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Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Facultad de Ciencias, Madrid, Spain
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Proteasomes are multicatalytic protease complexes that mediate most of the protein degradation in the cytosol. They are the main source of HLA class I-bound peptides and can directly generate natural MHC ligands in vitro. However, the contribution of additional proteases from the cytosol or other cell compartments is suggested by evidence that proteasome-specific inhibitors do not totally block class I expression or generation of particular class I ligands (2, 3). Recently, a subtilisin-like protease with the potential to substitute for some of the proteasome function has been reported (4). In addition, peptide trimming could occur in the cytoplasm and ER, generating peptides not produced directly by the proteasome (5, 6, 7, 8, 9, 10). Furin, a protease in the trans-Golgi network may contribute to the production of class I-restricted viral Ags (11). However, the actual contribution of nonproteasomal peptidases to shaping class I-bound peptide repertoires is not known.
Peptides generated in the cytosol are transported into the ER by the TAP transporter, an heterodimeric protein located in the membrane of the ER. Human TAP deficiency causes a severe drop in cell surface expression of class I proteins, indicating that transport of most peptides is TAP dependent (12, 13). Human TAP is less restrictive for peptides than rat and murine counterparts but may exhibit large peptide-binding differences, as revealed by combinatorial peptide libraries (14, 15).
Once into the ER, peptide binding to class I molecules occurs in a process that requires the cooperative activity of at least four chaperones: calnexin, calreticulin, Erp57, and tapasin. The first three proteins have a role in assembling and correct folding of the class I protein and are not known to introduce sequence-dependent restrictions to peptide loading. Tapasin directly mediates the association of TAP and the class I molecule during peptide loading (16, 17). Allelic variation in MHC class I molecules influences their dependence on tapasin for peptide loading and Ag presentation. In particular, HLA-B*2705 seemed able to efficiently form peptide complexes in the absence of tapasin (18).
Finally, peptide binding in the ER will be determined by MHC affinity. Peptides bind to class I molecules through conserved contacts involving the peptidic main chain and the peptide ends. Additional interactions take place between peptidic anchor residues and side chain pockets in the peptide-binding site of the class I molecule (19). Class I polymorphism affects mainly residues located in or close to the pockets, modulating in this way the nature of class I-bound peptide repertoires.
The experiments described in this study arose from the identification of two closely related natural ligands of B*2705: the RRFFPYYV octamer and the C-terminally extended nonamer RRFFPYYVY. These peptides derive from the C5 subunit of the proteasome. The octamer was the specific epitope of an alloreactive CTL clone raised against B*2705, which recognized the nonamer much less efficiently (20). Starting from these observations, we have now addressed the following issues: 1) Are both B*2705 ligands directly generated by the proteasome or require further processing steps? 2) Does the proteasome generate additional peptides from the same region of the parental protein with the capacity to bind HLA-B27? 3) If so, do they bind B*2705 in vivo? 4) What factors impair HLA-B27-binding peptides generated by the proteasome from becoming natural ligands? Together these questions attempt to define the influence of several structural and functional factors in shaping the B27-bound peptide repertoire.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The anti-B*2705 alloreactive CTL 27S69 clone and its culture conditions have been described (20, 21). Hmy2.C1R (C1R) is a human lymphoid cell line with low expression of its endogenous class I Ags. The transfectant expressing B*2705 was cultured in DMEM (Life Technologies, Paisley, U.K.) with 7.5% heat-inactivated FCS. T2 is a TAP-deficient human cell line of lymphoid origin (12). The B*2705-T2 transfectant was a kind gift from Dr. David Yu (University of California, Los Angeles, CA). It was cultured in DMEM supplemented with 5% FCS. RMA-S is a TAP-deficient murine cell line (22, 23).The B*2705-RMA-S transfectant was cultured in RPMI 1640 supplemented with 10% FCS. When cultured at 26°C, T2 and RMA-S transfectants express class I molecules presumably devoid of peptides. These molecules are unstable at 37°C, but their surface expression at this temperature can be stabilized by exogenous peptide ligands.
