The Lymphotoxin-alpha (LTalpha ) Subunit Is Essential for the Assembly, but Not for the Receptor Specificity, of the Membrane-anchored LTalpha 1beta 2 Heterotrimeric Ligand*

(Received for publication, March 26, 1997)

Laura Williams-Abbott , Barbara N. Walter Dagger , Timothy C. Cheung §, Cynthia R. Goh , Alan G. Porter and Carl F. Ware §par

From the § Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, the Dagger  Department of Biochemistry, University of California, Riverside, California 92021, and the  Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent S., 119260 Republic of Singapore

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The lymphotoxins (LT) alpha  and beta , members of the tumor necrosis factor (TNF) cytokine superfamily, are implicated as important regulators and developmental factors for the immune system. LTalpha is secreted as a homotrimer and signals through two TNF receptors of 55-60 kDa (TNFR60) or 75-80 kDa (TNFR80). LTalpha also assembles with LTbeta into a membrane-anchored, heterotrimeric LTalpha 1beta 2 complex that engages a distinct cognate receptor, the LTbeta receptor (LTbeta R). To investigate the role of the LTalpha subunit in the function of the membrane LTalpha 1beta 2 complex, gene transfer via baculovirus was used to assemble LTalpha and -beta complexes in insect cells. LTalpha containing mutations at D50N or Y108F are secreted as homotrimers that fail to bind either TNF receptor and are functionally inactive in triggering cell death of the HT29 adenocarcinoma cell line. In contrast, these mutant LTalpha proteins retain the ability to co-assemble with LTbeta into membrane-anchored LTalpha 1beta 2 complexes that engage the LTbeta R and trigger the death of HT29 cells. Membrane-anchored LTbeta expressed on the cell surface in absence of the LTalpha subunit binds the LTbeta R but is functionally inactive in the cell death assay. These results indicate that the TNF receptor-binding regions of the LTalpha subunit are not necessary for engagement of the LTbeta R, but the LTalpha subunit is required for the assembly of LTbeta into a functional heteromeric ligand.


INTRODUCTION

Lymphotoxins (LT)1 alpha  and beta  are structurally related to TNF, the prototypical member of a superfamily of type II transmembrane glycoproteins (1, 2). These cytokines also exist in soluble forms, although distinct mechanisms generate secreted and membrane-bound LTalpha and TNF. Secreted TNF is generated by proteolysis of the transmembrane protein (3-5), whereas LTalpha lacks a transmembrane domain and is exclusively secreted as a homotrimer (and in this form is also known as TNFbeta ). Unlike TNF, LTalpha also assembles with LTbeta into heteromeric complexes and is consequently localized to the cell surface by the transmembrane domain of LTbeta (6, 7). Substantial evidence indicates that membrane LT exists in two trimeric forms with either an alpha 1beta 2 or alpha 2beta 1 stoichiometry (6, 8). The secreted and membrane-bound forms of LT are further distinguished by their distinct specificities for cell surface receptors. LTalpha and TNF both bind and signal through two receptors, the 55-60-kDa TNF receptor (TNFR60; CD120a or type 1) (9, 10) and the 75-80-kDa TNFR (TNFR80; type 2 or CD120b) (11). By contrast, the surface LTalpha 1beta 2 complex binds a related but distinct receptor, termed LTbeta R, that does not bind either LTalpha or TNF, whereas both TNFRs bind the LTalpha 2beta 1 heterotrimer (8, 12). The LTalpha 1beta 2 complex is the most abundant form expressed by activated T cells (13), and unlike TNF, it is not produced naturally in soluble form (8, 12). The existence of a LTbeta homotrimer is uncertain, since LTbeta protein is apparently always associated with LTalpha in T cells, and a direct assessment has been hindered by unsuccessful attempts at stable expression of membrane-bound LTbeta in mammalian cells (8).

