The Endoplasmic Reticulum Lumenal Domain of the Ad
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病菌学杂志 2005年第21期
University of Connecticut, School of Pharmacy, Storrs, Connecticut 06269
Boston Biomedical Research Institute, Watertown, Massachusetts 02472
ABSTRACT
E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 ± 10 nM for peptide-filled molecules and of about 10 μM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.
INTRODUCTION
The ability of many large DNA viruses such as adenoviruses (Ads), herpesviruses, and poxviruses to establish persistent infections in mammalian cells implies that they have evolved mechanisms to circumvent host antiviral immune defenses. A thorough understanding of molecular and mechanistic aspects of these processes is fundamental to our ability to prevent, treat, and cure virus-induced immune dysfunctions.
It is estimated that Ads are responsible for ca. 30% of virus-induced respiratory diseases (19). In addition to genes involved in replicative functions, the Ad genome includes a number of genes that code for immunomodulatory proteins (reviewed in references 25 and 45). For example, the Ad early transcription unit 1A (E1A) codes for two proteins that suppress transcription of class I genes in host cells. Moreover, the Ad E3 codes for several proteins, of which the 19-kDa protein (E3-19K) is the most abundant, that are dedicated to modulate host cellular immune responses at the posttranslational level.
E3-19K is a type I membrane glycoprotein expressed by all human Ad serotypes with the exception of those from subgroups A and F (33). The basic structural organization of Ad E3-19K-like proteins consists of an N-terminal endoplasmic reticulum (ER) lumenal domain (107 residues), a transmembrane domain (23 residues), and a C-terminal cytosolic tail segment (14 residues) with an ER retention signal (17, 20). The lumenal domain of E3-19K has been subdivided into three distinct regions based on sequence alignment (23): (i) residues 1 to 84 are variable among Ad serotypes in different subgroups, (ii) residues 85 to 98 are conserved among Ad serotypes in different subgroups, and (iii) residues 99 to 107 serves as a linker. E3-19K has been shown to associate with class I major histocompatibility complex (MHC) molecules and prevent their egress from the ER in different Ad-infected cells, including human (8), rat (28), and mouse (9, 14). Two structural features of E3-19K are responsible for these observations: its lumenal domain associates with the lumenal domain of class I MHC molecules (3, 9, 16, 18), whereas its C-terminal cytosolic tail segment blocks the transport of class I MHC molecules to the cell surface by virtue of an ER retention motif (15, 20, 34). The retention of class I MHC molecules in the ER blocks presentation of antigenic peptides to CD8+ cytotoxic T lymphocytes (CTLs).
Although E3-19K is the first viral immunomodulatory protein identified and one of the most characterized, mechanistic aspects of its function remain unexplained. Using a series of biochemical and biophysical techniques, we show here that recombinant, soluble E3-19K is a monomeric protein that is thermally stable and that possesses a correctly folded tertiary structure. The results from in vitro reconstitution experiments revealed that soluble E3-19K associates with both peptide-filled and peptide-deficient HLA-A1101 (hereafter referred to as HLA-A11) molecules. Our studies provide direct insights into molecular aspects of the mechanism by which E3-19K retains class I MHC molecules in the ER. Our findings may have implications for other Ad E3-19K-like proteins given their shared immunomodulatory functions.
MATERIALS AND METHODS
Construction of a baculovirus expression system for E3-19K. The cDNA encoding the signal peptide and ER lumenal domain (residues 1 to 100) of the Ad type 2 E3-19K protein with a C-terminal His6 tag sequence was generated by the PCR. This was carried out with the plasmid pBSKSII (kindly provided by H.-G. Burgert) containing the cDNA of full-length Ad2 E3-19K as a template and the following synthetic primers (restriction sites are in italics, stop codon is underlined, and the His6 tag sequence is in parentheses): 5'-GGATCCATGAGGTACATGATTTTAGGCTTGC-3' (forward) and 5'-GGTACCTTA(GTGATGGTGATGGTGATG)CTTTTGTGGGGGCCACAA-3' (reverse). The amplified cDNA was ligated into the pFastBac-1 vector (Invitrogen) by using BamHI and KpnI restriction sites. Generation of recombinant baculovirus for the expression of soluble E3-19KHis6 was carried out by using the Bac-to-Bac baculovirus expression system (Invitrogen) as recommended by the manufacturer.
Expression and purification of E3-19K. High Five insect cells were cultured at 27°C in serum free Express Five medium (Gibco) supplemented with 1.5% fetal bovine serum (Gibco). Infection of High Five cells with recombinant baculovirus was carried out in 2-liter Delong flasks at a cell density of 1.8 x 106 to 2 x 106 cells/ml, followed by incubation at 27°C for 70 h. The culture medium was centrifuged (700 x g at 4°C for 10 min), the supernatants were adjusted to 0.2 mM phenylmethylsulfonyl fluoride, and centrifugation continued at 6,500 x g for an additional 30 min. Combined supernatants containing soluble E3-19K were filtered through a 0.45-μm-pore-size polyethersulfone disposable filter unit and concentrated ca. 30-fold by using a Prep/scale-TFF cartridge (Millipore). The concentrated supernatant was changed into a buffer containing 5 mM imidazole, 50 mM NaH2PO4, and 200 mM NaCl (pH 8.0) and centrifuged (6,500 x g at 4°C for 15 min). The supernatant was directly applied onto an equilibrated nickel-nitrilotriacetic acid (Ni-NTA) affinity column (QIAGEN). The column was washed with 5 mM imidazole, 50 mM NaH2PO4, and 200 mM NaCl (pH 8.0). The desired protein was eluted with the same buffer containing 200 mM imidazole. The eluate containing crude E3-19K was concentrated by using Centriprep-3 (Amicon), followed by purification on a Superdex 200 HR 10/30 column in 20 mM Tris plus 150 mM NaCl (pH 7.5). Stock solutions of purified E3-19K in the eluant were kept at –70°C.
PNGase F treatment. A sample of purified E3-19K (15 μg) was incubated in 0.5% sodium dodecyl sulfate (SDS) and 1% ?-mercaptoethanol at 100°C for 10 min. The mixture was adjusted to 0.05 M sodium phosphate plus 1% NP-40 (pH 7.5), followed by the addition of 1,000 U peptide:N-glycosidase F (PNGase F) (New England Biolabs). The mixture was incubated at 37°C for 16 h. Digest was terminated by the addition of SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer, followed by heating the sample at 100°C for 5 min and analysis on a 15% SDS-PAGE gel.
Gel filtration chromatography. Samples of purified E3-19K was analyzed at 10°C by using a Superdex 200 HR 10/30 column in 20 mM Tris plus 150 mM NaCl (pH 7.5).
CD. Circular dichroism (CD) experiments were done by using a Jasco-810 spectropolarimeter equipped with a thermoelectric temperature controller. The far- and near-UV spectra represents the average of 8 and 15 scans, respectively. A thermal denaturation curve was obtained in duplicate by monitoring the change in ellipticity at 213 nm in the range 20 to 100°C using a scan rate of 40°C/h. The thermal denaturation mid-point temperatures (Tm) were determined by fitting the averaged thermal denaturation curve to an equation describing a two-state denaturation process (5). Ellipticity values are expressed on a molar residue basis.
Assembly and purification of recombinant, soluble peptide-filled and peptide-deficient HLA-A11 molecules. Peptide-filled HLA-A11 molecules were assembled in vitro as described previously (30). In brief, refolding was initiated by diluting the urea-solubilized inclusion bodies of HLA-A11 heavy chain (residues 1 to 275) (1 μM) and ?2m (2 μM) in the presence of an excess of Nef(73-82) (QVPLRPMTYK) human immunodeficiency virus type 1 peptide (10 mM) (40) in a well-established oxidative refolding buffer (21). Stock solutions (20 to 40 mg/ml) of purified HLA-A11/Nef molecules in 20 mM Tris plus 150 mM NaCl (pH 7.5) were kept at –70°C. Peptide-deficient HLA-A11 molecules were assembled from the guanidinium denaturation of peptide-filled HLA-A11 molecules according to a previously described protocol (6). This strategy has proven to be an effective way of releasing bound peptides and of generating denatured HLA-A11 heavy chain and ?2m with native disulfide bonds. Purified peptide-deficient HLA-A11 molecules (1 to 2 mg/ml) in 20 mM HEPES, 150 mM NaCl, and 20% glycerol (pH 7.5) were kept at –70°C.