Isolation of HLA-B27-bound peptides
This was done as described previously (20), with minor modifications. Briefly, 1–1.5 x 1010 B*2705-C1R cells were lysed at 4°C in 20 mM Tris/HCl buffer, 150 mM NaCl, and 1% Nonidet P-40 (pH 7.5) with a mixture of protease inhibitors. Cell lysates were subjected to affinity chromatography using the W6/32 mAb (IgG2a, specific for a monomorphic HLA-A, HLA-B, and HLA-C determinant; see Ref. 24). HLA-B27-bound peptides were eluted from the column with 0.1% trifluoroacetic acid (TFA) in water at room temperature, filtered through Centricon 3 (Amicon, Beverly, MA), and concentrated to 100 µl for HPLC fractionation. This was conducted in a Waters alliance system (Waters, Milford, MA), using a Vydac C18 (0.21 x 25 cm) 5-µm particle size column (Vydac, Hesperia, CA), at a flow rate of 100 µl/min, as follows: isocratic conditions with buffer A (0.08% TFA in water) for 15 min, followed by a linear gradient of 0–44% buffer B (80% acetonitrile and 0.075% TFA in water) for 90 min, and a linear gradient of 44–100% buffer B for another 35 min. Peptide fractionation was simultaneously monitored at 210 and 280 nm. Fractions of 50 µl were collected and stored at -20°C.
Mass spectrometry (MS)
The peptide composition of individual HPLC fractions was
determined by matrix-assisted laser desorption/ionization time of
flight (MALDI/TOF) MS. A calibrated Kompact Probe instrument
(Kratos-Shimadzu, Manchester, U.K.) operating in the positive linear
mode was used. Dried fractions were resuspended in 5 µl
methanol/water (1:1) containing 0.1% formic acid, and 0.5 µl was
applied onto target and dried out. A total of 0.5 µl of saturated
-ciano-4-hydroxycinnamic acid matrix in water:acetonitrile (1:1)
containing 0.1% TFA was then added and dried out. Sometimes, 1 µl of
these samples was subjected to peptide sequencing in a LCQ
electrospray/ion trap mass spectrometer (Finnigan Thermoquest, San
Jose, CA), exactly as previously described (20).
Peptide synthesis and purification
Peptides were synthesized using standard F-moc chemistry and purified by HPLC to a purity >95%. The correct composition and molecular mass of purified peptides were confirmed by amino acid analysis using a 6300 amino acid analyzer (Beckman Coulter, Palo Alto, CA), which also allowed their quantification, and by MALDI-TOF and electrospray ion/trap MS.
Epitope stabilization assays
The quantitative epitope stabilization assay used to measure binding to B*2705 has been described (25). Briefly, B*2705-RMA-S cells were incubated for 24 h at 26°C. They were then incubated without FCS for 1 h at 26°C, with 10-4–10-9 M peptide, transferred to 37°C for 4 h, and collected for flow microcytometry analysis with the ME1 mAb (IgG1, specific for HLA-B27, HLA-B7, HLA-B22; see Ref. 26). Binding was expressed as the C50, which is the molar concentration of the peptide at 50% of the maximum fluorescence obtained with that peptide in the concentration range used. When multiple peptides were compared, the C50 value of a reference peptide was first obtained. Binding of other peptides was expressed as EC50, which is the molar concentration of a given peptide required to obtain the fluorescence value at the C50 of the reference peptide. EC50 values were calculated as described previously (25). Peptides with EC50 values <10 µM were considered to bind with high affinity. EC50 values between 10 and 50 µM were considered to reflect intermediate affinity, and EC50 > 50 µM indicated low affinity.
A previously described (27) cell surface MHC-peptide complex stability assay was used. Briefly, T2-B*2705 cells (105 cells/well) were incubated overnight at 37°C, in serum-free cell culture medium (Serotec, Oxford, U.K.), in the presence of 100 µM peptide and 100 nM ß2-microglobulin. After intensive washing, cells were incubated for 1 h at 37°C in RPMI 1640 containing 10% FCS and brefeldin A (10 µg/ml) to block egress of newly synthesized class I molecules. Cells were washed and incubation continued in the presence of 0.5 µg/ml of brefeldin A at 37°C. Cells were removed at various times and stained with the ME1 mAb as described above. The decay of B*2705-peptide complexes was determined as follows: % mean linear fluorescence (MLF) remaining = [(MLFt(+pep) - MLFt(-pep))/(MLFt = 0(+pep) - MLFt = 0(-pep))].