In tissue culture systems, TNF and LTalpha homotrimers are well recognized for their abilities to elicit a similar but not identical spectrum of cellular responses, including apoptosis and proinflammatory activities (14). Purified soluble recombinant LTalpha 1beta 2 (15) exhibits the ability to induce tumor cell death (16) and chemokine secretion (17) and activate NF-kappa B, a transcription factor that regulates inflammatory gene expression through the LTbeta R (18, 19), but may be less potent than LTalpha and TNF. Interestingly, membrane-anchored TNF is more active in signaling via TNFR80 than soluble TNF (20), raising the possibility that membrane-bound and soluble ligands may diverge in some of their functions. This possibility was suspected for the different forms of LT (7) and was brought into acute focus by the characterization of mice with an inactivated LTalpha gene (21, 22). LTalpha -deficient mice lack most lymph nodes and Peyer's patches, a phenotype not associated with deletions of TNF (23) or either of the TNF receptor genes (24-27). Placental transfer of an LTalpha 1beta 2 antagonist constructed as a fusion protein between LTbeta R extracellular domain and the Fc region of IgG (LTbeta R-Fc) results in lymph node-deficient offspring, which established a role for membrane-bound LTalpha 1beta 2 distinct from the LTalpha trimer (28). In addition, the formation of germinal centers during an immune response, a process critical for efficient antibody class switching, is also dramatically altered in mice that lack LTalpha (21, 22), TNF (23), TNFR60 (23, 29), or LTalpha 1beta 2 (29, 30) or express LTbeta R-Fc as a transgene (31). Thus, characterization of the membrane-anchored LT ligands will help elucidate their physiologic functions.

Here, we employ recombinant baculovirus to reconstitute LTalpha and LTbeta homo- and heteromeric complexes in insect cells to investigate the roles of the LTalpha and LTbeta subunits in activation of the cell death response in tumor cells. Using two loss of function mutations in LTalpha (32), aspartic acid 50 to asparagine (D50N) and tyrosine 108 to phenylalanine (Y108F), we show that these LTalpha mutant proteins co-assemble with LTbeta to form a ligand that binds LTbeta R but not TNFR. The membrane-bound mutant LTalpha ·LTbeta complexes, but not the secreted homotrimers, are active at inducing death of the HT29 colon carcinoma cell line. By contrast, LTbeta when expressed alone as a membrane protein binds the LTbeta R but is functionally inactive in inducing cell death. These results demonstrate that the LTalpha subunit is necessary for the LTalpha 1beta 2 complex to activate the LTbeta R cell death pathway.


EXPERIMENTAL PROCEDURES

Cells and Reagents

The insect cell line BTI Tn5B1-4 (Tn5B1-4), kindly provided by JRH Biosciences (Lenexa, KS), was cultured at 27 °C in ExCell 401 serum-free medium containing 10 µg/ml gentamycin. The human adenocarcinoma cell line, HT29.14S, was cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM glutamine, and antibiotics (penicillin and streptomycin, 100 µg/ml) (16). The anti-human LTalpha mAb, 9B9 (mouse IgG1), was purchased from Boehringer Mannheim. The anti-human LTalpha mAb, NC2 (mouse IgG2a), and anti-human LTbeta mAbs, B9 and B27 (mouse IgG1) (8), were generous gifts from Jeffrey Browning (Biogen, Inc.), as was the recombinant human LTalpha produced in Chinese hamster ovary cells and rabbit anti-LTalpha polyclonal serum (33). Construction, expression, and purification of the bivalent chimeric proteins formed with the Fc region of human IgG1 and the ligand binding domains of LTbeta R (12), TNFR60 (34), and TNFR80 (35) have been previously described.

Expression of LT Subunits in Insect Cells

The construction of recombinant baculoviruses expressing LTalpha or soluble LTbeta tagged with a Myc epitope (sLTbeta myc) has been described (12, 34). A cDNA encoding full-length membrane-bound LTbeta was isolated as an 860-base pair HindIII fragment from pCDM8/LTbeta (7), and HindIII/BamHI linkers were added. After restriction with BamHI, the LTbeta cDNA was ligated into the baculovirus transfer vector, pVL1393. Recombinant baculoviruses were produced by coinfection of pVL1393/LTbeta with baculovirus DNA as described (34). LTalpha Y108F and LTalpha D50N mutant cDNAs as originally constructed for expression in bacteria (32) lack the LTalpha signal sequence required for export; therefore, a 300-base pair Nsi1/Pfl M1 cassette, containing the Y108F or D50N mutation, was isolated from p8/3 and p11A/20, respectively, and then used to replace the corresponding region in wild-type LTalpha . The resulting mutant cDNAs containing the LTalpha signal sequence were isolated as NotI fragments and ligated into pVL1393. The mutant constructs were confirmed by sequence analysis of the baculovirus vector (U.S. Biochemical Corp. Sequenase version 2.0 sequencing kit).

