MHC (B9.12.1, first lane from the right) mAbs. The upperpart of the figure shows CD4 band intensities measured and calculated by densitometry. Panel B, MOLT-3 cells were incubated as in panel A, lysed, and immunoprecipitated with OKT4 orB9.12.1 mAbs. In vitro kinase assays on immunoprecipitates were performed as described under “Materials and Methods.” Immunoprecipitates obtained with B9.12.1 mAb did not show the presence of any kinase activity (see Fig. 9). One representative of three experiments is shown. 100

-

m

1 $

z

80 60 40

20 0

GM-1 Min.

-

+

+

+

5

10

15

+

30 FIG.8. Induction of ~56’”’ dissociation from CD4 by GMl. MOLT-3 cells were treated with 100 pg/ml G M for ~ the times indicated, and in vitro kinase assays were performed on anti-CD4 immunoprecipitates. CD4 expression was assayed by OKT4 mAb staining and is reported in the upper part of the figure. A representative of two experiments is shown.

30

cytoplasmic domain residueswhich areimportantinthis process (9). Our findings indicate that GM1 did not induce CD4 internalization in HeLa CD4 cyt- cells but masked the NH2-terminal portion of the molecule. This masking effect can explain &,-induced modulation of a glycosylphosphatidylinositol-anchored CD4/Thy-l chimeric molecule by GMl (31) without hypothesizing its internalization. In fact, this

FIG. 10. Staurosporine inhibits PMA-induced but not G M I induced dissociation of p56“’ from CD4. MOLT-3 cells were incubated with 100 ng/ml PMA or 100 pg/ml G M , for 60 or 30 min, respectively, and in the absence or thepresence of 50 nM staurosporine. CD4 was immunoprecipitated from cell lysates with the OKT4 mAb, and in vitro kinase assays were performed on the immunoprecipitates, in the absence of enolase, as described under “Materials and Methods.” The lane marked withan asterisk shows in vitro kinase assays on control (B9.12.1 mAb, anti-class I MHC antigens) immunoprecipitates. A representative of two experiments isshown.

included the first two CD4 domains and the Thy-1 domain. As we showed that GMl masks CD4 epitopesuptothat recognized by OKT4B mAb, which maps justbelow the second NHp-terminal domain, inhibition of anti-Thy-1 mAb binding may also be explained byhampered recognition of the appropriate epitope or by conformational changes in thechimeric molecule also involving the Thy-1domain. When cells were prebound with OKT4A mAb, which recognizes an epitope located in the first NH2-terminaldomain of CD4 (23-27), GMl could not displace the mAb from its binding site and trigger the series of events that eventually leads to CD4 internalization. Theeffects of cell preincubation with OKT4A were selective for GM1; in fact, PMA was still able to induce CD4 down-modulation in cells prebound with OKT4A. Significantly, OKT4B and E were less effective in preventing GMl effects since FITC-conjugated anti-mouse Ig staining was decreased. This observation suggests that GMl displaced OKT4B and E from their binding sites. It may be significant that prebound OKT4E was displaced more than OKT4B, in fact, the epitope recognized by OKT4E is closer to the OKT4A epitope(23-27), i.e. to a putative binding site for GMl. Taken together, these observations suggest that GM1 masks a discrete CD4 region located within or near epitope the recognized by OKT4A mAb and inducesaconformational change in the CD4 molecule that involves the first NH2terminal domains and prevents the binding of mAbs directed against epitopes located in this region. That the first action ofGMl is to interact with a discrete CD4 region was also indicated by studies inwhich a synthetic peptide derived from