Gel mobility shift assay. Samples of purified E3-19K (14 μg) and HLA-A11 molecules (20 μg; 2:1 molar ratio) were incubated in 20 mM Tris plus 150 mM NaCl (pH 7.5; the buffer contained 10% glycerol for peptide-deficient HLA-A11 molecules) on ice for 30 min. Controls and incubation mixtures were immediately assayed on a native PAGE gel (10%) at 4°C in a buffer containing 60 mM Tris plus 40 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) (pH 9.4) (for uncomplexed E3-19K) or 25 mM Tris-200 mM glycine (pH 8.3) (for uncomplexed and complexed HLA-A11 molecules). Proteins were visualized with Coomassie blue.
Analytical ultracentrifugation. Sedimentation velocity runs for E3-19K were carried out at 20°C and 60,000 rpm, with patterns being acquired every 7 s on a Beckman Instruments Optima XL-I analytical ultracentrifuge. A stock solution of E3-19K at ca. 2 mg/ml was dialyzed for 16 h against 20 mM HEPES plus 150 mM NaCl (pH 7.5). Dilutions in the range of 0.3 to 1.0 mg/ml were prepared by using the dialysate as diluent. Sedimentation boundaries were analyzed by using time derivative analysis as described previously (41, 42) and SEDANAL software (43). The value of the partial specific volume (V = 0.723 cm3/g) was calculated from the amino acid sequence of recombinant E3-19K (V = 0.7340 cm3/g), assuming an approximate carbohydrate content of 11% (V = 0.6250 cm3/g), using the consensus partial volumes of the amino acids (35). A value of 0.369 g of water/g of protein for the hydration of recombinant E3-19K was calculated from the amino acid composition. Sedimentation equilibrium runs for E3-19K were carried out at 4°C as described previously (7). A stock solution of E3-19K at ca. 2 mg/ml was dialyzed for 24 h against the same buffer as that for sedimentation velocity runs. A series of dilutions in the range of 0.1 to 1.0 mg/ml were prepared by using the dialysate as diluent. Sedimentation equilibrium data were analyzed by global fitting from combined data at three loading concentrations and two speeds as described previously (7), using the global equilibrium fitter included in the SEDANAL package (43).
Interaction studies between E3-19K and HLA-A11 molecules were carried out by sedimentation velocity at 20°C and 60,000 rpm. Separate stock solutions of E3-19K and HLA-A11 molecules at ca. 2 mg/ml were simultaneously dialyzed overnight against 20 mM HEPES plus 150 mM NaCl (pH 7.5) (buffer contained 20% glycerol for peptide-deficient HLA-A11 molecules). Values of the partial specific volume of HLA-A11/Nef and peptide-deficient HLA-A11 molecules were calculated from amino acid sequences and found to be 0.7240 and 0.723 cm3/g, respectively. Data from four nominal loading concentrations were used in these analyses: [E3-19K]:[HLA-A11 molecules] concentrations of 11:11, 3.7:3.7, 1.2:1.2, and 0.41:0.41 μM. The sedimentation velocity profiles were analyzed with the program SEDANAL (43) to produce the g(s) profiles, and were also globally fitted to a 1:1 association model of type A + B = C to obtain estimates of equilibrium constants. Analysis of peptide-deficient HLA-A11 molecules was also carried with the program SEDFIT using c(s) analysis (36) to reveal the likely existence of conformational isomers.
Temperature-dependent thermolysin digests. Digests of peptide-deficient HLA-A11 molecules and of E3-19K/peptide-deficient HLA-A11 complex were carried out by thermolysin at an enzyme/substrate ratio of 1:100 in 20 mM Tris, 150 mM NaCl, 10% glycerol, and 2 mM CaCl2 (pH 7.5). After a 15-min incubation, digests were terminated by the addition of EDTA (to 10 mM) and boiling samples. Products of digests were analyzed on a 15% SDS-PAGE gel. Digitalized signals of the Coomassie blue-stained band corresponding to HLA-A11 heavy chain were analyzed with 1D Software (Kodak, Rochester, New York).
N-terminal amino acid sequencing. N-terminal amino acid sequencing analyses of proteins were carried out (Tufts University, Boston, MA) from either a SDS-PAGE gel (for E3-19K) or a native PAGE gel (for E3-19K/HLA-A11 complexes). A sample of purified E3-19K was analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (Harvard University, Cambridge, MA).
RESULTS
Characterization of E3-19K. The ER lumenal domain (residues 1 to 100) of E3-19K tagged with a C-terminal His6 sequence was optimally harvested from High Five cell culture at 70 h postinfection (Fig. 1A, lane 1). The crude protein was purified by Ni-NTA affinity (Fig. 1A, lane 2) followed by gel filtration chromatography (Fig. 1A, lane 3) to remove impurities and soluble aggregates. Purified E3-19K migrated as a triplet at ca. 14 to 18 kDa (Fig. 1A, bands 1 to 3 in lane 3). These three bands were individually analyzed by N-terminal amino acid sequencing analysis, and the results confirmed the sequence of mature E3-19K for each band: A1KKVEFK. Analysis of purified E3-19K by MALDI mass spectrometry (Fig. 1B) revealed three distinct regions of peaks, a pattern that is consistent with the series of three bands observed on SDS-PAGE gel (Fig. 1A, lane 3): the single peak at 12.75 kDa is in agreement with the value of 12.74 kDa calculated from the amino acid sequence of unglycosylated E3-19K (including six His residues) (band 3 in Fig. 1A), whereas the two clusters of peaks at higher molecular masses (13.48 to 13.94 kDa and 14.51 to 15.04 kDa) are likely to reflect the differentially glycosylated nature of E3-19K (bands 2 and 1, respectively, in Fig. 1A). Two Asn residues (Asn12 and Asn61) have been identified as putative N glycosylation sites in the amino acid sequence of E3-19K (44). To firmly establish that the three bands observed on the SDS-PAGE gel correlates with the glycosylated nature of E3-19K, a sample of purified E3-19K was incubated with PNGase F under denaturing conditions (Fig. 1C). The results (lane 2) show that PNGase F treatment caused the disappearance of the slowest-migrating band in the control lane (lane 1) and the accumulation of two major products, one of which migrates at the same position as the fastest-migrating band in lane 1. Although the hydrolysis of glycans by PNGase F was only partial, the sensitivity of E3-19K to hydrolysis with this enzyme reflects the glycosylated nature of recombinant E3-19K. A similar pattern of two intense bands were obtained after incubating purified E3-19K with Endo H or with a mixture of PNGase F and Endo H (data not shown). That a subset of E3-19K molecules secreted from High Five cells is resistant to hydrolysis with both PNGase F and Endo H suggests that, in contrast to the ER-resident form of E3-19K, secreted E3-19K has undergone terminal modifications of its N-linked high-mannose carbohydrate moieties (27) in the secretory pathways. For example, it is possible that 1,3-fucose, which is often added to proteins that are secreted from High Five cells and is resistant to hydrolysis with PNGase F and Endo H (1), was incorporated at one of the two Asn residues of E3-19K. Overall, these data strongly suggest that the heterogeneity displayed by recombinant, soluble E3-19K (Fig. 1A and B) is due to N glycosylation. Consistent with our results, electrophoretic heterogeneity of full-length Ad2 E3-19K expressed in Sf9 insect cells was also shown to arise from variations in N glycosylation (29). Analysis of purified E3-19K by native PAGE gel (Fig. 1D) showed that the protein migrates as a single, compact band consistent with a well-behaved protein in solution.
E3-19K is a monomer in solution. The unaggregated nature of soluble E3-19K is reflected in its gel filtration chromatography profile, which shows a single symmetric peak (Fig. 2). Sedimentation equilibrium and velocity analysis of E3-19K unambiguously demonstrated the monodispersed and monomeric nature of the protein in solution, yielding a sedimentation coefficient (S020,w) of 1.56S and an apparent molecular mass of 14.2 kDa. Sedimentation equilibrium analysis showed that soluble E3-19K has a very weak tendency to form reversible dimers (Kd = 1.3 ± 0.1 mM) at high concentrations.