Recombinant baculoviruses containing LTalpha Y108F and LTalpha D50N were generated as described for wild-type LTalpha (34). At 6 days post-infection, supernatants from Tn5B1-4 insect cells infected with LTalpha , LTalpha Y108F, or LTalpha D50N recombinant baculovirus were harvested and clarified by centrifugation; protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml aprotinin, and 0.8 mg/ml benzamidine HCl) and fetal bovine serum (10%) were added before dialysis against Hanks' balanced salt solution. The supernatants were filter-sterilized before testing in cytotoxicity assays.

Radioimmunoassays

The concentration of LTalpha was determined by competitive radioimmunoassay using the anti-LTalpha mAbs NC2 and 125I-LTalpha . Anti-LTalpha NC2 was bound (50 ng/well) to plastic snap wells (Immulon 2, Dynatech, Chantilly, VA) precoated with goat anti-mouse Ig (500 ng/well) to capture 125I-LTalpha . The standard curve was generated with purified recombinant LTalpha (33) diluted in 100 µl of phosphate-buffered saline with 1% bovine serum albumin with a 30-min binding interval. LTalpha was radioiodinated to a specific activity of 126 µCi/µg by the IodoGen method (36). 125I-LTalpha in 10 µl was added to a final concentration of 0.2 nM and allowed to bind for an additional 30 min. Each well was washed five times, and the bound 125I-LTalpha in individual wells was detected using a gamma -counter. Each data point is the mean of duplicate wells from which the LTalpha concentration in supernatants was determined from the mean of four dilutions using the radioimmune assay template in Prism (GrapdPAD Software, San Diego, CA). The range was less than 5% for duplicate determinations.

Receptor binding activity of LTalpha and mutant proteins was assessed using a solid phase competitive radioligand binding assay with TNFR60-Fc as a surrogate receptor. The format was identical to the radioimmunoassay described above except that purified TNFR60-Fc was bound at 50 ng/well to wells previously coated with goat anti-human Ig at 500 ng/well.

Biosynthetic Labeling and Immunoprecipitation

Baculovirus-infected insect cells were labeled with [35S]methionine and [35S]cysteine as described (34). Briefly, 24 h after infection (multiplicity of infection was 10 at 105 cells/cm2), the cells were washed with buffered saline and incubated in medium deficient in methionine for 2 h before adding [35S]methionine and [35S]cysteine labeling mixture at 0.2 mCi/ml. After 20 h, the supernatants were cleared by centrifugation for 15 min at 23,000 × g, treated with protease inhibitors, and dialyzed against saline. Protein cross-linking was carried out by the addition of 10 µl of BSCOES, freshly dissolved in Me2SO at 100 mM, to 1.0 ml of serum-free culture supernatants from baculovirus-infected Tn5B1-4 cells (37). After a 30-min incubation on ice, the reaction was stopped by the addition of 25 µl of glycine (1 M) and subjected to immunoprecipitation as described below. The cellular fraction was extracted with Nonidet P-40 (1%) nonionic detergent in buffer with 50 mM Tris, pH 7.4, containing 10 mM iodoacetamide and protease inhibitors. The detergent-soluble fraction, obtained after centrifugation, was subjected to immunoprecipitation as described (13). Briefly, detergent extracts were precleared by the addition of 10 µg of normal mouse or rabbit IgG and 20 µl of protein G-Sepharose beads followed by the addition of 10 µg of either mouse anti-LTalpha or LTbeta antibodies or polyclonal rabbit anti-LTalpha and protein G beads. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (12% acrylamide) and detected by PhosphorImager analysis (Molecular Dynamics).

Flow Cytometry

Tn5B1-4 cells were harvested, washed, and incubated on ice in Hanks' balanced salt solution with 10% bovine calf serum, 0.1% sodium azide containing the antibodies or TNFR-Fc chimeras at 10 µg/ml. Phycoerythrin-conjugated affinity-purified goat anti-mouse or anti-human IgG (5 µg/ml) was used to stain for mAb or TNFR-Fc, respectively. Controls for nonspecific binding included normal mouse or human IgG and inclusion in the buffer of human (or mouse) heat-aggregated IgG at 10 µg/ml to block nonspecific binding when staining for mouse IgG. Immunofluorescence staining was detected by flow cytometry (FACScan, Becton-Dickenson) using forward and side scatter parameters to identify infected and noninfected cells. Each fluorescence histogram represents 1 × 104 events gated on infected cells. Fluorescence intensity = (mean fluorescent channel) × (percentage of positive fluorescent events), where a positive event has a fluorescence value >98% of the value for normal IgG. Specific fluorescence intensity represents the fluorescence intensity after subtraction of the value for normal IgG.