Mechanism of Action of Gnnl as ModulatorCD4 of

Expression

1375

the principal neutralizing domain of HIV-1 was able to inhibit nificance in the modulation of T lymphocytes responses deserves further investigation. GMl effects (32). The possible effect of gangliosides in regulating CD4 Although the thesis that GMl acts by inducing conformational changes in theCD4 moleculeis attractive, other expla- expression and T cell responses in uiuo remains to be deternations may be advanced. Indeed, GM1 might cover the first mined, especially in light of the evidence that serum albumin two NH2-terminal domains uniformly, and the different ex- can block GMl effects (17). However, it may be significant tent of displacement of various prebound mAbs might be that the amount of sialic acid on the cell surface of myeloid because of their intrinsic avidity. Alternatively, initial G M ~cells was recently shown to be regulated by a mobilization of interaction with a discrete CD4 region, which appears to be sialidases from an intracellular compartment (34). Previous located within or around that recognized by OKT4A mAb, studies showed that only sialyated gangliosides are able to might favor the assembly of GM1 micelles which eventually modulate CD4 expression (16). Could desialyation of gangliosides by secreted sialidase, or redistribution of gangliosides cover a large part of CD4. GMl capacity to induce CD4 modulation was also affected on the surface of antigen-presenting cells, regulate T cell by ligation of the domain proximal to the plasma membrane activation? by OKT4 mAb. To cells that were prebound with OKT4 mAb, Acknowledgments-Wethank Patricia Segato for assistance in GM1 could inhibit DAKO T4 mAb binding; however, after 4 h of incubation, a time which causes CD4 internalization, the preparing this manuscriptand Dr. Carlo Chizzolini of the Fidia Research Laboratoriesfor the critical reading of the manuscript. prebound OKT4 mAb was still present on the cell surface. REFERENCES Again, the effect was selective, as PMA induced internaliza1. Dalgleish, A. G. (1986) Immunol. Today 7,142-144 tion of the prebound OKT4 mAb. This experimental obser2. Parnes, J. R. (1989) Ado. Immunol. 44,265-311 vation also suggests that changes in CD4 conformation may 3. Travers, P., and Zamoyska, R. (1992) Current Biol. 2,38-40 4. Sattentau, Q. J., and Weiss, R. A. (1988) CeU 62,631-633 underlie GM1 effects, and mAb binding may prevent or inhibit 5. Pelchen-Matthews,A., Armes, J. E.,Griftiths, G., and Marsh, M. (1991) J. conformational changes, either directly or through formation Exp. Med. 173,575-587 6. Hoxie, J. A,, Rackowski, J. L., Haggarty, B. S., and Gaulton, G. N. (1988) of CD4 aggregates. J. Immunol. 1 4 0 , 786-795 The results presented in thispaper demonstrate that after 7. Acres, R. B., Conlon, P. J., Mochizuki, D. Y., and Gallis, B. (1986) J. Biol. Chem. 2 6 2 , 16210-16214 GM1 masks CD4 epitopes located within the first two NH28. Maddon, P. J., McDougal, J. S., Clapham, P. R., Dalgleish, A. G., Jamal, terminal domains, it induces CD4 internalization in an enS., Weiss, R. A., and Axel, R. (1988) Cell 64,865-874 docytic compartment where it is eventually degraded. Staining 9. Shin, J., Dunbrack, R. L., Jr., Lee, S., and Strominger, J. L. (1991) J. Biol. Chem. 266,10658-10665 with anti-CD4 antibody of saponin-permeabilized cells dis- 10. Weyand, C. M., Goronzy, J., and Fathman, C. G. (1987) J. Immunol. 1 3 8 , closed newlyinternalized CD4 in (&-treated cells (20). That 1351-1354 M. L., Hafler, D. A., Craig, K. A,, Levine, H., and Schlossman, S. F. cycloheximide (20), as well as sodium azide (15), inhibited 11. Blue, (1987) J. Immunol. 139,3949-3954 CD4 antigen re-expression indirectly indicated that following 12. Blue, M. L., Daley, J. F., Levine, H., Branton, K. R., Jr., and Schlossman, S. F. (1989) J. +aunol. 