E3-19K adopts a well-defined structure in solution. The far-UV CD spectrum (Fig. 3A) of E3-19K shows a positive maximum at 230 nm and a negative maximum at 210 nm with a discernible shoulder at 215 nm. These spectral features suggest that soluble E3-19K possesses predominantly ?-sheets with little -helices. The thermal denaturation curve (Fig. 3B) of E3-19K shows a single transition centered at a Tm of 62.7°C. The near UV-CD spectrum (Fig. 3C) shows strong spectral features at around 280 nm that essentially disappear at 80°C. These results suggest that aromatic side chains in E3-19K are located in an asymmetrical environment consistent with the protein having a well-defined three-dimensional structure. Overall, CD results indicate that soluble E3-19K possesses conformational properties that can be attributed to a correctly folded three-dimensional structure.
E3-19K associates with peptide-filled and peptide-deficient HLA-A11 molecules. Complex formation between soluble E3-19K and soluble HLA-A11 molecules was monitored by using a gel mobility shift assay. For this experiment, we first reconstituted a soluble form of peptide-filled HLA-A11 molecules that consists of the ER lumenal domain of HLA-A11 heavy chain, ?2m, and the decamer Nef peptide (see Materials and Methods). These HLA-A11/Nef molecules were also used to generate peptide-deficient HLA-A11 molecules according to a well-established protocol (see Materials and Methods). These peptide-deficient class I MHC molecules have been convincingly shown to be devoid of synthetic peptides and to display an unstable peptide-binding groove (6). Moreover, these peptide-deficient class I MHC molecules can be loaded efficiently with antigenic peptides, indicating that they represent a functional, intermediate form of class I MHC molecules (6).
For interaction between E3-19K and peptide-filled HLA-A11 molecules (Fig. 4, lanes 1 and 2, respectively), a native PAGE gel shows a new band (lane 3) that migrates at a different position relative to uncomplexed proteins. This new band was analyzed by N-terminal amino acid sequencing and results confirmed the presence of each component in the complex: E3-19K (A1KKVEFK), HLA-A11 heavy chain [G1SH(_)M(_)Y], ?2m (M1IQRTPK), and Nef peptide [Q1VP(_)(_)P(_)]. Similarly, for interaction between E3-19K and peptide-deficient HLA-A11 molecules (Fig. 4, lanes 1 and 4, respectively), the results show a new band (lane 5) that migrates at a different position relative to uncomplexed proteins. N-terminal amino acid sequencing of this new band confirmed the presence of each protein making up the complex: E3-19K (A1KKVEFK), HLA-A11 heavy chain [G1SHSM(_)Y], and ?2m (M1IQRTPK). Together, these results clearly indicate that recombinant, soluble E3-19K retains a native structure that mediates direct interaction with both peptide-filled and peptide-deficient HLA-A11 molecules.
The stoichiometry and equilibrium constants of E3-19K/HLA-11 molecule complexes were determined by sedimentation velocity experiments (Fig. 5). E3-19K and HLA-A11 molecules were mixed at different ratios (see Materials and Methods) and sedimentation velocity profiles analyzed with the program SEDANAL (43) to fit a 1:1 (A + B = C) association model. A global fit of all data sets (see Materials and Methods) gave a Kd of 50 ± 10 nM for the E3-19K/HLA-A11/Nef complex (curve C in upper panel) and of Kd of about 10 μM for the E3-19K/peptide-deficient HLA-11 molecule complex (curve C in lower panel). Thus, the results from analytical ultracentrifugation studies revealed that although E3-19K associates with peptide-filled and peptide-deficient HLA-A11 molecules, it displays a differential affinity for these two forms: the E3-19K/HLA-A11/Nef complex is energetically considerably more stable than the E3-19K/peptide-deficient HLA-A11 complex. Interestingly, peptide-deficient class I MHC molecules appeared by sedimentation analysis, both with c(s) analysis using SEDFIT (36) and with Lamm equation modeling using SEDANAL (43), to be comprised of at least four conformational isomers that were either distinct or very slowly interconvertible. This finding is consistent with earlier data showing that peptide-deficient class I MHC molecules exist in a partially unfolded molten globule-like state (6). Because of this conformational heterogeneity, only a semiquantitative estimate for the Kd of E3-19K/peptide-deficient HLA-A11 complex could be computed. Whether or not peptide-deficient HLA-A11 molecules in the E3-19K/peptide-deficient HLA-A11 complex also exhibit conformational heterogeneity could not be determined from the data with confidence.
E3-19K stabilizes peptide-deficient HLA-A11 molecules. In previous studies, we showed that peptide-deficient class I MHC molecules have a distinct peptide-binding groove from that of peptide-filled class I MHC molecules (6): the peptide-binding groove is more unstable, it lacks native tertiary packing, and it is considerably more susceptible to cleavage by proteases. To assess whether the association of E3-19K with peptide-deficient HLA-A11 molecules confers stability to the peptide-binding groove, we compared the relative susceptibilities of HLA-A11 heavy chain in peptide-deficient HLA-A11 molecules and in the E3-19K/peptide-deficient HLA-A11 complex to digest with thermolysin at different temperatures (Fig. 6A). SDS-PAGE analysis of products from the digest of peptide-deficient HLA-A11 molecules at 15°C and 25°C showed several new bands that are absent in the control (lane C1). These bands arise from the cleavage of HLA-A11 heavy chain. At 37°C, HLA-A11 heavy chain is nearly completely digested by thermolysin. In contrast, the incubation of E3-19K/peptide-deficient HLA-A11 complex with thermolysin shows that HLA-A11 heavy chain is considerably more resistant to digest at 25 and 37°C, as evidenced by the more intense HLA-A11 heavy-chain bands at these temperatures. At 45°C, the HLA-A11 heavy chain is nearly completely digested by thermolysin, an effect which was observed at 37°C for peptide-deficient HLA-A11 molecules. As expected, ?2m is stable in the range of 15 to 37°C in both experiments, a finding consistent with the thermal stability of ?2m (5). A quantitative analysis (Fig. 6B) of the HLA-A11 heavy chain in the thermolysin gel (Fig. 6A) clearly indicated that the heavy chain is more stable in the E3-19K/peptide-deficient HLA-A11 complex relative to peptide-deficient HLA-A11 molecules. This trend was consistently reproducible in independent experiments. Together, these results indicate that E3-19K confers a stabilizing effect to HLA-A11 heavy chain in the E3-19K/peptide-deficient HLA-A11 complex. Given that the peptide-binding groove is the region of HLA-A11 heavy chain that is destabilized in the peptide-deficient form (6), our results therefore implicate a region of the binding groove as part of the E3-19K interaction surface on class I MHC molecules.
The association of E3-19K with peptide-deficient HLA-A11 molecules is compatible with the binding of antigenic peptides. In previous studies, we showed that peptide-deficient class I MHC molecules are competent in binding antigenic peptides (6; see also lanes 1 and 2 in Fig. 7). Here, we have examined if the association of E3-19K with peptide-deficient HLA-A11 molecules blocks the binding of antigenic peptides. Our results from native PAGE gel (Fig. 7) clearly show that the E3-19K/peptide-deficient HLA-A11 complex (lane 3) is capable of binding Nef peptide, as evidenced by the formation of a new band (lane 4) that migrates at the same position on the gel as preformed E3-19K/HLA-A11/Nef complex (lane 5). N-terminal amino acid sequencing of this new band (lane 4) confirmed the presence of three proteins, E3-19K (A1KKVEFK), HLA-A11 heavy chain [G1SHSM(_)Y], and ?2m [M1IQ(_)TPK], and of Nef peptide [Q1V(_)L(_)P]. These results provide convincing evidence that peptide-deficient class I MHC molecules sequestered by E3-19K can mature into peptide-filled molecules. On the basis of these results, we conclude that E3-19K does not block the binding of antigenic peptides. Based on results shown in Fig. 6, which implicate a region of the binding groove as part of the E3-19K interaction surface, the results presented in Fig. 7 suggest that E3-19K binds away from the interior of the peptide-binding groove.
