Eur. J. Immunol. 1992. 22: 447-455

Wil A. M. Loenen., Evert De Vries., Loes A. Gravestein., Rogier Q. HintzenA, Rene A. W.Van Lier. and Jannie Borst. Division of Immunology, The Netherlands Cancer Institute. and Central Laboratory of the Netherlands Red Cross Blood Transfusion ServiceA, Amsterdam

CD27: biosynthesis and generation of a soluble form

The CD27 membrane receptor, a lymphocytespecific member of the nerve growth factor receptor family, gives rise to a soluble form by protein processing that does not involve receptor endocytosis* CD27 is a transmembrane glycoprotein found exclusively on human T and B lymphocytes. It belongs to a recently identified receptor family, whose members are involved in cell differentiation and survival. This family includes the nerve growth factor receptor, two different types of tumor necrosis factor, receptors the Fas antigen, and the B cell-specific protein CD40.Tcell activation via the antigen receptor strongly enhances CD27 membrane expression, suggesting a role for CD27 duringTcel1 differentiation. A soluble form of CD27 (sCD27) is released into the supernatant of activated Tcells, and detected in serum and urine of healthy individuals and patients. We have investigated the mechanism underlying the generation of sCD27. One mRNA encodes both the transmembrane receptor and sCD27, as shown by cDNA transfection. In line with this, only one CD27 precursor protein is found, that is processed to the mature receptor by extensive 0-linked glycosylation. All newly synthesized protein is rapidly transported to the plasma membrane; no internal pool of mature protein is detectable. The transmembrane form gives rise to sCD27 after arrival at the cell surface, most likely via a proteolytic event, that does not involve receptor internalization.

1 Introduction CD27 is a membrane glycoprotein, selectively expressed on mature thymocytes, the majority of peripheral blood T cells [l-31 and a small proportion of B cells [4].Tcell activation, by binding of ligand to the Tcell receptorKD3 complex, enhances CD27 membrane expression about fivefold [2,3, 51.This enhancement is transient, reaching a maximum 3 to 4 days after activation, and is regulated at the mRNA level [6]. On thymocytes, CD27 expression is regulated in a similar manner [7]. The function of CD27 is unknown. Since the T cell activation-related increase in protein expression occurs relatively late after antigen recognition. CD27 is not likely to contribute to the initial interaction of restingT cells with antigen-presenting cells. Rather, it may be important in the events following antigen recognition. On resting T cells, CD27 is a disulfide-linked homodimer with subunits of 50-55 kDa (p55) [3,5, 81. Sequence analysis of a cDNA clone encoding p55 [9, 101 has identified CD27 as a member of a novel family of cysteine-rich receptors [ l l ] , including the nerve growth factor receptor (NGF-R; [12, 13]), two TNF-R [14-171, the Fas antigen [18], the Bcell antigen CD40 [19], the rat Tcell antigen

[I 99721

*

This work was supported by the Netherlands Organization for Scientific Research (NWO), grant 900-509-128to W.A.M.L. and grant H93-155 to J.B. and E. d.V

Correspondence: Jannie Borst, Division of Immunology. The Netherlands Cancer Institute, Plesmanlaan 121, NL-1066 CX Amsterdam, The Netherlands Abbreviations: Endo-F growth factor s: soluble

447

Endoglycosidase-F

NGF

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1992

Nerve

OX40 [20] and the murine Tcell antigen 4-1BB [21]. Members of the NGF-R family regulate cell differentiation and survival [18, 22-24]. The extracellular region of CD27 can be divided into two domains: the “Cys” domain and the “stalk” domain. The “Cys” domain contains a large number of Cys residues, occurring in a repetitive pattern, that is shared among the members of this receptor family. Such domains probably have a globular conformation, due to extensive intramolecular disulfide bonding, and are involved in ligand binding, as shown for the NGF-R [25]. The membrane-proximal “stalk” domain is rich in Ser,Thr and Pro residues, and most likely forms an extended structure. This domain is present in some, but not all family members. Such regions are found in many receptors and are often 0-glycosylated [26]. A soluble form of CD27 (sCD27) has recently been detected in the supernatant of activated Tcells, as well as in serum and urine of healthy individuals and patients. This sCD27 has an M, of 28-32 kDa and is structurally related to the p55 transmembrane form [27]. Also, NGF-R [28, 291, TNF-RI, and TNF-RII [17,30-351 can occur in truncated, soluble forms that can be purified from serum and urine. Soluble forms have been described for many IL receptors, such as IL 2R [36], IL 4R [37],IL 6R [38] and IL 7R [3Y]. Many soluble receptors retain their ligand binding site, and thus may prevent ligands from reaching their cell surface receptors. In this way, they can regulate cellular responses. Little is known about the way in which soluble receptors are generated. Soluble IL 4R [37] and IL 7R [39] are encoded by separate mRNA species. However, often, only one mRNA is present. It has been postulated, that soluble receptors can be derived from the transmembrane forms by 0014-2980/92/0202-0447$3.50+ .25/0

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W. A. M. Loenen, E. DeVries, L. A. Gravestein et a1

proteolysis [40]. Here, we have addressed the relationship between the transmembrane and sCD27 proteins. The CD27 cDNA directs the synthesis of a 31-kDa precursor, that is 0-glycosylated to give rise to the p55 membrane receptor. Our studies indicate that sCD27 is generated from this pS5 form by proteolysis, that takes place after cell surface expression and does not require receptor internalization.