374-380 GMl treatment internalized CD4 was degraded. We observed 13. Bedinger, P., Monarty, A., von Boestel, R. C., Donovan, N. J., Steimer, K. that degradation of the internalized CD4 was, at least in S., and Littman,D. R. (1988) Nature 334,162-165 14. Sleckman, B. P., Peterson, A., Foran, J. A., Gorga, J. C., Kara, C. J., MOLT-3 cells, relatively rapid; some degradation was evident Strominger, J. L., Burakoff, S. J.,and Greenstein, J. L. (1988) J . after 30 min ofGM1 treatment, and it was almost complete Immunol. 141,49-54 H., Thieme, T., and Vandenbark, A.A. (1987) J. Immunol. 1 3 9 , after 1-2 h. A comparison of internalized CD4 degradation, 15. Offner, 3295-3305 and ~ 5 6 association "~ with CD4, suggested that ~ 5 6 ' disso'~ Martino, 16. Grassi, F., Lopalco, L., Lanza, P., Ciccomascolo, F., Cazzola, F., Di A., Kirschner, G., Callegaro, L., Chieco-Bianchi, L., and Siccardi, A. G. ciation from CD4 precedes CD4 degradation (Fig. 7); short (1990) Eur. J. Immunol. 20,145-150 term GM1 treatment of MOLT-3 cells clearly showed that 17. Chieco-Bianchi, L., Calabrb, M. L., Panozzo M. De Rossi A., Amadori, A,, Callegaro, L., and Siccardi, A. (1989) A h 3,501-50; dissociation of ~ 5 6 ' precedes "~ CD4 internalization and deg- 18. Nakakuma, H., Kawaguchi, T., Koito, A,, Hattori, T., Kagimoto, T., and radation (Fig. 8). It was advanced that p56Ickassociation with Takatsuki, K. (1989) J n J Cancer Res. 80,702-705 A., Hoshino, H., Rakajima, K., Adachi, M., Ikeda, K., Achiwa, K., CD4 might be responsible for the maintenance of the molecule 19. Handa, Itoh, T., and Suzuki, Y. (1991) Biochem. Biophys. Res. Commun. 1 7 6 , 1-9 at the plasma membrane level and its very low extent of chi T.,Nakakuma, H., Ka 'moto, T., Shirono, K., Horikawa, K., constitutive internalization in T cells (5). PMA-triggered 20. Kawa Hi&a, h., Iwamori, M., Nagai,$., and Takatsuki, K. (1989) Biochem. Biophys. Res. Conmun. 168,1050-1059 delivery of the CD4 antigen to anendocytic compartment was 21. Laske R A , and Mills, A.D. (1975) Eur. J. B k h e m . 66 335-341 also explained on the basis of its capacity to induce p56Ick 22. Poli, E:, Sori0 C., and Berton, G. (1991) J. Cell Sci. 100,633-840 23. Sattentau, Q. j . , Dalgleish, A. G., Weiss, R. A,, and Beverley, P. C. (1986) dissociation from CD4 (30). Our results would be consistent Science 234,1120-1123 with these observations; however, GMl also induces CD4 mod- 24. Mizukami, T., Fuerst, T. R., Berger, E. A,, and Moss, B. (1988) Proc. Natl. Acad. Sci. U. S. A. 86,9273-9277 ulation in the myeloid cell line U9375 and HeLa CD4+ cells D. Capon D. J. Karp D. R. Gregory, T., Long, E. O., and (Fig. 1). Moreover, CD4 immunoprecipitation from U937 25. Lamarre, Sekal , R 'P (1989) EMEO J. 8,3271->277 cells5 (33) did not reveal the presence of any kinase activity 26. Kieber-gmmons, T., Jameson B. A,, and Morrow, W. J. W. (1989) Biochim. Biophys. Acta 989,281-306 in the immunoprecipitate. Detailed studies demonstrated re- 27. Merkenschlager, M., Buck, D.,Beverley, P. C. L., and Sattentau, Q. J. (1990) J. Immunol. 146,2839-2845 cently that CD4 cytoplasmic tail residues required for PMAS., Moretta, A., Pantaleo, G., Tambussi, G., Mer, P., Perussia, B., induced internalization might indeed be distinct from those 28. Carrel, and Cerottini,J. C. (1988) Eur. J . Immunol. 18,333-339 involved in ~ 5 6 ' binding '~ (9). Therefore, the rapid dissocia- 29. Morrison, W. J., Offner, H., and Vandenbark, A.A. (1991) Immunopharmocology 2 2 , 77-84 tion of p56Ickfrom CD4, induced by GMI, might not be causally 30. Hurley, T. R., Luo, K., and Sefton,B. M. (1989) Science 246,407-410 31. Jasin, M., Pagpe, K: A., Littman, D. R. (1991) J. Virol. 66,440-444 related to endocytosis andeventual CD4 degradation but 32. De Rossi, A., astl, M., Mammano, F., Panozzo, M., Dettin, M., Di Bello, could represent an additional GMl effect whose possible sigC., and Chieco-Biancbi, L. (1991) Virolo 1 8 4 , 187-196 .E

C. Sorio and G. Berton, unpublished observation.

33. Rudd, C. E., Trevillyan, J. M., Dasgupta, PD., Wong, L. L., and Schlossman, S. F. (1988) Proc. Natl. Acad. Sci. U. S. A. 86,5190-5194 34. Cross, A. S., and Wright, D. G. (1991) J. Clin. Inuest. 88,2067-2076