DISCUSSION
Initial attempts to express the ER-lumenal domain of Ad2 E3-19K were carried out in bacterial cells and yielded high levels of inclusion bodies (13; H. Liu and M. Bouvier, unpublished results). In vitro refolding of these urea-solubilized inclusion bodies under a variety of conditions consistently resulted in large amounts of aggregates (Liu and Bouvier, unpublished). In contrast, expression of the ER lumenal domain of Ad2 E3-19K in baculovirus-infected insect cells reproducibly yielded over 1 mg of secreted protein per liter of cell culture. Characterization of recombinant, soluble E3-19K by SDS-PAGE gel, N-terminal amino acid sequencing, and MALDI mass spectrometry confirmed the correct identity of the recombinant protein. CD studies showed that soluble E3-19K adopts a stable tertiary structure characterized by a relatively high content of ?-sheets. These characteristics are consistent with predictions that E3-19K may be a member of the immunoglobulin superfamily of proteins (12). Sedimentation equilibrium and velocity experiments clearly established the monomeric nature of soluble E3-19K, although a very weak tendency to form reversible dimers (Kd = 1.3 ± 0.1 mM) was evident from our data. It was previously demonstrated from an SDS-PAGE analysis of immunoprecipitates of E3-19K-infected cells (15) and mentioned as "unpublished observations" (32) that full-length E3-19K is capable of forming dimers intracellularly. The physiological significance of these putative E3-19K dimers is undetermined.
In vitro reconstitution experiments showed that soluble Ad2 E3-19K associates with peptide-filled HLA-A11 molecules. This is consistent with numerous cell-based studies which demonstrated that the peptide-filled form of class I MHC molecules is the cellular target of E3-19K (8, 15, 29). Here, we provide conclusive evidence that E3-19K sequesters peptide-deficient class I MHC molecules as well. Our results therefore establish that E3-19K recognizes class I MHC molecules at more than one stage during their biogenesis: after (late stage) and before (early stage) the step of peptide binding. Although cell-based studies also provided evidence that E3-19K associates with class I heavy chain prior to the association of class I heavy chain with ?2m (14, 26, 38, 39), it is of note that we were unable to refold urea-solubilized inclusion bodies of HLA-A11 heavy chain in the presence of E3-19K (data not shown). The association of full-length E3-19K with class I MHC molecules has been shown to occur independently of glycans (8, 9). In view of this, the differently glycosylated nature of secreted E3-19K relative to the ER-resident form of E3-19K is unlikely to have functional implications on our in vitro results.
Sedimentation velocity studies firmly established that E3-19K associates with class I MHC molecules in a 1:1 molar ratio with a Kd of 50 ± 10 nM for the E3-19K/peptide-filled HLA-A11 complex and a Kd of about 10 μM for the E3-19K/peptide-deficient HLA-A11 complex. It cannot be ruled out that E3-19K and HLA-A11 molecules may display a higher affinity in their full-length forms. It is well-known that the transmembrane and cytoplasmic domains of interacting membrane-anchored proteins can contribute in stabilizing complex formation (11), and circumstantial evidence have been provided that this may be the case in the E3-19K/class I MHC system (9, 10, 32, 34).
From a structural point of view, the differential affinity of E3-19K for peptide-filled and peptide-deficient HLA-A11 molecules can be accounted for on the basis of the distinct maturation levels of these molecules. In vivo and in vitro studies have provided evidence that maturation of class I MHC molecules is accompanied by significant peptide-induced conformational changes in the binding groove (37). Specifically, the binding groove is altered from a destabilized and partially folded structure in peptide-deficient molecules to a more stable and native structure in peptide-filled molecules (6). In keeping with this view, and with our results indicating that the peptide-binding groove is implicated in the E3-19K binding site on class I MHC molecules, two potential modes of interaction can be proposed for these 1:1 complexes. (i) E3-19K uses the same binding site on both peptide-filled and peptide-deficient HLA-A11 molecules. The E3-19K binding site is structurally more immature in the peptide-deficient form but, as a result of peptide binding, the site undergoes conformational rearrangements and acquires a native structure. These structural changes optimize interactions between MHC residues and E3-19K, thereby increasing the avidity of E3-19K for the peptide-filled form. (ii) E3-19K recognizes different surfaces on peptide-filled and peptide-deficient HLA-A11 molecules: a high-affinity site on peptide-filled HLA-A11 molecules and a low-affinity site on peptide-deficient HLA-A11 molecules. Here again, we suggest that peptide-induced structural changes within the binding groove leads to formation of the structurally more mature, high-affinity site in peptide-filled molecules and the concomitant elimination of the low-affinity site. We therefore propose that the higher affinity of E3-19K for peptide-filled compared to peptide-deficient HLA-A11 molecules reflects the different conformational states of the peptide-binding groove in these two forms of class I MHC molecules, independently of whether or not these molecules share a common binding site for E3-19K. Crystallization studies will reveal the precise interaction surface of E3-19K on peptide-filled and peptide-deficient class I MHC molecules.
That the affinity of E3-19K for peptide-filled HLA-A11 molecules is in the nanomolar range is entirely consistent with the biological function of E3-19K, which is to tightly retain host proteins in the ER. Although the intrinsic propensity of E3-19K for peptide-deficient HLA-A11 molecules is lower than for peptide-filled HLA-A11 molecules, our results clearly showed that peptide-deficient HLA-A11 molecules sequestered by E3-19K are more stable and, importantly, retain the ability to bind antigenic peptides. These findings may have functional implications since they suggest that, in sequestering and stabilizing peptide-deficient class I MHC molecules, E3-19K not only allows peptides to bind and peptide-filled molecules to be formed but may also actually facilitate the maturation process. The association of E3-19K with peptide-deficient class I MHC molecules may therefore serve to sustain the function of E3-19K, which is directed preferentially at peptide-filled molecules. In spite of the lower affinity of the E3-19K/peptide-deficient class I MHC complex, this association may be important at the beginning of natural infection by Ads when immature class I molecules are more abundant in the ER, before the virus impairs host protein synthesis (2, 24). Overall, we therefore suggest that the ability of E3-19K to act at the late and early stages of the class I assembly pathway is likely to provide a functional advantage to E3-19K. Future studies that examine whether peptide-deficient class I MHC molecules held in association with E3-19K are concomitantly associated with class I assembly proteins will provide important insights into whether E3-19K may play a direct role in maturation of class I MHC molecules in Ad-infected cells. In this context, it was recently shown that the US2 protein from cytomegalovirus uses a binding surface on peptide-filled HLA-A2 molecules that is distinct from the putative binding surface of class I assembly proteins (22). This suggests that, in principle, US2 and class I assembly proteins could simultaneously associate with class I MHC molecules (22). Unlike E3-19K, however, no evidence have been provided that US2 binds to peptide-deficient class I MHC molecules.
In summary, our studies have provided new insights into molecular aspects of the E3-19K immune evasion mechanism. The results showed that the ER lumenal domain of Ad2 E3-19K expressed in insect cells retains a native structure that mediates stable interaction with class I MHC molecules. Importantly, E3-19K sequesters ?2m-associated class I heavy chains independently of their maturation states, although it shows a preference for peptide-filled molecules. This is the first example of a viral immunomodulatory protein that associates with conformationally distinct forms of class I MHC molecules. It was recently shown that E3-19K binds directly to the transporter associated with antigen processing (4). A functional relationship has also been identified between E3-19K and APLP2, a protein presumed to act as a chaperone in the maturation of some, but not all, class I MHC molecules in the ER (31, 38). Together, these mechanisms expand the means by which E3-19K interferes with class I antigen presentation and underline the efficacy with which this immunomodulatory protein retains class I MHC molecules in the ER. Since the identification of immunomodulatory gene products of other large DNA viruses is intensively being pursued, their characterization will eventually reveal whether viruses that directly interfere with the class I antigen presentation pathway share common strategies with E3-19K.
ACKNOWLEDGMENTS
We thank Mingnan Chen for helpful discussions and for valuable comments on the manuscript.
This study was funded by a fellowship from the Outstanding Scholars Program of the University of Connecticut (to H.L.), grant BIR-953060 from the National Science Foundation (to W.F.S.), and grant AI055717 from the NIH/National Institute of Allergy and Infectious Diseases (to M.B.).