Eur. J. Immunol. 1992. 22: 447-455 3.3 x lo4 cells/cm2 (33% confluence) on day0 in DMEM + 10% FCS; on day 1, cells were washed three times with DMEM, and transfected with the cDNA constructs using the lipofectin method [43], with modifications suggested by the supplier (BRL, Breda,The Netherlands); a mixture of 1-1.5 pg DNA in 10 pl 10 mM TrisHC1, pH 8.0, 1 mM EDTA/10 pl lipofectin was added per 106cells and 1 ml DMEM. After incubation for 5 h at 37 "C, 5 ml DMEM 20% FCS/106cells was added and cells were cultured for 24 h. Next, cells were split as needed.

+

2 Materials and methods 2.5 I n vitro transcriptiotdtranslation 2.1 Isolation and activation of Tcells PBMC were isolated from buffy coats derived from healthy volunteer donors by density centrifugation on Ficoll (Nycomed, Oslo, Norway). For activation, they were cultured at 2 x lo6 cells/ml in culture medium for 3 or 4 days, in the presence of 0.4 pglml PHA (Wellcome Foundation Ltd., Dartford, GB).

2.2 mAb and immunofluorescence mAb used for immunofluorescence and ELISA were: CLB-CD27/1 (clone 9F4; IgGz,), CLB-CD2712 (clone 3A12; IgGz,), CLB-CD27/3 (clone 2E4; IgG2,) and antiIL 2R mAb CLB-IL2R/1 (IgGZb) [3]. CLB-CD2711 was routinely used for immunoprecipitation. Indirect immunofluorescence was performed according to standard procedures using FITC-conjugated goat anti-mouse F(ab')z Ig (Tago, Burlingame, CA) for staining and a FACScan (Becton Dickinson, Mountain View, CA) for analysis. 2.3 ELISA sCD27 was measured by ELISA, according to Hintzen et al. [27]. Plates were coated with CLB-CD27/3 mAb, nonspecific binding sites were blocked with normal goat serum, and wells were washed and incubated with sample dilutions. After washing, biotinylated CLB-CD27/1 mAb was added. Next, wells were washed, avidin-peroxidase (Sigma Chemical Co., St. Louis, MO) was added, followed, after washing, by 3,3',5,5'-tetramethylbenzidine(Merck, Darmstadt, FRG) and 0.06% hydrogen peroxide dissolved in 0.1 M sodium acetate buffer, pH 5.5. The peroxidase reaction was stopped by adding 1M H2S04 and the absorbance at 450 nm was determined with a Titertek ELISA reader (Flow Laboratories, Rockville, MD).

2.4 cDNA constructs and transfection The CD27 cDNA was isolated from a plasmid library constructed from PHA-activated PBMC, as described [9]. It has been cloned in the CDM8 vector [41]; CDM8 contains the SV40 origin of replication, allowing a very high copy number during transient transfection assays in COS7 cells. The cDNA is expressed from the cytomegalovirus promoter in vivo, but can also be transcribed in vitro using the bacteriophage T7 promoter. DNA manipulations were carried out according to standard procedures, as described in Sambrook et al. [42]. COS7 cells were seeded at

In vitro transcription was carried out according to the protocol of Melton et al. [44],with modifications suggested by the suppliers (Promega Corporation, Madison, WI). Briefly, 2 pg plasmid DNA, linearized with Bam HI, was transcribed with 40 U T7 RNA polymerase in the presence of 100 U RNasin ribonuclease inhibitor in a 100-p1volume. After phenol extraction and ethanol precipitation, 0.3 pg RNA was translated in a reticulocyte lysate [4S] in a total volume of 25 pl, in the presence of 2.5 pCi [3sS]Cys.Samples were prepared in parallel in the presence or absence of canine pancreas microsomes [46].