Cytotoxicity Assays

Cytotoxicity of soluble LTalpha produced by insect cells was determined using a colorimetric assay with (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) as described (38). Briefly, the HT29.14S (a TNF/LT-sensitive subclone of HT29 human adenocarcinoma) (16) or murine L929 fibrosarcoma cells (104 cells/well in 96-well flat bottom microtiter plates) were incubated in medium with serial dilutions of supernatants from infected insect cells. For HT29.14S cells, human interferon-gamma was included in the medium at 80 units/ml. After 3 days of incubation, viable cells were detected by the addition of the MTT dye. Cytotoxicity assays with insect cells were performed using paraformaldehyde-fixed insect cells. Tn5B1-4 cells were infected with LTalpha and/or LTbeta recombinant baculoviruses at a multiplicity of infection of 10 for each virus. After 2 days, Tn5B1-4 cells were harvested, washed twice in phosphate-buffered saline, and incubated for 15 min on ice with 1% paraformaldehyde in phosphate-buffered saline and then washed four times with RPMI 1640 containing 10% fetal bovine serum. The fixed insect cells were added at various ratios to HT29.14S cells (104 cells/well) and incubated for 3 days, and viability was detected by reduced MTT dye. The percentage of cell viability was calculated as a ratio of the absorbance of reduced MTT dye at 570 nm for cytokine (or insect cell)-treated cells to the absorbance of dye by cells in medium (with interferon-gamma ) times 100. Each data point represents the mean ± S.D. of triplicate wells.


RESULTS

Characterization of Secreted LTalpha Mutants

The D50N and Y108F mutations in LTalpha were identified as cytotoxicity loss mutants that failed to bind to L929 cells (32). Both LTalpha D50N and Y108F mutants, like wild type LTalpha , are secreted by Tn5B1-4 cells, typically to 20-40 µg/ml (34). When treated with BSOCOES (1 mM), a homobifunctional protein cross-linking reagent, LTalpha and the two mutant proteins formed a ladder of three bands consistent with predicted sizes for trimers, dimers, and monomers of LTalpha (Fig. 1a). The ladder is created by the incomplete cross-linking of LTalpha subunits by BSCOES (37). The less selective cross-linker glutaraldehyde (0.1%) forms a 65-70-kDa adduct in similar preparations (data not shown, and see Ref. 33), indicating that the majority of the LTalpha subunits exist as trimers. Both LTalpha mutants were ineffective as competitors for binding to TNFR60-Fc (Ki = >700 nM) when compared with wild type LTalpha (Ki = 10 nM) (Fig. 1b). Wild type LTalpha induces death in HT29 adenocarcinoma cells (IC50 = 100-200 pM), but both mutants were inactive when tested on HT29 cells (Fig. 1c) or L929 cells (data not shown).


Fig. 1. LTalpha mutants form homotrimers but induce cytotoxicity poorly. a, homotrimer formation. Supernatants from [35S]methionine- and [35S]cysteine-labeled Tn5B1-4 insect cells (lanes 4 and 8) or cells infected with LTalpha (lanes 1 and 5), LTalpha Y108F (lanes 2 and 6), or LTalpha D50N (lanes 3 and 7) baculoviruses were incubated without (lanes 1-4) or with (lanes 5-8) cross-linking reagent BSOCOES. The supernatants were precleared with normal rabbit serum and then immunoprecipitated with rabbit anti-LTalpha antiserum and analyzed by SDS-polyacrylamide gel electrophoresis and PhosphorImager analysis (pixel range 21-307). b, LTalpha mutants compete weakly for TNFR60 binding. The concentration of LTalpha in supernatants from the indicated baculovirus-infected Tn5B1-4 cells was determined by radioimmunoassay. These supernatants were used to compete for 125I-LTalpha binding (0.2 mM) to plate-bound TNFR60-Fc. Each data point is the mean of duplicate wells. The total radioactivity bound in the absence of competing ligand was 2800 ± 125 cpm. c, cytotoxic effect of LTalpha mutants on HT29 cells. HT29.14S cells were incubated with graded concentrations of LTalpha produced by Tn5B1-4 insect cells. Cell viability was measured by MTT dye assay.
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Reconstitution of LTalpha beta Heteromers on the Surface of Tn5B1-4 Cells