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Boston Biomedical Research Institute, Watertown, Massachusetts 02472
ABSTRACT
E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 ± 10 nM for peptide-filled molecules and of about 10 μM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.
INTRODUCTION
The ability of many large DNA viruses such as adenoviruses (Ads), herpesviruses, and poxviruses to establish persistent infections in mammalian cells implies that they have evolved mechanisms to circumvent host antiviral immune defenses. A thorough understanding of molecular and mechanistic aspects of these processes is fundamental to our ability to prevent, treat, and cure virus-induced immune dysfunctions.
It is estimated that Ads are responsible for ca. 30% of virus-induced respiratory diseases (19). In addition to genes involved in replicative functions, the Ad genome includes a number of genes that code for immunomodulatory proteins (reviewed in references 25 and 45). For example, the Ad early transcription unit 1A (E1A) codes for two proteins that suppress transcription of class I genes in host cells. Moreover, the Ad E3 codes for several proteins, of which the 19-kDa protein (E3-19K) is the most abundant, that are dedicated to modulate host cellular immune responses at the posttranslational level.
E3-19K is a type I membrane glycoprotein expressed by all human Ad serotypes with the exception of those from subgroups A and F (33). The basic structural organization of Ad E3-19K-like proteins consists of an N-terminal endoplasmic reticulum (ER) lumenal domain (107 residues), a transmembrane domain (23 residues), and a C-terminal cytosolic tail segment (14 residues) with an ER retention signal (17, 20). The lumenal domain of E3-19K has been subdivided into three distinct regions based on sequence alignment (23): (i) residues 1 to 84 are variable among Ad serotypes in different subgroups, (ii) residues 85 to 98 are conserved among Ad serotypes in different subgroups, and (iii) residues 99 to 107 serves as a linker. E3-19K has been shown to associate with class I major histocompatibility complex (MHC) molecules and prevent their egress from the ER in different Ad-infected cells, including human (8), rat (28), and mouse (9, 14). Two structural features of E3-19K are responsible for these observations: its lumenal domain associates with the lumenal domain of class I MHC molecules (3, 9, 16, 18), whereas its C-terminal cytosolic tail segment blocks the transport of class I MHC molecules to the cell surface by virtue of an ER retention motif (15, 20, 34). The retention of class I MHC molecules in the ER blocks presentation of antigenic peptides to CD8+ cytotoxic T lymphocytes (CTLs).
Although E3-19K is the first viral immunomodulatory protein identified and one of the most characterized, mechanistic aspects of its function remain unexplained. Using a series of biochemical and biophysical techniques, we show here that recombinant, soluble E3-19K is a monomeric protein that is thermally stable and that possesses a correctly folded tertiary structure. The results from in vitro reconstitution experiments revealed that soluble E3-19K associates with both peptide-filled and peptide-deficient HLA-A1101 (hereafter referred to as HLA-A11) molecules. Our studies provide direct insights into molecular aspects of the mechanism by which E3-19K retains class I MHC molecules in the ER. Our findings may have implications for other Ad E3-19K-like proteins given their shared immunomodulatory functions.
MATERIALS AND METHODS
Construction of a baculovirus expression system for E3-19K. The cDNA encoding the signal peptide and ER lumenal domain (residues 1 to 100) of the Ad type 2 E3-19K protein with a C-terminal His6 tag sequence was generated by the PCR. This was carried out with the plasmid pBSKSII (kindly provided by H.-G. Burgert) containing the cDNA of full-length Ad2 E3-19K as a template and the following synthetic primers (restriction sites are in italics, stop codon is underlined, and the His6 tag sequence is in parentheses): 5'-GGATCCATGAGGTACATGATTTTAGGCTTGC-3' (forward) and 5'-GGTACCTTA(GTGATGGTGATGGTGATG)CTTTTGTGGGGGCCACAA-3' (reverse). The amplified cDNA was ligated into the pFastBac-1 vector (Invitrogen) by using BamHI and KpnI restriction sites. Generation of recombinant baculovirus for the expression of soluble E3-19KHis6 was carried out by using the Bac-to-Bac baculovirus expression system (Invitrogen) as recommended by the manufacturer.
Expression and purification of E3-19K. High Five insect cells were cultured at 27°C in serum free Express Five medium (Gibco) supplemented with 1.5% fetal bovine serum (Gibco). Infection of High Five cells with recombinant baculovirus was carried out in 2-liter Delong flasks at a cell density of 1.8 x 106 to 2 x 106 cells/ml, followed by incubation at 27°C for 70 h. The culture medium was centrifuged (700 x g at 4°C for 10 min), the supernatants were adjusted to 0.2 mM phenylmethylsulfonyl fluoride, and centrifugation continued at 6,500 x g for an additional 30 min. Combined supernatants containing soluble E3-19K were filtered through a 0.45-μm-pore-size polyethersulfone disposable filter unit and concentrated ca. 30-fold by using a Prep/scale-TFF cartridge (Millipore). The concentrated supernatant was changed into a buffer containing 5 mM imidazole, 50 mM NaH2PO4, and 200 mM NaCl (pH 8.0) and centrifuged (6,500 x g at 4°C for 15 min). The supernatant was directly applied onto an equilibrated nickel-nitrilotriacetic acid (Ni-NTA) affinity column (QIAGEN). The column was washed with 5 mM imidazole, 50 mM NaH2PO4, and 200 mM NaCl (pH 8.0). The desired protein was eluted with the same buffer containing 200 mM imidazole. The eluate containing crude E3-19K was concentrated by using Centriprep-3 (Amicon), followed by purification on a Superdex 200 HR 10/30 column in 20 mM Tris plus 150 mM NaCl (pH 7.5). Stock solutions of purified E3-19K in the eluant were kept at –70°C.
PNGase F treatment. A sample of purified E3-19K (15 μg) was incubated in 0.5% sodium dodecyl sulfate (SDS) and 1% ?-mercaptoethanol at 100°C for 10 min. The mixture was adjusted to 0.05 M sodium phosphate plus 1% NP-40 (pH 7.5), followed by the addition of 1,000 U peptide:N-glycosidase F (PNGase F) (New England Biolabs). The mixture was incubated at 37°C for 16 h. Digest was terminated by the addition of SDS-polyacrylamide gel electrophoresis (PAGE) loading buffer, followed by heating the sample at 100°C for 5 min and analysis on a 15% SDS-PAGE gel.
Gel filtration chromatography. Samples of purified E3-19K was analyzed at 10°C by using a Superdex 200 HR 10/30 column in 20 mM Tris plus 150 mM NaCl (pH 7.5).
CD. Circular dichroism (CD) experiments were done by using a Jasco-810 spectropolarimeter equipped with a thermoelectric temperature controller. The far- and near-UV spectra represents the average of 8 and 15 scans, respectively. A thermal denaturation curve was obtained in duplicate by monitoring the change in ellipticity at 213 nm in the range 20 to 100°C using a scan rate of 40°C/h. The thermal denaturation mid-point temperatures (Tm) were determined by fitting the averaged thermal denaturation curve to an equation describing a two-state denaturation process (5). Ellipticity values are expressed on a molar residue basis.
Assembly and purification of recombinant, soluble peptide-filled and peptide-deficient HLA-A11 molecules. Peptide-filled HLA-A11 molecules were assembled in vitro as described previously (30). In brief, refolding was initiated by diluting the urea-solubilized inclusion bodies of HLA-A11 heavy chain (residues 1 to 275) (1 μM) and ?2m (2 μM) in the presence of an excess of Nef(73-82) (QVPLRPMTYK) human immunodeficiency virus type 1 peptide (10 mM) (40) in a well-established oxidative refolding buffer (21). Stock solutions (20 to 40 mg/ml) of purified HLA-A11/Nef molecules in 20 mM Tris plus 150 mM NaCl (pH 7.5) were kept at –70°C. Peptide-deficient HLA-A11 molecules were assembled from the guanidinium denaturation of peptide-filled HLA-A11 molecules according to a previously described protocol (6). This strategy has proven to be an effective way of releasing bound peptides and of generating denatured HLA-A11 heavy chain and ?2m with native disulfide bonds. Purified peptide-deficient HLA-A11 molecules (1 to 2 mg/ml) in 20 mM HEPES, 150 mM NaCl, and 20% glycerol (pH 7.5) were kept at –70°C.