2.6 Radiolabeling For cell surface iodination, SO x lo6-100 X lo6 activated Tcells were washed and resuspended in PBS and labeled with 1-2 mCi NalZ5I (Amersham Co., Amersham, GB) using lactoperoxidase as a catalyst. For metabolic pulsechase labeling, activated T cells or transfected COS7 cells were washed in PBS, resuspended at 5 X lo6 cells/ml (Tcells) or 2 x 106 cells/ml (COS7 cells) in L-Met-free RPMI 1640 medium (Selectamine kit, Gibco, Grand Island, NY), supplemented with 10% dialyzed FCS, L-G~u, and pyruvate, and preincubated for 3 h at 37°C in a 5% COz atmosphere. Subsequently, cells were resuspended at 50 X 106/ml(Tcells) or 2 x 106/ml(COS7 cells) and SO pCi [35S]Metand 50 pCi [3sS]Cys(Amersham Co.) were added per 10 x lo6 T cells or 2 x lo6 COS7 cells. After a defined pulse time, ten volumes Iscove's medium were added containing 0.15 mg/ml unlabeled L-Met and L-CYS,and 5% FCS. After defined chase periods, cell samples (10 x lo6 Tcells, 2 x lo6 COS7 cells) were resuspended in ice cold PBS, harvested and lysed in immuno-precipitation buffer (IPB). For overnight labeling, cells were washed, starved for Met and Cys for 1 h, resuspended at 5 X lo6 cells/ml for activated Tcells or 2 x lo6 cells/ml for COS7 cells in Met/Cys-free medium, radioactivity was added as above, and cells were cultured for 14 h at 37"C, 5% COz. COS7 cells were used for pulse-chase experiments 60 h after transfection and for overnight labeling 48 h after transfection.

2.7 Immunoprecipitation and gel electrophoresis After labeling, cells were lysed in IPB, consisting of 0.01 M triethanolamine-HC1, pH 7.8, 0.15 M NaCl, 5 mM EDTA, 1 mM PMSF, 0.02 mg/ml ovomucoid trypsin inhibitor, 1 mM Na-p-tosyl-L-lysine chloromethyl ketone, 0.02 mg/ml leupeptin, 1% NP40. Cell supernatants were collected after

labeling, protease inhibitors were added, and centrifugation took place for 30 min at 100000 x g. Immunoprecipitation from lysates and supernatants was carried out as described [8].For SDS-PAGE, 10%-15% polyacrylamide gradient gels were used, according to a modification of the Laemmli procedure. Samples were analyzed under reducing conditions (5% 2-ME in SDS-sample buffer). Gels with ?S-labeled material were treated with 1 M sodium salicylate, pH 5.4, prior to autoradiography, that took place at -70"C, using Kodak XAR-5 film in combination with intensifier screens (Cronex, Dupont Chem. Co., Newtown, CT) .

A

(a1

Mr

B

C

D

E

F

G

H

I

J

K

L

20097-

68-

D 43-

29

-

18

-

14

-

2.8 Deglycosylation procedures Chase:

Immunoprecipitates were suspended in 50 pl buffer, containing 0.05 M sodium acetate, pH 5.5, 0.9% NaCl, 0.1% CaC12, and protease inhibitors, and incubated with 0.1 U neuraminidase type VIII (Sigma) per sample, for 3 h at 37°C. Intact cells were suspended at 10 x 1O6/100 pl PBS, 1 mM CaC12, and incubated with 0.5 U neuraminidase type V (Sigma) per sample for 30 min on ice. Cells were washed twice in PBS, 20% FCS and lysed in IPB, supplemented with 100 pg/ml fetuin [47]. Treatment of immunoprecipitates with 0-glycanase (0-glycopeptide-endo-Dgalactosyl-N-acetyl-a-galactosaminohydrolase; Genzyme, Boston, MA) was preceded by neuraminidase treatment as outlined above. Next, immunoprecipitates were washed and resuspended in 100 pl 100 mM potassium phosphate buffer, pH 6.0, containing protease inhibitors, 0-glycanase was added at 0.04 mU/sample and incubation took place for 20 h at 37 "C. For endoglycosidase-F (Endo-F) treatment, immunoprecipitates were resuspended in 20 pl 0.5 M sodium phosphate buffer, pH 5.0, 0.2% SDS and boiled for 5 min. Next, proteins were reduced and alkylated by incubation with 10 mM dithiothreitol for 30 min at 45°C and 20 mM iodoacetamide for 20 min at room temperature. NP40 was added to 1% and EDTA to 20mM. Protease inhibitors were also added. Incubation took place for 18 h in the presence of 10 mU Endo-F (Boehringer Mannheim GmbH, Mannheim, FRG). For SDS-PAGE following Endo-F digestion, sample buffer was added directly followed by electrophoresis. For inhibition of 0-linked glycosylation, activated T cells were cultured overnight in RPMI 1640 medium, containing 10% of the prescribed concentration of glucose. Next, they were labeled for 4 h with [3s5SIMet and [3sS]Cys at 5 x loh cells/ml in glucose-poor RPMI 1640 medium lacking L-Met and L-Cys, in the presence of 1 mg/ml phenyl N-acetyl a-D-galactosaminide [48] (Sigma).