Inhibition of peptide presentation assay
N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal (LLnL), which reversibly inhibits proteasomes, cysteine proteases, and calpains (28, 29), was purchased from Sigma (St. Louis, MO). Lactacystin (LCT) was purchased from E. J. Corey (Harvard University, Cambridge, MA) and irreversibly inhibits proteasomes (30, 31) and cathepsin A (29, 32). About 5 x 105 B*2705-C1R cells were preincubated for 18–20 h at 37°C in RPMI 1640 medium supplemented with 10% FCS in the presence of LLnL or LCT. Then cells were labeled for 90 min at 37°C with 50 µCi of 51Cr in the continuous presence of proteasome inhibitor, washed (four times) with the same medium plus 4 µg/ml brefeldin A (Sigma), and seeded in 96-well plates with or without 10 µM of the RRFFPYYV octamer. CTL 27S69 was added, and incubation at 37°C for 4 h was conducted in the presence of 2 µg/ml brefeldin A. Specific 51Cr in the supernatants was calculated as follows: (experimental lysis - spontaneous lysis)/(maximum release - spontaneous lysis) x100.
Purification of 20S proteasome and digestion assays
About 3 x 109 B*2705-C1R cells were potter lysed in 50 ml of 50 mM Tris/HCl and 25 mM KCl (pH 8). The homogenate was centrifuged at 1500 x g for 10 min. The supernatant was further centrifuged at 100,000 x g for 1 h, and fractionated on a 35-ml DEAE-cellulose DE52 column (Whatman, Maidstone, U.K.) equilibrated in homogenization buffer. Proteins were eluted with a linear gradient of 0.025–0.6 M KCl in 50 mM Tris/HCl buffer (pH 8). Fractions were analyzed by Western blot using an anti-C2 polyclonal Ab (33). Proteasome-containing fractions were pooled and concentrated in an 8-ml DEAE-cellulose DE52 column equilibrated in homogenization buffer. Bound proteins were eluted with 50 mM Tris/HCl and 300 mM KCl (pH 8). Protein-containing fractions were scanned using the Bradford method (Bio-Rad, Munich, Germany) and further subjected to centrifugation at 200,000 x g for 18 h in a gradient of 10–30% glycerol in 1 M urea, 50 mM Tris/HCl, and 50 mM KCl (pH 8). Proteasome-containing fractions were identified by 12% SDS-PAGE (34) and further subjected to anion-exchange chromatography in a MonoQ SR5/5 column (Pharmacia, Uppsala, Sweden), at a flow rate of 0.5 ml/min, as follows: isocratic conditions with buffer A [50 mM Tris/HCl and 50 mM KCl (pH 8)] for 10 min, followed by a linear gradient of 0–30% buffer B [50 mM Tris/HCl and 0.5 M KCl (pH 8)] for 5 min and a linear gradient of 30–100% buffer B for another 30 min. Purity of the fractions was assessed by denaturing SDS-PAGE as above. Aliquots of purified proteasome were stored at -80°C.
Proteasome digestions were performed at 37°C in 20 mM HEPES buffer (pH 7.4). The peptide substrate was incubated with 4 x 106 cell-equivalents of 20S proteasome/µg at a final substrate concentration of 250 µg/ml. Digestions were stopped by adding 1 vol of 0.1% aqueous TFA and stored at -80°C.
HPLC fractionation of the proteasomal digestions was conducted in a Waters 625LC system using the same column and flow rate as for B*2705-bound peptides (see above) and the following chromatographic gradient: isocratic conditions with buffer A (0.1% TFA in water) for 20 min, followed by a linear gradient of 0–44% buffer B (80% acetonitrile and 0.1% TFA in water) for 80 min, and a linear gradient of 44–100% buffer B for another 40 min.
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Results |
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In preliminary experiments, we tested whether lysis of B*2705-C1R
targets could be affected by the proteasome inhibitors LLnL and the
more specific LCT. As shown in Fig. 1
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cells previously treated with LLnL were not lysed by CTL 27S69 upon
adding brefeldin A to prevent egress of newly synthesized molecules.
The effect was not due to brefeldin A, and lysis was restored upon
addition of the synthetic RRFFPYYV peptide. The same result was
obtained with LCT. This indicates that expression of the
RRFFPYYV epitope is critically dependent on proteasome
activity.