Gel mobility shift assay. Samples of purified E3-19K (14 μg) and HLA-A11 molecules (20 μg; 2:1 molar ratio) were incubated in 20 mM Tris plus 150 mM NaCl (pH 7.5; the buffer contained 10% glycerol for peptide-deficient HLA-A11 molecules) on ice for 30 min. Controls and incubation mixtures were immediately assayed on a native PAGE gel (10%) at 4°C in a buffer containing 60 mM Tris plus 40 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) (pH 9.4) (for uncomplexed E3-19K) or 25 mM Tris-200 mM glycine (pH 8.3) (for uncomplexed and complexed HLA-A11 molecules). Proteins were visualized with Coomassie blue.
Analytical ultracentrifugation. Sedimentation velocity runs for E3-19K were carried out at 20°C and 60,000 rpm, with patterns being acquired every 7 s on a Beckman Instruments Optima XL-I analytical ultracentrifuge. A stock solution of E3-19K at ca. 2 mg/ml was dialyzed for 16 h against 20 mM HEPES plus 150 mM NaCl (pH 7.5). Dilutions in the range of 0.3 to 1.0 mg/ml were prepared by using the dialysate as diluent. Sedimentation boundaries were analyzed by using time derivative analysis as described previously (41, 42) and SEDANAL software (43). The value of the partial specific volume (V = 0.723 cm3/g) was calculated from the amino acid sequence of recombinant E3-19K (V = 0.7340 cm3/g), assuming an approximate carbohydrate content of 11% (V = 0.6250 cm3/g), using the consensus partial volumes of the amino acids (35). A value of 0.369 g of water/g of protein for the hydration of recombinant E3-19K was calculated from the amino acid composition. Sedimentation equilibrium runs for E3-19K were carried out at 4°C as described previously (7). A stock solution of E3-19K at ca. 2 mg/ml was dialyzed for 24 h against the same buffer as that for sedimentation velocity runs. A series of dilutions in the range of 0.1 to 1.0 mg/ml were prepared by using the dialysate as diluent. Sedimentation equilibrium data were analyzed by global fitting from combined data at three loading concentrations and two speeds as described previously (7), using the global equilibrium fitter included in the SEDANAL package (43).
Interaction studies between E3-19K and HLA-A11 molecules were carried out by sedimentation velocity at 20°C and 60,000 rpm. Separate stock solutions of E3-19K and HLA-A11 molecules at ca. 2 mg/ml were simultaneously dialyzed overnight against 20 mM HEPES plus 150 mM NaCl (pH 7.5) (buffer contained 20% glycerol for peptide-deficient HLA-A11 molecules). Values of the partial specific volume of HLA-A11/Nef and peptide-deficient HLA-A11 molecules were calculated from amino acid sequences and found to be 0.7240 and 0.723 cm3/g, respectively. Data from four nominal loading concentrations were used in these analyses: [E3-19K]:[HLA-A11 molecules] concentrations of 11:11, 3.7:3.7, 1.2:1.2, and 0.41:0.41 μM. The sedimentation velocity profiles were analyzed with the program SEDANAL (43) to produce the g(s) profiles, and were also globally fitted to a 1:1 association model of type A + B = C to obtain estimates of equilibrium constants. Analysis of peptide-deficient HLA-A11 molecules was also carried with the program SEDFIT using c(s) analysis (36) to reveal the likely existence of conformational isomers.
Temperature-dependent thermolysin digests. Digests of peptide-deficient HLA-A11 molecules and of E3-19K/peptide-deficient HLA-A11 complex were carried out by thermolysin at an enzyme/substrate ratio of 1:100 in 20 mM Tris, 150 mM NaCl, 10% glycerol, and 2 mM CaCl2 (pH 7.5). After a 15-min incubation, digests were terminated by the addition of EDTA (to 10 mM) and boiling samples. Products of digests were analyzed on a 15% SDS-PAGE gel. Digitalized signals of the Coomassie blue-stained band corresponding to HLA-A11 heavy chain were analyzed with 1D Software (Kodak, Rochester, New York).
N-terminal amino acid sequencing. N-terminal amino acid sequencing analyses of proteins were carried out (Tufts University, Boston, MA) from either a SDS-PAGE gel (for E3-19K) or a native PAGE gel (for E3-19K/HLA-A11 complexes). A sample of purified E3-19K was analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (Harvard University, Cambridge, MA).
RESULTS
Characterization of E3-19K. The ER lumenal domain (residues 1 to 100) of E3-19K tagged with a C-terminal His6 sequence was optimally harvested from High Five cell culture at 70 h postinfection (Fig. 1A, lane 1). The crude protein was purified by Ni-NTA affinity (Fig. 1A, lane 2) followed by gel filtration chromatography (Fig. 1A, lane 3) to remove impurities and soluble aggregates. Purified E3-19K migrated as a triplet at ca. 14 to 18 kDa (Fig. 1A, bands 1 to 3 in lane 3). These three bands were individually analyzed by N-terminal amino acid sequencing analysis, and the results confirmed the sequence of mature E3-19K for each band: A1KKVEFK. Analysis of purified E3-19K by MALDI mass spectrometry (Fig. 1B) revealed three distinct regions of peaks, a pattern that is consistent with the series of three bands observed on SDS-PAGE gel (Fig. 1A, lane 3): the single peak at 12.75 kDa is in agreement with the value of 12.74 kDa calculated from the amino acid sequence of unglycosylated E3-19K (including six His residues) (band 3 in Fig. 1A), whereas the two clusters of peaks at higher molecular masses (13.48 to 13.94 kDa and 14.51 to 15.04 kDa) are likely to reflect the differentially glycosylated nature of E3-19K (bands 2 and 1, respectively, in Fig. 1A). Two Asn residues (Asn12 and Asn61) have been identified as putative N glycosylation sites in the amino acid sequence of E3-19K (44). To firmly establish that the three bands observed on the SDS-PAGE gel correlates with the glycosylated nature of E3-19K, a sample of purified E3-19K was incubated with PNGase F under denaturing conditions (Fig. 1C). The results (lane 2) show that PNGase F treatment caused the disappearance of the slowest-migrating band in the control lane (lane 1) and the accumulation of two major products, one of which migrates at the same position as the fastest-migrating band in lane 1. Although the hydrolysis of glycans by PNGase F was only partial, the sensitivity of E3-19K to hydrolysis with this enzyme reflects the glycosylated nature of recombinant E3-19K. A similar pattern of two intense bands were obtained after incubating purified E3-19K with Endo H or with a mixture of PNGase F and Endo H (data not shown). That a subset of E3-19K molecules secreted from High Five cells is resistant to hydrolysis with both PNGase F and Endo H suggests that, in contrast to the ER-resident form of E3-19K, secreted E3-19K has undergone terminal modifications of its N-linked high-mannose carbohydrate moieties (27) in the secretory pathways. For example, it is possible that 1,3-fucose, which is often added to proteins that are secreted from High Five cells and is resistant to hydrolysis with PNGase F and Endo H (1), was incorporated at one of the two Asn residues of E3-19K. Overall, these data strongly suggest that the heterogeneity displayed by recombinant, soluble E3-19K (Fig. 1A and B) is due to N glycosylation. Consistent with our results, electrophoretic heterogeneity of full-length Ad2 E3-19K expressed in Sf9 insect cells was also shown to arise from variations in N glycosylation (29). Analysis of purified E3-19K by native PAGE gel (Fig. 1D) showed that the protein migrates as a single, compact band consistent with a well-behaved protein in solution.
E3-19K is a monomer in solution. The unaggregated nature of soluble E3-19K is reflected in its gel filtration chromatography profile, which shows a single symmetric peak (Fig. 2). Sedimentation equilibrium and velocity analysis of E3-19K unambiguously demonstrated the monodispersed and monomeric nature of the protein in solution, yielding a sedimentation coefficient (S020,w) of 1.56S and an apparent molecular mass of 14.2 kDa. Sedimentation equilibrium analysis showed that soluble E3-19K has a very weak tendency to form reversible dimers (Kd = 1.3 ± 0.1 mM) at high concentrations.