449

CD27: biosynthesis and generation of a soluble form

Eur. J. Immunol. 1992. 22: 447-455

-

(bl Mr

0

A

lh

- -

2h 4h 85h 2Oh Cells

B

C

0

lh

2h 4h SUP

-

85h 20h

Pulse: l h

D

97-

68

-

D

43

-

29

-

1814-

-

Cells

-- Sup

Figure 1 . (a) Kinetics of release of sCD27 into the supernatant of activated T cells. Activated T cells were pulse-labeled with L["sS]Met and -Cys for 1 h . Samples wcrc taken from cells (lanes A-F) and supernatants (lanes G-L) directly after the pulse (lanes A, G), and at chase times of 1 h (lanes B, H), 2 h (lanes C. I), 4 h (IanesD, J), 8.5 h (lanesE,K)and20 h(lanesF,L).Thisfigureand all following show SDS-PAGE of anti-CD27 immunoprecipitates under reducing conditions. (b) sCD27 is released into the supernatant of COS7 cells transfected with CD27 cDNA. COS7 cells were transfected with the cDNA encoding the CD27 membrane form (p55) and labeled overnight with and -Cys 2 days later. CD27 was recovered from the cell lysate (lane B) and supernatant (lane D). Lanes A and C show immunoprecipitates made with normal mouse Ig (control).

met

3 Results 3.1 CD27 cDNA directs the synthesis of both membrane and soluble protein forms

2 h of biosynthesis (lane H) and accumulated in the supernatant during the following hours (lanes I-L).

Fig. 1a illustrates the occurrence of sCD27 in the supernatant of activated T cells. PBMC were cultured with PHA for 3 days and pulse labeled for 1 h. Subsequently, radioactivity was chased for various time periods and CD27 was isolated from cell lysates and supernatants. Lanes A-D show the p55 CD27 membrane form (open arrow). sCD27, migrating at 31-33 kDa, (closed arrow) was detected after

Since CD27 is encoded by a single-copy gene [9], and only one CD27 mRNA is found in activated Tcells [6], it was considered likely that the transmembrane and soluble receptor forms are generated from a common precursor protein.Therefore, it was tested whether the CD27 cDNA. previously shown to encode the cell surface receptor [9], directs synthesis of sCD27. COS7 cells were transiently

450

Eur. J. Immunol. 1992. 22: 447-455

W. A. M. Loenen, E. DeVries, L. A. Gravestein et al.

Table 1. sCD27 in the supernatant of COS7 cells Supernatant

sCD27 (units/ml)a)

COS7/CD27 COS7 Activated T

c 10

75

100

a) sCD27 release was assayed 2 days after transfection with the CD27 cDNA by ELISA.

were compared in size with the CD27 proteins synthesized in vivo in COS7 cells, transfected with the CD27 cDNA, and in activated Tcells. After a 45 rnin pulse, a protein of 31 kDa (closed arrow) was observed in COS7 cells (lane C) and activated Tcells (lane D), that had the same M, as the in vitro processed product (lane B). After a 2-h chase, the 31-kDa protein had almost disappeared, while p55 was prominent (lanes E , F; open arrow). (The mature CD27 (a)

A

C

B

D

E

F

G

H

0

15’

15. lh

I

J

2h

4h

Mr

transfected with the CD27 cDNA. Two days later, CD27 expression was detected at the cell surface by immunofluorescence, while a sample of the culture medium gave a positive signal in the sCD27 ELISA [27]. As a positive control, sCD27 was detected in the supernatant of activated Tcells (Table 1). In addition, COS7 cells, transfected with the CD27 cDNA, were labeled biosynthetically and CD27 was immunoprecipitated from both cells and supernatant. The CD27 membrane form was present in the cell lysate (Fig. l b , laneB, open arrow), as expected. From the supernatant (lane D, closed arrow), a protein of 31-33 kDa was recovered, consistent with the identity of sCD27 found in the supernatant of activated Tcells (Fig. 1a). Thus, the cDNA that encodes the cell surface receptor can also give rise to a soluble CD27 species. 3.2 The CD27 transmembrane form is generated from a 31-kDa precursor The transfection experiments indicated that the transmembrane and soluble forms of CD27 arise from a common precursor protein. We have studied the biosynthesis of CD27, in order to determine whether sCD27 is derived from the mature CD27 membrane form or its precursor. Activated Tcells were pulse labeled for either 2, 5 or 15 min. After a pulse of 2 rnin (Fig. 2 a , lane A) or 5 min (lane B), a 31-kDa protein (closed arrow) was isolated with anti-CD27 mAb. After a 15-min pulse, in addition, small amounts of the p55 CD27 form were recovered (lane C; open arrow). Upon a 3-h chase, the 31-kDa form was virtually absent and p55 was predominant. These data suggest that the 31-kDa protein is a CD27 precursor, that is processed to p55 between 5 and 15 min after synthesis. To demonstrate that the 31-kDa protein is a precursor of p55, COS7 cells were transfected with the CD27 cDNA and pulse labeled. As in activated T cells, after a 15-min pulse (Fig. 2 a, lane F) the 31-kDa protein was predominant, while a small amount of p55 could be detected. During the next 4 h of biosynthesis, the 31-kDa species gradually disappeared, while p55 became prominent (lanes F-J).