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An examination of the primary structure of the C5 protein around
the core sequence of the CTL 27S69 epitope revealed a nested set of
sequences ranging from 8 to 11 residues with the HLA-B27 peptide motif
R2 (Fig. 2). Thus, we asked whether the
known natural ligands RRFFPYYV and its C-terminally extended nonamer
RRFFPYYVY as well as the other peptides of the nested set (designated
J1-J6) were directly generated in vitro by the 20S proteasome from
B*2705-C1R cells. A precursor peptide spanning residues 120/146 of the
C5 protein was digested for 4, 8, and 24 h at the same
enzyme:substrate ratio. Approximately 30, 82, and 97%, respectively,
of the substrate was digested at these three times on the basis of the
absorbance of the HPLC peak corresponding to the undigested 27-mer at
210 nm. Unfractionated digests at both 4 and 24 h were analyzed by
MALDI-TOF MS (Fig. 3). At both time
points, the maximal signal among the digestion products corresponded to
the RRFFPYYVY nonamer, and two other major peaks corresponded to
RRFFPYYV and to J3. J2 and J5 yielded minor signals at 4 h, the
former of which became more significant at 24 h. Other peptides,
unrelated to the nested set, were also detectable. As a control for the
absence of proteasomal autolysis in vitro, the 20S proteasome was
incubated for 24 h in the same conditions, but in the
absence of the 27-mer substrate. No peptide peaks were observed upon
MALDI-TOF analysis (Fig. 3B).
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This revealed a very complex digestion pattern and the direct
generation of the two natural HLA-B27 ligands plus J1-J5, but not
J6.
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A total of 55 molecular species were detected in the HPLC-fractionated
proteasomal digest (Fig. 4
A), 34 of which were recovered
with a yield 
0.5% (Fig. 4B). The most prominent peptide
was RRFFPYYVY, accounting for 21.4% of the total digest. The second
most abundant peptide was J3 (4.4%), followed by the RRFFPYYV octamer
(2.5%) and J5 (2.3%). J2 and J4 were below 1% (0.5 and 0.8%,
respectively), J1 was obtained with very low yield (0.05%), and J6 was
not detected (Table I). The identity of
the octamer, nonamer, and J1-J5 was confirmed by fragmentation analysis
using electrospray MS/MS.
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B). Most peptides from the RRFFPYYV-related set bind B*2705 in vitro
Binding of J1-J6 to B*2705 in vitro was compared with that of the
octamer and nonamer ligands using a peptide stabilization assay (Fig. 5). The RRFFPYYVY nonamer, J2, J5, and J6
bound with the highest affinity (EC50, 1–2
µM). J3 and the RRFFPYYV octamer bound well but somewhat less
efficiently (EC50, 6–7 µM). J1 and J4 bound
weakly (EC50, >50 µM). Thus, four of the six
peptides in the J1-J6 set bound B*2705 in vitro similarly as the two
natural ligands.
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On the basis of their efficient binding in vitro (Fig. 5), J2, J3,
J5, and J6 had the potential of being natural B*2705 ligands. To search
for their putative presence in the B*2705-bound peptide pool, we first
determined the retention time of the corresponding synthetic peptides
in HPLC. Then the B*2705-bound peptide pool from B*2705-C1R cells was
fractionated in exactly the same chromatographic conditions, and the
HPLC fractions at the retention time of each peptide, as well as
neighbor ones, were analyzed by MALDI-TOF and, in some cases, also by
electrospray ion trap MS (Figs. 6-8). The
natural octamer and nonamer ligands provided a control for the
reproducibility of retention times between the synthetic peptides and
the peptide pool.
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A). However, whereas the nonamer was clearly detected
in HPLC fractions 194–195 of the B*2705-bound peptide pool, no signal
corresponding to J3 was obtained in these fractions or in the following
one (Fig. 6B). This was not due to deficient detection of J3
by MALDI-TOF MS, since in a mixture of the synthetic J3 and nonamer
both peptides were detected with similar sensitivity by this technique
(data not shown).