The D50N and Y108F LTalpha mutants were tested for their ability to assemble into membrane LTalpha beta complexes by coinfection of Tn5B1-4 cells with recombinant LTbeta baculovirus. Reciprocal co-immunoprecipitations with antibodies to individual alpha  and beta  subunits were used to detect the formation of LTalpha beta complexes. Anti-LTalpha specifically immunoprecipitated major bands at 18, 21, and 22-23 kDa, consistent with precursor and glycosylated forms of LTalpha from baculovirus-infected Tn5B1-4 cells labeled with [35S]methionine and [35S]cysteine (Fig. 2, lanes 1-4). A band at 31-33 kDa expected for LTbeta was also immunoprecipitated by anti-LTalpha . Similarly, anti-LTbeta co-immunoprecipitated two major bands: LTalpha at 22-23 kDa and LTbeta at 31-33 kDa (Fig. 2, lanes 5-7). The LTalpha mutants associated with LTbeta equally as well as wild type LTalpha as judged by the volume-density of the phosphor image. These results indicate that these mutations do not disrupt assembly of LTalpha beta heteromers. Also, note that LTbeta is not associated with the secreted form of LTalpha (see Fig. 1a), indicating that insect cells, like mammalian T lymphocytes, do not cleave LTbeta .


Fig. 2. LTalpha mutants form heteromeric complexes with LTbeta . Tn5B1-4 insect cells were infected with LTalpha (lanes 1, 2, and 5), LTalpha Y108F (lanes 3 and 6) or LTalpha D50N (lanes 4 and 7) baculoviruses in combination with LTbeta baculovirus. Following biosynthetic labeling with [35S]methionine and [35S]cysteine, cell extracts were immunoprecipitated with normal mouse IgG (lane 1), anti-LTalpha (lanes 2-4), or anti-LTbeta (lanes 5-7). Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis and visualized by PhosphorImager analysis (pixel range 53-703).
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In the Tn5B1-4 cells, anti-LTbeta primarily immunoprecipitated the 22-23-kDa mature form of LTalpha and not the smaller 18- and 21-kDa forms recognized by anti-LTalpha (Fig. 2, lanes 4-7). These two smaller LTalpha bands match reasonably well with the predicted sizes of the nascent and signal peptidase-cleaved LTalpha polypeptides of 22.2 and 18.6 kDa, respectively. LTalpha produced by insect cells is glycosylated (39), which indicates that LTbeta assembles with LTalpha soon after the initial processing steps. Precursor-product analysis of LTalpha beta synthesis in T-lymphocytes by pulse-chase methods revealed that LTbeta initially associates with a 21-22-kDa LTalpha precursor that matures to a 25-kDa form (6, 8). Glycosylation (N- and O-linked) of LTalpha also occurs in mammalian cells (40), although in T lymphocytes LTalpha shows more extensive change in molecular mass compared with the protein produced by Chinese hamster ovary cells (21 kDa).

Tn5B1-4 cells singly infected with LTbeta baculovirus express LTbeta protein on the cell surface as detected by immunofluorescence staining with anti-LTbeta , whereas anti-LTalpha did not stain (Fig. 3, a and b). Surface expression of LTalpha requires infection with both LTalpha and LTbeta recombinant baculoviruses (Fig. 3c). The level of LTbeta protein is approximately the same on both singly and coinfected cells, indicating that the presence of the LTalpha subunit does not modify surface expression of LTbeta protein. Receptor binding function, assessed by staining with Fc fusion proteins as surrogate receptors, revealed that LTbeta R-Fc, but not TNFR60-Fc, stained cells expressing LTbeta (Fig. 3d). However, coinfection with the LTalpha baculovirus dramatically increased the LTbeta R-Fc-specific fluorescence staining. Half-maximal binding of the LTbeta R-Fc to LTalpha beta -expressing cells occurred at 0.8 µg/ml (~6 nM), similar to the binding to LTbeta alone (Fig. 4). However, the total LTbeta R-Fc bound is substantially greater (~100-fold) in cells coinfected with LTalpha and LTbeta . This result would be consistent with LTalpha increasing the number of binding sites for LTbeta R. Specific binding of TNFR60-Fc occurred only with LTalpha coinfection, a result consistent with the formation of the LTalpha 2beta 1 ligand (Figs. 3e and 4) with half-maximum binding at 0.6 µg/ml, although the total TNFR60-Fc bound was substantially less (~10-fold) than the LTbeta R-Fc.