Zn vitro transcription/translation of the CD27 cDNA indicated that the 31-kDa molecule is indeed the initial CD27 translation product. A protein of 30 kDa was synthesized (Fig. 2 b, lane A), in agreement with the cDNA sequence, that predicts an open reading frame for 260 amino acids. Processing of the 30-kDa protein in vitro by dog pancreas microsomes, led to a shift to 31 kDa (lane B), consistent with removal of the signal peptide and addition of one N-linked sugar onThr5’ in the “Cys”domain.The p55 CD27 protein was not observed. The in vitro translation products

- 6 8 -

U

Pulse : Chase:

-

2’ 0

97

-

68

-

B

43

-

-

29

-

14

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15‘ 3h

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T

A

(b) Mr

5’ 15. 0 0

-

C

D

cos

L

E

-

F

d 43-

29-

18

-

-

-

+ IVT

C

--

T C T Pulse Chase +

Figure 2. (a) Biosynthesis of CD27 in activated Tcells and transfected COS7 cells. Activated Tcells (T) were pulse labeled with and -Cys for 2 min (lane A), 5 rnin (lane B), 15 rnin (lane C), or pulse labeled for 15 min and chased for 3 h (lane D). COS7 cells (COS), transfected with the CD27 cDNA were pulse labeled with met and -Cys for 15 min (lane F) and chased for 15 min (lane G), 1 h (lane H), 2 h (lane I), or 4 h (lane J). Lane E shows a control immunoprecipitate. (b) The p55 CD27 molecule is derived from a 31-kDa precursor. CD27 was synthesized by in vitro transcriptionltranslation (IVT) in the absence (-, lane A) or presence (+, lane B) of dog pancreas microsomes, or in vivo, in transfected COS7 cells (C, lanes C, E ) or activated Tcells (T, lanes D, F). COS7 cells and Tcells were pulse labeled with and -Cys for 45 min and samples were taken directly (lanes C, D), or after a 2-h chase period (lanes E, F). In vitro translation products were analyzed directly and in vivo translation products after immunoprecipitation.

met

met

Eur. J. Immunol. 1992. 22: 447-455

CD27: biosynthesis and generation of a soluble form

membrane protein of COS7 cells has a slightly lower mobility in SDS-PAGE than that of Tcells, which is probably due to differences in glycosylation). These experiments confirm that the 31-kDa molecule is the precursor of the p55 CD27 form. 3.3 Processing of the 31-kDa CD27 precursor includes addition of 0-linked carbohydrate The CD27 cDNA sequence predicts one attachment site for N-linked carbohydrate, on the “Cys” domain. The “stalk” domain contains a large number of Ser and Thr residues, that may be subject to 0-linked glycosylation.This has been investigated, in order to establish the biosynthetic relationship between the 31-kDa precursor and p5S. Activated T cells were pulse labeled for 15 min and radioactivity was chased for 3 h. CD27, isolated from cell samples taken at both time points of biosynthesis,was either treated with neuraminidase, that removes sialic acid residues, or with Endo-F, that removes all N-linked carbohydrate. The 31-kDa precursor, predominant directly after the pulse (Fig. 3 a, lane A, closed arrow), was resistant to neuraminidase (lane C), while Endo-F digestion caused a small shift in M, to 30 kDa (lane D). p55, predominant after the 3-h chase (lane B, open arrow), was sensitive to neuraminidase (lane E) and shifted slightly in M, after Endo-F digestion (lane F).This experiment indicates that the 31-kDa precursor and p55 CD27 carry the same amount of N-linked carbohydrate, consistent with cotranslational addition of this type of sugar moiety. However, only p5S carries sialic acids, that are not solely attached to complex N-linked carbohydrate, since removal of sialic acids (lane E) gave a larger shift in M, than removal of N-linked carbohydrate (lane F). Therefore, p55, but not the 31-kDa precursor must carry 0-linked carbohydrate. This was confirmed by 0-glycanase treatment. Activated Tcells were pulse labeled for 15 min and radioactivity was chased for 30 min. CD27 was isolated and treated with a (b) Mr (

a

)

A

B

C

D

E

A

B

451

combination of neuraminidase and 0-glycanase, to give optimal digestion (Fig. 3 b, lane C), or mock incubated (lane B). Removal of both sialic acids and 0-linked carbohydrate from p55 led to a shift in M, to 33-35 kDa, while neuraminidase treatment alone gave a shift to 36-37 kDa (Fig. 3 a , lane E). The 31-kDa precursor was clearly resistant to enzyme treatment, as predicted. Thus, conversion of the 31-kDa precursor to pSS primarily entails addition of 0-linked sugars. This was confirmed with the use of phenyl-N-acetyl-a-D-galactosaminide, which inhibits 0-glycosylation by acting as an intracellular acceptor for 0-linked carbohydrate groups [48] (results not shown). 3.4 The transmembrane form gives rise to sCD27 after arrival at the cell surface, by an event that does not involve receptor internalization Peptide mapping had indicated that sCD27 and p55 are structurally related, but not identical [27]. IEF analysis demonstrated that p55 and sCD27 carry similar amounts of 0-linked carbohydrate (results not shown). Treatment of purified sCD27 with neuraminidase/O-glycanase reduced its M, from 33 kDa to 20 kDa (Fig. 4), while the same treatment of p55 resulted in an M, of about 35 kDa (Fig. 3 b). These data suggest that sCD27 is derived from the membrane receptor by a proteolytic clip in the “stalk” domain at a site beyond the region carrying 0-linked sugars. The observed 15-kDa difference in M, between the deglycosylated membrane receptor and sCD27 is consistent with this idea. The time lapse between p.55 synthesis and cell surface expression was determined. Activated T cells were pulse labeled, and prior to lysis, treated with neuraminidase. In this way, CD27 that had arrived at the plasma membrane was desialated, while intracellular CD27 was protected. After a 15-min pulse, the 31-kDa precursor (closed arrow) and a small amount of p5S (open arrow) were present (Fig. 5 a, lane A). The amount of pSS increased during the chase period (lanes B, C). The 31-kDa precursor was