Similarly, J2 showed the same retention time as the natural
octamer ligand (Fig. 7A) and
both peptides generated similarly intense signals in MALDI-TOF spectra
(data not shown). The octamer was detected in HPLC fractions 189–193
of the B*2705-bound peptide pool corresponding to retention times of
93.5–95.5 min. A prominent peak similar to the molecular mass of J2
(M+H+: 1235.4 Da) was seen in HPLC fraction 192
(Fig. 7B) and, in smaller amounts, in adjacent fractions
(data not shown). When subjected to electrospray MS/MS fragmentation
analysis, the fragmentation pattern was unrelated to J2, as none of the
fragmentation ions of this peptide were observed, but was compatible
with RRFVNVVPTF. This peptide was originally sequenced from B*2702
(35) and subsequently from B*2705 (36). It
has the same molecular mass as J2 and coelutes with the octamer in the
HPLC conditions used (our unpublished observations). Thus, J2 was not
found in this analysis in the B*2705-bound peptide pool.
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A) and similar sensitivity by
MALDI-TOF (data not shown), but they were not present in the
corresponding HPLC fractions of the B*2705-bound peptide pool (Fig. 8B). By the same analysis, we also failed to find J6 in this
pool (data not shown).
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B and 8B, the intensity of the signal for each
individual peptide in a given HPLC fraction is relative to the maximal
signal from that fraction. For instance, in Fig. 6B, the
maximal signal (100%) corresponded to the RRFFPYYVY nonamer and
background noise was about 1%. Thus, absence of a signal corresponding
to J3 suggests that, if present, J3 should be <1% of the nonamer.
This is considerably lower than the proteasomal yield of J3, relative
to the nonamer, in vitro (see above). Similarly, J4 and J5 would
probably be <1% the amount of the peptides that give the maximal
signals in Fig. 8B. Thus, we cannot exclude that J1-J6 might
be present in amounts <100-fold lower than the reference nonamer or
the most abundant peptides in the corresponding HPLC fractions. Stability of the J1-J6 peptides in complex with B*2705
In a final set of experiments, we analyzed peptide stability in
complex with B*2705. The natural octamer and nonamer ligands and J1-J6
were separately incubated in the presence of human
ß2-microglobulin with B*2705-T2 transfectant
cells. After loading the exogenous peptides on cell surface-expressed
B*2705, the cells were treated with brefeldin A, to prevent B*2705
egress, and the decay of HLA-B27-associated fluorescence at the cell
surface was measured as a function of time. The stability of each
peptide in complex with B*2705 was expressed as the time required to
obtain 50% of the maximal fluorescence (DT50), measured just
before adding brefeldin A. The results are shown in Table I. The
natural octamer and nonamer ligands had DT50
values of 13.2 and 17.9 h, respectively, indicating that
DT50 values in this range or higher are
appropriate for natural ligands. Thus, absence of J5 and J6
(DT50, 15.2 and 17.4 h, respectively) in
vivo was not explained by low stability. The DT50
value of J4 (9.9 h) was somewhat lower than for the octamer. However,
its binding in vitro was weak (EC50, 61 µM),
suggesting that J4 associates inefficiently with B*2705. The lowest
DT50 value corresponded to J1 (7.6 h), a peptide
that bound very weakly in vitro (EC50, >100
µM), J2 (8.5 h), which nevertheless bound efficiently in vitro
(EC50, 2 µM), and J3 (9.1 h). It is unclear
whether any of the DT50 values of J1-J6 are
outside the range allowed for natural ligands. However, J2 and J3,
which differ from the octamer and nonamer, respectively, by one
additional N-terminal Ser residue, showed lower stability than the
respective natural ligands. This might be related to the absence of J2
and J3 in vivo (see Discussion).
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Discussion |
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8.5:1 ratio. This is not very different from the
estimated 4:1 ratio of these peptides in the B*2705-bound pool
(20). It has been pointed out that in vitro digestions of
relatively short peptides with the 20S proteasome do not resemble the
situation in vivo where ubiquitinated proteins are digested by the 26S
proteasome (2). However, generation of the octamer and
nonamer in vitro in a ratio comparable to that found in the
B*2705-bound peptide pool suggests that their generation in vivo may be
similar and a major determinant of their presence in that pool, with
transport and tapasin-mediated loading not having a major influence on
their relative abundance. Second, that the nonamer was the major digestion product after 4 h, when only 30% of the precursor peptide was digested, and the octamer was also in significant amounts indicates that proteasomal generation of these peptides is strongly favored. That their yields were maintained after 24 h indicates that their kinetics of generation and stability with the 20S proteasome is similar. Although the proteasome can destroy class I epitopes (37) and despite cleavage within the core octamer sequence, the stability of both peptides in the presence of the proteasome might favor their abundance in vivo.