Fig. 3. Reconstitution of surface LTalpha beta complexes. Tn5B1-4 insect cells were infected with the baculoviruses containing LTalpha (a), LTbeta (b and d), or both LTalpha and LTbeta (c and e). LTalpha and LTbeta antigens were detected with anti-LTalpha (9B9) or anti-LTbeta (B9) monoclonal antibodies and goat anti-mouse IgG-conjugated PE. Ligand formation was measured in panels d and e with LTbeta R-Fc or TNFR60-Fc and detected with goat anti-human IgG-PE. Normal mouse (a-c) or human IgG (d and e) was used as controls for nonspecific staining.
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Fig. 4. Saturation binding of LTbeta R and TNFR60 fusion proteins. Insect cells infected with LTbeta (upper panel) or coinfected with LTalpha and LTbeta (lower panel) were incubated with graded concentrations of LTbeta R-Fc or TNFR60-Fc for 1 h, washed twice, and stained with goat anti-human IgG-PE. The specific fluorescence intensity was calculated from histograms similar to those shown in Fig. 3.
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As expected, both the LTalpha D50N and Y108F mutant proteins were retained on the surface of insect cells coinfected with LTbeta baculovirus, consistent with the ability of these mutants to assemble with LTbeta into heteromers (Fig. 5, a and b). Tn5B1-4 insect cells infected with mutant LTalpha baculoviruses specifically bound to the LTbeta R-Fc fusion protein (Fig. 5, d and f). However, binding interactions with TNFR60-Fc were dramatically reduced by the LTalpha mutants (Fig. 5, e and g). The D50N mutant retained a some capacity to bind TNFR60-Fc compared with Y108F, with a half-maximum binding at 80-100 nM for D50N and >200 nM for Y108F (data not shown). TNFR80-Fc binding to insect cells expressing either LTalpha mutants was also decreased (data not shown).


Fig. 5. Cell surface LTalpha beta heteromers formed with LTalpha mutants reduce TNFR60-Fc binding. Tn5B1-4 insect cells were infected with LTbeta and LTalpha , LTalpha Y108F, or LTalpha D50N baculoviruses. Protein expression was detected with anti-LTalpha (9B9) or anti-LTbeta (B9) and goat anti-mouse IgG-PE (panels a-c). In panels d-g, staining is with receptor-Fc fusion proteins, LTbeta R-Fc (d and f), or TNFR60-Fc (e and g) as detected with goat anti-human IgG-PE. Background staining was assessed with either mouse (a-c) or human IgG (d-g).
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These results indicated that the D50N and Y108F residues in the LTalpha subunit are not directly involved in interactions with the LTbeta R, although the LTalpha subunit dramatically enhanced binding of the LTbeta R-Fc. The binding of LTbeta R-Fc to LTbeta expressed alone suggested the possibility that, as a resident membrane protein, LTbeta could activate the LTbeta R.

Cell Death-inducing Activity of Membrane-anchored LTalpha beta Complexes

To investigate the role of the LTalpha and LTbeta subunits in the activation of the LTbeta R, insect cells infected with LTbeta or coinfected with LTalpha baculoviruses were fixed and used as effector cells in cytotoxicity assays to measure the functional capacity of the surface ligands. LTbeta -infected cells displayed no significant cytotoxic activity for HT29.14S cells when compared with uninfected Tn5B1-4 cells (Fig. 6a). By contrast, LTalpha and LTbeta co-expressing cells were highly effective at killing HT29.14S cells, typically with a 50% reduction in viability at an effector:target cell ratio of 0.5 (Fig. 6b). Supernatants from fixed cells were not active in this assay, demonstrating that cell death requires cell contact. In striking contrast to the soluble LTalpha mutants, insect cells infected with either D50N or Y108F mutants and LTbeta were completely functional in this assay (Fig. 6, c and d). That the effect of cell death was mediated by LTalpha 1beta 2 is indicated by the ability of the LTbeta R-Fc, but not TNFR60-Fc, to block the death-inducing activity of these killer insect cells. Together, these results indicate that the LTalpha subunit is required for functional conformation of the LTalpha 1beta 2 but not for specificity of binding to the LTbeta R.