E3-19K adopts a well-defined structure in solution. The far-UV CD spectrum (Fig. 3A) of E3-19K shows a positive maximum at 230 nm and a negative maximum at 210 nm with a discernible shoulder at 215 nm. These spectral features suggest that soluble E3-19K possesses predominantly ?-sheets with little -helices. The thermal denaturation curve (Fig. 3B) of E3-19K shows a single transition centered at a Tm of 62.7°C. The near UV-CD spectrum (Fig. 3C) shows strong spectral features at around 280 nm that essentially disappear at 80°C. These results suggest that aromatic side chains in E3-19K are located in an asymmetrical environment consistent with the protein having a well-defined three-dimensional structure. Overall, CD results indicate that soluble E3-19K possesses conformational properties that can be attributed to a correctly folded three-dimensional structure.
E3-19K associates with peptide-filled and peptide-deficient HLA-A11 molecules. Complex formation between soluble E3-19K and soluble HLA-A11 molecules was monitored by using a gel mobility shift assay. For this experiment, we first reconstituted a soluble form of peptide-filled HLA-A11 molecules that consists of the ER lumenal domain of HLA-A11 heavy chain, ?2m, and the decamer Nef peptide (see Materials and Methods). These HLA-A11/Nef molecules were also used to generate peptide-deficient HLA-A11 molecules according to a well-established protocol (see Materials and Methods). These peptide-deficient class I MHC molecules have been convincingly shown to be devoid of synthetic peptides and to display an unstable peptide-binding groove (6). Moreover, these peptide-deficient class I MHC molecules can be loaded efficiently with antigenic peptides, indicating that they represent a functional, intermediate form of class I MHC molecules (6).
For interaction between E3-19K and peptide-filled HLA-A11 molecules (Fig. 4, lanes 1 and 2, respectively), a native PAGE gel shows a new band (lane 3) that migrates at a different position relative to uncomplexed proteins. This new band was analyzed by N-terminal amino acid sequencing and results confirmed the presence of each component in the complex: E3-19K (A1KKVEFK), HLA-A11 heavy chain [G1SH(_)M(_)Y], ?2m (M1IQRTPK), and Nef peptide [Q1VP(_)(_)P(_)]. Similarly, for interaction between E3-19K and peptide-deficient HLA-A11 molecules (Fig. 4, lanes 1 and 4, respectively), the results show a new band (lane 5) that migrates at a different position relative to uncomplexed proteins. N-terminal amino acid sequencing of this new band confirmed the presence of each protein making up the complex: E3-19K (A1KKVEFK), HLA-A11 heavy chain [G1SHSM(_)Y], and ?2m (M1IQRTPK). Together, these results clearly indicate that recombinant, soluble E3-19K retains a native structure that mediates direct interaction with both peptide-filled and peptide-deficient HLA-A11 molecules.
The stoichiometry and equilibrium constants of E3-19K/HLA-11 molecule complexes were determined by sedimentation velocity experiments (Fig. 5). E3-19K and HLA-A11 molecules were mixed at different ratios (see Materials and Methods) and sedimentation velocity profiles analyzed with the program SEDANAL (43) to fit a 1:1 (A + B = C) association model. A global fit of all data sets (see Materials and Methods) gave a Kd of 50 ± 10 nM for the E3-19K/HLA-A11/Nef complex (curve C in upper panel) and of Kd of about 10 μM for the E3-19K/peptide-deficient HLA-11 molecule complex (curve C in lower panel). Thus, the results from analytical ultracentrifugation studies revealed that although E3-19K associates with peptide-filled and peptide-deficient HLA-A11 molecules, it displays a differential affinity for these two forms: the E3-19K/HLA-A11/Nef complex is energetically considerably more stable than the E3-19K/peptide-deficient HLA-A11 complex. Interestingly, peptide-deficient class I MHC molecules appeared by sedimentation analysis, both with c(s) analysis using SEDFIT (36) and with Lamm equation modeling using SEDANAL (43), to be comprised of at least four conformational isomers that were either distinct or very slowly interconvertible. This finding is consistent with earlier data showing that peptide-deficient class I MHC molecules exist in a partially unfolded molten globule-like state (6). Because of this conformational heterogeneity, only a semiquantitative estimate for the Kd of E3-19K/peptide-deficient HLA-A11 complex could be computed. Whether or not peptide-deficient HLA-A11 molecules in the E3-19K/peptide-deficient HLA-A11 complex also exhibit conformational heterogeneity could not be determined from the data with confidence.
E3-19K stabilizes peptide-deficient HLA-A11 molecules. In previous studies, we showed that peptide-deficient class I MHC molecules have a distinct peptide-binding groove from that of peptide-filled class I MHC molecules (6): the peptide-binding groove is more unstable, it lacks native tertiary packing, and it is considerably more susceptible to cleavage by proteases. To assess whether the association of E3-19K with peptide-deficient HLA-A11 molecules confers stability to the peptide-binding groove, we compared the relative susceptibilities of HLA-A11 heavy chain in peptide-deficient HLA-A11 molecules and in the E3-19K/peptide-deficient HLA-A11 complex to digest with thermolysin at different temperatures (Fig. 6A). SDS-PAGE analysis of products from the digest of peptide-deficient HLA-A11 molecules at 15°C and 25°C showed several new bands that are absent in the control (lane C1). These bands arise from the cleavage of HLA-A11 heavy chain. At 37°C, HLA-A11 heavy chain is nearly completely digested by thermolysin. In contrast, the incubation of E3-19K/peptide-deficient HLA-A11 complex with thermolysin shows that HLA-A11 heavy chain is considerably more resistant to digest at 25 and 37°C, as evidenced by the more intense HLA-A11 heavy-chain bands at these temperatures. At 45°C, the HLA-A11 heavy chain is nearly completely digested by thermolysin, an effect which was observed at 37°C for peptide-deficient HLA-A11 molecules. As expected, ?2m is stable in the range of 15 to 37°C in both experiments, a finding consistent with the thermal stability of ?2m (5). A quantitative analysis (Fig. 6B) of the HLA-A11 heavy chain in the thermolysin gel (Fig. 6A) clearly indicated that the heavy chain is more stable in the E3-19K/peptide-deficient HLA-A11 complex relative to peptide-deficient HLA-A11 molecules. This trend was consistently reproducible in independent experiments. Together, these results indicate that E3-19K confers a stabilizing effect to HLA-A11 heavy chain in the E3-19K/peptide-deficient HLA-A11 complex. Given that the peptide-binding groove is the region of HLA-A11 heavy chain that is destabilized in the peptide-deficient form (6), our results therefore implicate a region of the binding groove as part of the E3-19K interaction surface on class I MHC molecules.
The association of E3-19K with peptide-deficient HLA-A11 molecules is compatible with the binding of antigenic peptides. In previous studies, we showed that peptide-deficient class I MHC molecules are competent in binding antigenic peptides (6; see also lanes 1 and 2 in Fig. 7). Here, we have examined if the association of E3-19K with peptide-deficient HLA-A11 molecules blocks the binding of antigenic peptides. Our results from native PAGE gel (Fig. 7) clearly show that the E3-19K/peptide-deficient HLA-A11 complex (lane 3) is capable of binding Nef peptide, as evidenced by the formation of a new band (lane 4) that migrates at the same position on the gel as preformed E3-19K/HLA-A11/Nef complex (lane 5). N-terminal amino acid sequencing of this new band (lane 4) confirmed the presence of three proteins, E3-19K (A1KKVEFK), HLA-A11 heavy chain [G1SHSM(_)Y], and ?2m [M1IQ(_)TPK], and of Nef peptide [Q1V(_)L(_)P]. These results provide convincing evidence that peptide-deficient class I MHC molecules sequestered by E3-19K can mature into peptide-filled molecules. On the basis of these results, we conclude that E3-19K does not block the binding of antigenic peptides. Based on results shown in Fig. 6, which implicate a region of the binding groove as part of the E3-19K interaction surface, the results presented in Fig. 7 suggest that E3-19K binds away from the interior of the peptide-binding groove.