The J1-J6 set provided the opportunity to follow, in a defined set of related peptides, the influence of different factors on shaping the HLA-B27-bound peptide repertoire. Although our MS analysis does not rule out that the J1-J6 peptides could be present in very low amounts, failure to detect them in the B*2705-bound pool suggested a significant restriction on the number of putative ligands arising around a core sequence from a given protein, a situation quite different from the nested peptide sets frequent among class II Ags (38, 39, 40).
Only the absence of J1 and J4 could be explained on the basis of low binding to B*2705 in vitro, as all other peptides bound similarly as natural ligands. Proteasome processing accounted only partially for further limitation, since only J6 was undetected, and J1 was generated with very low yield. The possibility that processing in vivo may yield a different peptide pattern is clearly open. Indeed, since proteasome activity is highly regulated in vivo, it is possible that the pattern of proteolytic products, especially for those reflecting minor cleavage specificities, may not be conserved in vivo. However, on the basis of the correlation between in vitro processing and in vivo occurrence of the octamer and nonamer ligands, our results suggest that at least J1-J5 might be generated in vivo and some (J3 and J5) perhaps in an amount comparable to the octamer epitope.
The efficiency with which these peptides bind TAP could influence their presence in the B*2705-bound repertoire. TAP-mediated transport was not addressed in this study. However, previous analyses using combinatorial peptide libraries have established the importance of the three N-terminal residues and, especially, the C-terminal one for human TAP binding, and defined the effect of different amino acid residues at these positions (14, 15). On that basis, J1-J3 and J6 have favored TAP-binding motifs and would presumably be transported in a similar way. In contrast, C-terminal Asn is a disfavored TAP-binding motif, and this might limit availability of J4 and J5 in the ER.
Thus, despite the limitations of in vitro assays, which cannot obviously reproduce the situation in vivo, it was possible to correlate low proteasomal digestion yields, disfavored TAP-binding motifs, and/or low B27-binding efficiency with the absence of J1, J4, J5, and J6 in the B*2705-bound pool. However, the absence of J3, and perhaps also J2, was not satisfactorily explained by these criteria.
A possible explanation for the absence of J3 among natural ligands might be its low stability (about 50% in our assay), relative to the natural nonamer ligand, from which it differs only by an N-terminal Ser extension. There is evidence for non-proteasomal cytosolic and ER proteases capable of trimming the N-terminal residues of proteasomal peptide products (2, 5, 7, 9, 10). It is conceivable that trimming of J3 to generate the RRFFPYYVY nonamer might be favored by the higher stability of this peptide in complex with B*2705. A similar mechanism might explain the absence of J2, an N-terminal extension of the octamer, which also binds HLA-B27 in vitro with lower stability (64%) than the octamer ligand.
Thus, of a nested set of eight peptides with size and peptide motifs
appropriate for binding to HLA-B27, only two become bound in vivo. This
limitation cannot be explained by a single bottleneck feature, but
rather by a combination of requirements including efficient proteasomal
processing, appropriate TAP-binding motifs, high affinity for HLA-B27,
and sufficient stability of the B27-peptide complex (Table I). In
addition, N-terminal trimming might contribute to further reduce the
number of closely related ligands toward those forming more stable
complexes with HLA-B27. A role of tapasin in limiting loading of these
peptides into HLA-B27, although not ruled out, does not need to be
invoked.
In conclusion, in contrast to class II molecules, nested peptide sets are unlikely to exist in class I-bound repertoires. The more strict length requirements for class I-ligands do not fully account for this absence, since in the allowed size range there is room for nested peptide sets with the potential to bind the class I molecule, as in the case analyzed here. Rather, besides size and MHC affinity, the specificity features of the processing and transport systems for class I-bound peptides and the stability of the MHC-peptide complex are critical limiting factors.