Fig. 6. Insect cells expressing LTalpha beta heteromers are cytotoxic for human HT29.14S cells. Tn5B1-4 cells, infected for 48 h with LTbeta or coinfected with either LTbeta and LTalpha or LTalpha mutant baculoviruses, or uninfected Tn5B1-4 cells were fixed and then incubated with HT29.14S cells at the indicated ratios for 3 days. Cell viability was measured by the MTT dye assay. LTbeta R-Fc or TNFR60-Fc was added to wells at 5 µg/ml (panels b-d). Each data point is the mean ± S.D. of triplicate determinations and is representative of four similar experiments.
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DISCUSSION

The crystal structures of LTalpha and TNFR60 provide a conceptual framework to model interactions between LTalpha beta ligands and their receptors (41, 42). Aspartic acid 50 located in the A-A" loop and Y108 in the D-E loop are solvent-exposed residues positioned on opposite sides of the LTalpha monomer, although in the native trimer both residues from different subunits localize to the same receptor binding site (Fig. 7, a and b). Our results indicate that the D50N and Y108F mutations probably cause a local distortion of the TNFR binding site, and not disruption of trimeric architecture, that results in the loss of cytotoxic activity. Additional support for this conclusion is seen in the ability of these LTalpha mutants to assemble with membrane-bound or soluble forms of LTbeta (15). Furthermore, these LTalpha mutants form a functional ligand with LTbeta that activates the LTbeta R cell death pathway.


Fig. 7. Lymphotoxin-alpha structure and theoretical receptor binding sites in LTalpha beta complexes. Shown is a structural model of LTalpha viewed as a ribbon diagram showing beta -strands for a single subunit (side view, solvent-exposed surface; N and C termini are located at the bottom) (a) or as a trimer (view from the base) (b) showing the positions of tyrosine 108 (Y108, red) and aspartic acid 50 (D50, blue) on opposite sides of the subunit. R denotes the position of the TNFR60 binding sites. The schematics are based on the crystal structure of the LTalpha -TNFR60 complex solved by Banner et al. (42) using Protein Data Bank file 1TNR as visualized with RasMol, version 2.6. Diagrams show the theoretical positions of tyrosine 108 (red) and aspartic acid 50 (blue) of LTalpha in the heterotrimeric ligands, LTalpha 1beta 2 (c), and LTalpha 2beta 1 (d). X and Z in LTbeta denote residues in LTbeta equivalent to Asp50 (a-a" loop) and Tyr108 (d-e loop).
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The TNFR60 binding site lies along the cleft formed by adjacent LTalpha subunits, an "alpha alpha " cleft (42). Based on this model, the interface between two adjacent LTbeta subunits is hypothesized to create an analogous beta beta (z-x) site that forms the major LTbeta R binding site within the LTalpha 1beta 2 complex (Fig. 7c). This model is consistent with high affinity binding observed between the LTbeta R and the LTalpha 1beta 2 complex and LTbeta but not the LTalpha 2beta 1 complex, which lacks a beta beta interface. Similarly, the major TNFR60 binding site on LTalpha 2beta 1 would be at the alpha alpha interface (Fig. 7d). The single LTalpha subunit within the LTalpha 1beta 2 complex creates two nonequivalent alpha beta interfaces (x-Y108 and z-D50), where the D50N and Y108F mutations reside in different alpha beta clefts.

Theoretically, binding to one or both of the heteromeric alpha beta interfaces must occur in order for LTalpha 1beta 2 to cluster (aggregate) receptors. Receptor clustering is necessary to recruit signaling molecules, such as TRAF3, to activate the cell death pathway (43), or TRAF5 that can activate NF-kappa B (18). The degree of receptor clustering appears to have a profound effect on the type of cellular responses. In the LTbeta R system, NF-kappa B activation, but not cell death, occurs when bivalent anti-LTbeta R monoclonal antibody is added to the culture medium (16, 19), although cell death occurs when the same monoclonal antibody is immobilized to a surface (16) or the receptor is ligated with soluble polyclonal antibodies (43). Presumably, a higher ordered aggregation of receptors, or stabilization of the receptor signaling complex, sufficient for cell death is achieved with immobilized or polyclonal antibodies. This implies that occupation of all binding sites on the LTalpha 1beta 2 ligand is important to signal cell death and predicts that both alpha beta and the beta beta binding sites are important to achieve this conformation. Either of these LTalpha mutants should create a ligand with two normal binding sites, which might not be sufficient to form complexes capable of signaling cell death. Our results, in fact, show that neither mutation affects cell death signaling by LTalpha 1beta 2. This indicates that LTbeta R clustering sufficient to signal death of HT29.14S cells depends upon contact with LTbeta subunit, and not the LTalpha subunit. An alternate possibility is that residues other than Asp50 and Tyr108 in LTalpha might be involved in binding to LTbeta R. Further mutational analysis may distinguish between these possibilities.