DISCUSSION
Initial attempts to express the ER-lumenal domain of Ad2 E3-19K were carried out in bacterial cells and yielded high levels of inclusion bodies (13; H. Liu and M. Bouvier, unpublished results). In vitro refolding of these urea-solubilized inclusion bodies under a variety of conditions consistently resulted in large amounts of aggregates (Liu and Bouvier, unpublished). In contrast, expression of the ER lumenal domain of Ad2 E3-19K in baculovirus-infected insect cells reproducibly yielded over 1 mg of secreted protein per liter of cell culture. Characterization of recombinant, soluble E3-19K by SDS-PAGE gel, N-terminal amino acid sequencing, and MALDI mass spectrometry confirmed the correct identity of the recombinant protein. CD studies showed that soluble E3-19K adopts a stable tertiary structure characterized by a relatively high content of ?-sheets. These characteristics are consistent with predictions that E3-19K may be a member of the immunoglobulin superfamily of proteins (12). Sedimentation equilibrium and velocity experiments clearly established the monomeric nature of soluble E3-19K, although a very weak tendency to form reversible dimers (Kd = 1.3 ± 0.1 mM) was evident from our data. It was previously demonstrated from an SDS-PAGE analysis of immunoprecipitates of E3-19K-infected cells (15) and mentioned as "unpublished observations" (32) that full-length E3-19K is capable of forming dimers intracellularly. The physiological significance of these putative E3-19K dimers is undetermined.
In vitro reconstitution experiments showed that soluble Ad2 E3-19K associates with peptide-filled HLA-A11 molecules. This is consistent with numerous cell-based studies which demonstrated that the peptide-filled form of class I MHC molecules is the cellular target of E3-19K (8, 15, 29). Here, we provide conclusive evidence that E3-19K sequesters peptide-deficient class I MHC molecules as well. Our results therefore establish that E3-19K recognizes class I MHC molecules at more than one stage during their biogenesis: after (late stage) and before (early stage) the step of peptide binding. Although cell-based studies also provided evidence that E3-19K associates with class I heavy chain prior to the association of class I heavy chain with ?2m (14, 26, 38, 39), it is of note that we were unable to refold urea-solubilized inclusion bodies of HLA-A11 heavy chain in the presence of E3-19K (data not shown). The association of full-length E3-19K with class I MHC molecules has been shown to occur independently of glycans (8, 9). In view of this, the differently glycosylated nature of secreted E3-19K relative to the ER-resident form of E3-19K is unlikely to have functional implications on our in vitro results.
Sedimentation velocity studies firmly established that E3-19K associates with class I MHC molecules in a 1:1 molar ratio with a Kd of 50 ± 10 nM for the E3-19K/peptide-filled HLA-A11 complex and a Kd of about 10 μM for the E3-19K/peptide-deficient HLA-A11 complex. It cannot be ruled out that E3-19K and HLA-A11 molecules may display a higher affinity in their full-length forms. It is well-known that the transmembrane and cytoplasmic domains of interacting membrane-anchored proteins can contribute in stabilizing complex formation (11), and circumstantial evidence have been provided that this may be the case in the E3-19K/class I MHC system (9, 10, 32, 34).
From a structural point of view, the differential affinity of E3-19K for peptide-filled and peptide-deficient HLA-A11 molecules can be accounted for on the basis of the distinct maturation levels of these molecules. In vivo and in vitro studies have provided evidence that maturation of class I MHC molecules is accompanied by significant peptide-induced conformational changes in the binding groove (37). Specifically, the binding groove is altered from a destabilized and partially folded structure in peptide-deficient molecules to a more stable and native structure in peptide-filled molecules (6). In keeping with this view, and with our results indicating that the peptide-binding groove is implicated in the E3-19K binding site on class I MHC molecules, two potential modes of interaction can be proposed for these 1:1 complexes. (i) E3-19K uses the same binding site on both peptide-filled and peptide-deficient HLA-A11 molecules. The E3-19K binding site is structurally more immature in the peptide-deficient form but, as a result of peptide binding, the site undergoes conformational rearrangements and acquires a native structure. These structural changes optimize interactions between MHC residues and E3-19K, thereby increasing the avidity of E3-19K for the peptide-filled form. (ii) E3-19K recognizes different surfaces on peptide-filled and peptide-deficient HLA-A11 molecules: a high-affinity site on peptide-filled HLA-A11 molecules and a low-affinity site on peptide-deficient HLA-A11 molecules. Here again, we suggest that peptide-induced structural changes within the binding groove leads to formation of the structurally more mature, high-affinity site in peptide-filled molecules and the concomitant elimination of the low-affinity site. We therefore propose that the higher affinity of E3-19K for peptide-filled compared to peptide-deficient HLA-A11 molecules reflects the different conformational states of the peptide-binding groove in these two forms of class I MHC molecules, independently of whether or not these molecules share a common binding site for E3-19K. Crystallization studies will reveal the precise interaction surface of E3-19K on peptide-filled and peptide-deficient class I MHC molecules.
That the affinity of E3-19K for peptide-filled HLA-A11 molecules is in the nanomolar range is entirely consistent with the biological function of E3-19K, which is to tightly retain host proteins in the ER. Although the intrinsic propensity of E3-19K for peptide-deficient HLA-A11 molecules is lower than for peptide-filled HLA-A11 molecules, our results clearly showed that peptide-deficient HLA-A11 molecules sequestered by E3-19K are more stable and, importantly, retain the ability to bind antigenic peptides. These findings may have functional implications since they suggest that, in sequestering and stabilizing peptide-deficient class I MHC molecules, E3-19K not only allows peptides to bind and peptide-filled molecules to be formed but may also actually facilitate the maturation process. The association of E3-19K with peptide-deficient class I MHC molecules may therefore serve to sustain the function of E3-19K, which is directed preferentially at peptide-filled molecules. In spite of the lower affinity of the E3-19K/peptide-deficient class I MHC complex, this association may be important at the beginning of natural infection by Ads when immature class I molecules are more abundant in the ER, before the virus impairs host protein synthesis (2, 24). Overall, we therefore suggest that the ability of E3-19K to act at the late and early stages of the class I assembly pathway is likely to provide a functional advantage to E3-19K. Future studies that examine whether peptide-deficient class I MHC molecules held in association with E3-19K are concomitantly associated with class I assembly proteins will provide important insights into whether E3-19K may play a direct role in maturation of class I MHC molecules in Ad-infected cells. In this context, it was recently shown that the US2 protein from cytomegalovirus uses a binding surface on peptide-filled HLA-A2 molecules that is distinct from the putative binding surface of class I assembly proteins (22). This suggests that, in principle, US2 and class I assembly proteins could simultaneously associate with class I MHC molecules (22). Unlike E3-19K, however, no evidence have been provided that US2 binds to peptide-deficient class I MHC molecules.
In summary, our studies have provided new insights into molecular aspects of the E3-19K immune evasion mechanism. The results showed that the ER lumenal domain of Ad2 E3-19K expressed in insect cells retains a native structure that mediates stable interaction with class I MHC molecules. Importantly, E3-19K sequesters ?2m-associated class I heavy chains independently of their maturation states, although it shows a preference for peptide-filled molecules. This is the first example of a viral immunomodulatory protein that associates with conformationally distinct forms of class I MHC molecules. It was recently shown that E3-19K binds directly to the transporter associated with antigen processing (4). A functional relationship has also been identified between E3-19K and APLP2, a protein presumed to act as a chaperone in the maturation of some, but not all, class I MHC molecules in the ER (31, 38). Together, these mechanisms expand the means by which E3-19K interferes with class I antigen presentation and underline the efficacy with which this immunomodulatory protein retains class I MHC molecules in the ER. Since the identification of immunomodulatory gene products of other large DNA viruses is intensively being pursued, their characterization will eventually reveal whether viruses that directly interfere with the class I antigen presentation pathway share common strategies with E3-19K.
ACKNOWLEDGMENTS
We thank Mingnan Chen for helpful discussions and for valuable comments on the manuscript.
This study was funded by a fellowship from the Outstanding Scholars Program of the University of Connecticut (to H.L.), grant BIR-953060 from the National Science Foundation (to W.F.S.), and grant AI055717 from the NIH/National Institute of Allergy and Infectious Diseases (to M.B.).
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