A final word should be devoted to the experimental strategy used in this study. It was based on in vitro analysis of proteasome cleavage patterns, peptide binding to "empty" HLA-B27 on the cell surface, stability of such complexes, and a consideration of TAP-binding motifs as defined with peptide libraries. Obviously, in vitro assays do not reproduce the conditions of peptide-processing, transport and MHC binding in vivo. Therefore, our results do not exclude that processing and binding of the peptides analyzed in this study may be different in vivo. Despite these limitations, our analysis explains the presence of two natural ligands of HLA-B27 on the basis of their processing and binding features in vitro. Similarly, failure to detect various related peptides in the B27-bound pool correlated with one or more unfavorable features related to their processing, transport, binding, or stability in vitro. This close correlation cannot be ignored and strongly suggests that some major factors influencing the composition of class I-bound peptide repertoires can be dissected and characterized in vitro.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. José A. López de Castro, Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid, Facultad de Ciencias, Cantoblanco, 28049 Madrid, Spain. E-mail address:
3 Abbreviations used in this paper: ER, endoplasmic reticulum; TFA, trifluoroacetic acid; C1R, Hmy2.C1R; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption/ionization time of flight; MLF, mean linear fluorescence; LLnL, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal; LCT, lactacystin; DT50, decay time at 50% of the maximal fluorescence.
Received for publication June 25, 1999. Accepted for publication October 19, 1999.
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L. Sesma, I. Alvarez, M. Marcilla, A. Paradela, and J. A. L. de Castro Species-specific Differences in Proteasomal Processing and Tapasin-mediated Loading Influence Peptide Presentation by HLA-B27 in Murine Cells J. Biol. Chem., November 21, 2003; 278(47): 46461 - 46472. [Abstract] [Full Text] [PDF]
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M. Ramos, I. Alvarez, L. Sesma, A. Logean, D. Rognan, and J. A. Lopez de Castro Molecular Mimicry of an HLA-B27-derived Ligand of Arthritis-linked Subtypes with Chlamydial Proteins J. Biol. Chem., September 27, 2002; 277(40): 37573 - 37581. [Abstract] [Full Text] [PDF]
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M. Ramos, A. Paradela, M. Vazquez, A. Marina, J. Vazquez, and J. A. Lopez de Castro Differential Association of HLA-B*2705 and B*2709 to Ankylosing Spondylitis Correlates with Limited Peptide Subsets but Not with Altered Cell Surface Stability J. Biol. Chem., August 2, 2002; 277(32): 28749 - 28756. [Abstract] [Full Text] [PDF]
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L. Sesma, V. Montserrat, J. R. Lamas, A. Marina, J. Vazquez, and J. A. L. de Castro The Peptide Repertoires of HLA-B27 Subtypes Differentially Associated to Spondyloarthropathy (B*2704 and B*2706) Differ by Specific Changes at Three Anchor Positions J. Biol. Chem., May 3, 2002; 277(19): 16744 - 16749. [Abstract] [Full Text] [PDF]
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J. Yague, A. Marina, J. Vazquez, and J. A. Lopez de Castro Major Histocompatibility Complex Class I Molecules Bind Natural Peptide Ligands Lacking the Amino-terminal Binding Residue in Vivo J. Biol. Chem., November 16, 2001; 276(47): 43699 - 43707. [Abstract] [Full Text] [PDF]
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 B. Korber, B. Gaschen, K. Yusim, R. Thakallapally, C. Kesmir, and V. Detours Evolutionary and immunological implications of contemporary HIV-1 variation Br. Med. Bull., September 1, 2001; 58(1): 19 - 42. [Abstract] [Full Text] [PDF]
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I. Alvarez, L. Sesma, M. Marcilla, M. Ramos, M. Marti, E. Camafeita, and J. A. L. de Castro Identification of Novel HLA-B27 Ligands Derived from Polymorphic Regions of Its Own or Other Class I Molecules Based on Direct Generation by 20 S Proteasome J. Biol. Chem., August 24, 2001; 276(35): 32729 - 32737. [Abstract] [Full Text] [PDF]
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I. Alvarez, M. Marti, J. Vazquez, E. Camafeita, S. Ogueta, and J. A. Lopez de Castro The Cys-67 Residue of HLA-B27 Influences Cell Surface Stability, Peptide Specificity, and T-cell Antigen Presentation J. Biol. Chem., December 21, 2001; 276(52): 48740 - 48747. [Abstract] [Full Text] [PDF]
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, Percent specific lysis of
target cells either untreated, incubated with 4 µg/ml brefeldin A, or
brefeldin A plus proteasome inhibitor. 
, Lysis of the same targets
in the presence of brefeldin A, proteasome inhibitor, and 10 µM
RRFFPYYV peptide. Specific lysis of untransfected C1R cells at the E:T
ratio used (2:1) was 0%. Data are means of two (for LLnL) or three
(for LCT) independent experiments.