The D50N and Y108F mutations dramatically affected the binding of TNFR60-Fc to secreted LTalpha and membrane-anchored LTalpha 2beta 1. As a soluble protein, LTalpha 2beta 1 does not elicit cellular responses akin to TNF and LTalpha ; rather, it functions as a weak antagonist for TNF (19). Presumably, the reason for the inability of LTalpha 2beta 1 to activate TNFR60 is that the alpha beta interfaces are not sufficient to promote TNFR60 clustering in the same way as LTalpha or TNF homotrimers. Our results show no significant gain (or loss) of cytotoxic activity by killer insect cells that express the membrane-anchored LTalpha mutant-LTbeta complexes. This result indicates that LTalpha 2beta 1 as a membrane protein is unlikely to activate the TNFR60 or LTbeta R cell death pathways. This result is further supported by the complete blocking effect of the LTbeta R-Fc, which should have revealed any putative killing activity by the LTalpha 2beta 1 complex if this ligand could independently activate TNFR60. Thus, the LTalpha 2beta 1-TNFR60 interaction does not mirror the receptor-activating function of LTalpha 1beta 2 binding with LTbeta R and suggests a significant distinction in the way these two receptors are activated by their respective ligands.

The physiologically relevant location of LTalpha 1beta 2 complex is presumed to be at the cell surface, since soluble forms are not naturally produced by lymphocytes. Soluble LTalpha and TNF bind to cell surface receptors with high affinity, typically with an observed Kd of 10-100 pM (37), whereas LTalpha 1beta 2 binding to LTbeta R is in the 1-10 nM range (15).2 The restricted diffusion of a membrane-bound ligand should enhance binding to cell surface receptors so that relatively weak interactions (Kd = ~10-100 nM) may become highly relevant in the context of cell to cell contact. The finding that LTbeta expressed alone does not induce cell death, although it is expressed in a form capable of binding the surrogate LTbeta R-Fc, indicates that the presence of the LTalpha subunit is critical for the conformation that activates the LTbeta R. In previous studies (12), soluble LTbeta protein (generated by deletion of the cytosolic and transmembrane domains) bound to LTbeta R-Fc, but weakly compared with LTalpha 1beta 2. Soluble LTbeta is polydisperse in the absence of LTalpha , forming aggregates of high molecular mass based on elution through gel filtration matrix (15) or by protein cross-linking.3 These biochemical findings suggest that the LTalpha subunit may restrict the assembly of LTbeta oligomers to dimers. The critical role of the LTalpha subunit is further revealed by the lymph node deficiency and germinal center failure in mice genetically deficient in LTalpha subunit (21, 22). These observations indicate that LTbeta as a single subunit ligand is insufficient to signal these developmental and physiologic processes. Rather, LTalpha is essential to form the biologically active LTalpha 1beta 2 ligand.


FOOTNOTES

*   This work was supported in part by National Institutes of Health Grants AI33068 and PO1 CA69381, American Cancer Society Grant IM663, and funding provided by the Institute of Molecular and Cell Biology, National University of Singapore.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121. Fax: 619-558-3526; E-mail: carl_ware{at}liai.org.
1   The abbreviations used are: LT, lymphotoxin; LTbeta R, LTbeta receptor; BSOCOES, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone; MTT, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; PE, phycoerythrin; mAb, monoclonal antibody; TNF, tumor necrosis factor; TNFR, TNF receptor.
2   C. Ware, unpublished observations.
3   C. Ware and P. Crowe, unpublished observations.

ACKNOWLEDGEMENTS

We are grateful to Dr. Jeffrey Browning and colleagues at Biogen, Inc. for gifts of reagents.


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