C

F 200-

Mr

97-

68-

68-

D 43

-

43

Q

-

b

29

-

18-

14

4

29-

18

-

14

-

-

--

N

E

N

E

-

0

Figure3. (a) The pS5 CD27 form, but not its 31-kDa precursor, is sensitive to neuraminidase. Activated Tcells were pulse-labeled for 15 min and cell samples were taken directly (lanes A, C, D), or after a 3-h chase period (lanes B, E, F). CD27 was recovered by immunoprecipitation and analyzed directly (-, lanes A, B), or after digestion with neuraminidase (N, lanes C, E), or Endo-F (E, lanes (D, F). (b) The p55 CD27 form, but not its 31-kDa precursor, is sensitive to 0-glycanase. Activated Tcells were pulse-labeled for 15 min and chased for 30 min. CD27 was isolated by immunoprecipitation and subjected to neuraminidase digestion, followed by treatment with 0-glycanase (0).Lane A: control immunoprecipitate. Lane B: CD27 untreated. Lane C: CD27 after deglycosylation.

452

W. A. M. Loenen, E. De Vries, L. A. Gravestein et al.

A

B

Eur. J. Immunol. 1992. 22: 447-4.55 (a)

C

A

Mr

Mr

B

C

D

E

F

G

200-

68-

97-

68-

43

-

29

-

43-

29

-

18-

18

-

14

-

Figure 4 . sCD27 contains 0linked sugars and has a backbone of 20 kDa. i251-labeled

14-

-

sCD27, purified from activated T cell supernatant [27], analyzed by SDS-PAGE without treatment (-, lane A), and NK)

after treatment with neuraminidase (N, lane B), or a combination of neuraminidase and 0-glycanase (N/O, lane C).

I bl M,

The transmembrane receptor might be subject t o proteolysis by a cell surface-associated protease, o r processing may occur in an endosomal compartment after receptor internalization. It was investigated whether endocytosis is required for generation of sCD27. Activated Tcells were surface iodinated at 0°C and incubated for 4 h , at either 4"C, 12°C o r 37°C. Next, cells and supernatants were harvested. Receptor internalization, that might have taken place during this time period, was monitored by neuraminidase treatment of intact cells. Internalized molecules would be resistant t o the enzyme. CD27 molecules were isolated from cells (Fig. 5 b, lanes A-F) and supernatants (lanes G-I) and analyzed by SDS-PAGE.The p55 form was fully sensitive to neuraminidase, also after incubation of the cells for 4 h at 37"C, indicating that no significant receptor endocytosis had taken place in this time period (or that all internalized species were rapidly broken down). As a control, internalization of the transferrin receptor was monitored. After 4 h of incubation at 37 "C, about half of the transferrin receptor molecules had become resistant to neuraminidase, while they remained sensitive after incubation at 12°C and 4°C (results not shown). Despite inhibition of all endocytotic activity at 12 "C and 4 "C, sCD27 was produced in significant amounts (lanes G, H), suggesting that endocytosis is not required for the generation of sCD27. Next, cells were iodinated at 0 "C and incubated at 37 "C for 2 h in the presence of chloroquine, that neutralizes acidic

B

C

14

37'

- + + +

D

E

F

G

H

I

NaNa J

K

L

M

N

O

P

O

97 68 -

200

43-

29

resistant t o neuraminidase, as expected, but p55 was completely sensitive (lanes D-F), even after only 15 min of labeling (lane D). Apparently, p55 arrives at the cell surface within 15 min of synthesis. Since there is no evidence for proteolysis in this and other pulse-chase experiments, proteolytic processing of p55 t o yield sCD27 probably does not take place during biosynthesis o r intracellular transport, but after the receptor has reached the cell surface.

A

-

-

1814-

40

--

cells

&

- - -

8 120

370

40

+ + -

SUP

---

120 370

-

-

-

cells

_-

- + + -

sup

- + + - + - + -

-

+

cq +Nab

Figure5. (a) CD27 arrives at the cell surface within 1.5min of

biosynthesis. Activated T cells were pulse-labeled with [3sS]Met and -Cys for 1.5min. Cell samples were taken directly after the pulse (lane A, D), after 15 min (lanes B, E), and after 30 min (lanes C, F) chase time. Intact cells were mock incubated (-, lanes A-C) or treated with neuraminidase (NaNa) at 0°C ( f,lanes D-F). Cells were lysed and CD27 was recovered by immunoprecipitation.Lane G shows a control precipitate. (b) Generation of sCD27 does not require receptor endocytosis.For the experiment analyzed in lanes A-I, activated Tcells were surface iodinated at 0°C and incubated for 4 h at either 4 "C (lanes A, D, G), 12°C (lanes B, E, H) or 37 "C (lanes C, F, I). Next, cells and supernatants were harvested and intact cells were mock incubated (lanes A, B, C), or treated with neuraminidase (NaNa, lanes D, E, F). CD27 was isolated from cell lysates (lanes A-F) and supernatants (lanes G-I) and analyzed by SDS-PAGE. For the experiment analyzed in lanes J-Q, cells were surface iodinated at 0°C and incubated for 2 h at 37"C, in the absence (lanes J, K , N, 0) or presence (lanes L, M, P, Q) of chloroquine (Cq). Next, cells were mock incubated (lanes J, L, N, P) or treated with neuraminidase (lanes K , M, 0, Q) and cultured for another 2 h at 37°C. Cells (lanes J-M) and supernatants (lanes N-Q) were harvested, CD27 was isolated and analyzed.

endosomal compartments. Receptor internalization was monitored by neuraminidase treatment of cells after the 2-h incubation period. Cells were cultured for another 2 h without chloroquine at 37 "C t o allow reappearance of internalized CD27 molecules at the cell surface and/or release into the supernatant. CD27 was isolated from lysates (Fig. 5 b, lanes J-M) and supernatants (lanes N-Q) and analyzed by SDS-PAGE. Neutralization of endosomes did not affect the generation of sCD27 (lanes P, Q).

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Moreover, neuraminidase treatment of intact cells, after the 2-h internalization period, also resulted in desialylation of sCD27 (lanes 0 , Q), indicating that sCD27 is not derived from a pool of internalized p55 molecules.

4 Discussion We have demonstrated, that the CD27 cDNA encodes both transmembrane and soluble receptor forms. Thus, these proteins share a common mRNA and probably a common protein precursor. This agrees with our inability to identify more than one mRNA species in activated Tcells. The transfection experiments also exclude that sCD27 is derived from a membrane form with a glycosylphosphatidylinositol lipid anchor that can be released by phospholipases [49]. The CD27 cDNA does not encode the conserved amino acid sequence necessary for addition of such an anchor and yet gives rise to the soluble form. Recently, it has been shown that the cDNA encoding other members of the NGF-R family, TNF-RI and TNF-RII, can also direct synthesis of a transmembrane receptor and a soluble form [17, 351. Perhaps protein processing is a common mechanism for the generation of soluble forms in this receptor family. A soluble receptor form could be generated by (a) differential processing of a common precursor protein leading to both cell surface and soluble receptor, (b) intracellular cleavage of the transmembrane form, or (c) a proteolytic clip of the mature receptor at the cell surface, as proposed f0re.g. soluble IL 2R [4O],TNF-R [14,15,35], NGF-R [29] and MEL-14 [SO].We had considered the possibility that the sCD27 was generated intracellularly from the same precursor as the membrane receptor. A protein of 31 kDa, i.e. similar in size to sCD27, was observed very early in biosynthesis [S]. I n this report, it has been established that this early 31-kDa protein is distinct from sCD27. It is the precursor of pSS, that is converted to the mature form within minutes after biosynthesis. This was demonstrated by in vitvo transcription/translation of the CD27 cDNA, which yielded the 31-kDa protein, but not pSS. Also, inhibition of 0-linked glycosylation during cell culture allowed synthesis of the 31-kDa precursor protein, but blocked appearance of pSS. Thus, it was clear that the 31-kDa form was not sCD27 on its way to the cell surface. The other two models imply cleavage of pS5, either intracellularly or after appearance at the cell surface. pSS and sCD27 are structurally highly related as demonstrated by peptide mapping [27] and IEEThey also contain similar amounts of 0-linked carbohydrate. This indicates that sCD27 is generated from the mature receptor and contains most or all of the pSS extracellular domain.The 20-kDa M, of the sCD27 backbone agrees with proteolysis of the mature receptor at a site close to the transmembrane segment. We have addressed the question of whether cleavage takes place before or after the receptor has reached the cell surface. sCD27 was not detected intracellularly in pulsechase experiments, indicating that sCD27 is not generated in the biosynthetic pathway. Moreover, neuraminidase protection experiments showed that all of the pS5 protein is transported to the cell surface immediately after its maturation. Apparently, pSS is not retained in an intracellular

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compartment, that would allow processing to sCD27. The remaining option is that sCD27 is derived from the membrane receptor after it has reached the cell surface. This might involve proteolytic processing in endosomes. However, this is contradicted by three observations. First, sCD27 was detected in the supernatant immediately after cell labeling at 0°C. Second, inhibition of endocytosis at low temperatures did not affect the appearance of sCD27. Third, neutralization of endosomes with chloroquine, or NHdC1 (not shown) had no effect on the generation of sCD27. If endosomal activity does not play a role, the protease involved could either be membrane bound or be present in the extracellular milieu. Previously, we had observed an alternative protein form of CD27 at the surface of activated Tcells [8]. It had an M, of 28-32 kDa (p32) and was highly related to p55 in the extracellular domain. p32 and pSS contained a similar amount of sialic acids and had a similar basic PI after neuraminidase treatment. In short, p32 had the same structural properties as sCD27. We now postulate that p32 and sCD27 are identical. The presence of sCD27 at the cell surface might be explained by the fact that the pS5 chains form a homodimer. If only one chain of the dimer is cleaved to yield sCD27, it remains attached to its transmembrane partner chain and, thus, stays associated with the plasma membrane. By which mechanism does sCD27 arise? A protease capable of releasing a soluble CD27 form is present not only in activated Tcells, but also in C0S7 cells, that do not normally express CD27. Similar results were obtained for TNF-RI [3S] and TNF-RII [17]. Either one specific protease, that has a wide tissue distribution, is responsible for generation of sCD27, or the nature of this receptor is such that it is readily prone t o proteolytic degradation by a variety of enzymes. The extended conformation of the protein due to the high proline content and extensive 0-glycosylation [26] might contribute to such susceptibility. The natural occurrence of three different truncated forms of the NGF-R [29] argues in favor of multiple proteasesensitive sites in the receptor. In the case of CD27, we have also noticed two additional truncated forms in minor amounts (results not shown). The nature of the protease(s) involved in the cleavage of CD27 remains to be established. The Tcell activation molecule CD26 is a transmembrane protease, but its specificity as a dipeptidyl aminopeptidase IV [Sl] is not in line with a role in cleavage of CD27. We have not been able to inhibit generation of sCD27 with a cocktail of protease inhibitors, that readily blocked endosoma1 degradation of the MHC class I1 invariant chain (Neefjes et al., unpublished). Proteases involved in the release of soluble receptors could either be constitutively expressed or induced. If they are induced, a signal could be relayed after ligand binding to the receptor itself, or after signaling via a different receptor. In the case of the TNF-R, various stimuli, including activators of PKC, rapidly induce release of sTNF-R [S2]. Also, shedding of MEL-14 is induced by PKC activation [SO]. Tcell activation via the TcR/CD3 complex leads to a strong increase in CD27 membrane expression and occurrence of the soluble form. We cannot exclude that sCD27 is also produced in resting Tcells below the level of detectability. Alternatively, T cell activation may induce the

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machinery that generates sCD27. Activation of PKC, resulting from TcR/CD3 triggering does not seem to be involved, since direct activation of PKC with phorbol ester inTcell blasts, does not induce release of sCD27 (Hintzen et al., unpublished). Shedding of cell surface receptors becomes more rule than exception. It is clearly a mechanism for receptor downregulation. On the other hand, the soluble receptors can have a function themselves, Many have been identified because they retain ligand binding capacity, e.g. IL R , and the growth hormone receptor [S3], but also NGF-R [28,29],TNF-RI and TNF-RII [17,31,32,34,35]. According to our studies, sCD27 would also contain the putative ligand-binding “Cys” domain. Soluble receptors may prevent ligands from reaching their cell surface receptors, carry ligands to their site of action, or interact with cell-bound ligands. The latter possibility is not imaginary, since TNF occurs not only as a secretory component, but also in a transmembrane form [54]. Membrane-bound and soluble forms may not always have the same affinity for ligand, which could be important in the regulatory network. One of the twoTNF inhibitory proteins found in urine, apparently affects the activity of TNF-a more strongly than that of TNF-fi [17, 311, though the corresponding cell surface receptors appear to bind each with equal affinity [15]. Perhaps, more than one receptor protein participates in ligand binding at the cell surface. For the NGF-R, a model has been proposed in which interaction between the NGF-R and a second NGF binding protein would constitute a high-affinity receptor [S]. There is increasing support for a functional role of soluble receptors in vivo. sCD25 (sIL2R) levels are used to measure T cell activation in vivo. In patients suffering from autoimmune diseases, serum levels of sCD25 increase during clinical relapse and are normal during remissions [56, 571. TNF inhibitory protein turned out to be soluble TNF-R and levels of this protein are increased in serum infiltrates of cancer patients [32]. It has recently been found that sCD27 levels are increased in patients with Tcellmediated diseases [58].Therefore, monitoring sCD27 levels in vivo may be relevant for diagnosis of human diseases, that involve activation of the immune system. We thank Dr. J. J. Neefjes for valuable discussions and critical reading of the manuscript. Received September 20, 1991

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