Key words:HLA-B39; B*3909; HLA-B27; HLA polymorphism;mass spectrometry; peptides; peptide motifs
Acknowledgments:We thank Masafumi Takiguchi (University ofTokyo) for the B*3901 construct, Fernado Roncal(Centro Nacional de Biotecnologıa, Madrid) forhelp in peptide synthesis, and FranciscoGavilanes (Universidad Complutense de Madrid)for amino acid analyses. We also acknowledgethe help of our colleague Fernando Garcıa andthe excellent technical assistance of JuanaBustos, Josefa Gonzalez-Nicolas (ProteinChemistry Department, CBMSO, Madrid), andAlicia Prieto (Centro de InvestigacionesBiologicas, Madrid) in tissue culture, Edmansequencing, and MALDI-TOF analyses,respectively. This work was supported by grantsSAF97/0182 from the Plan Nacional de IπD andPM95-002 from the Spanish Ministry ofEducation, and by an Institutional grant fromthe Fundacion Ramon Areces to the CBMSO.
Received 24 November,accepted for publication 30 December 1999
Copyright c Munksgaard 1999Tissue Antigens . ISSN 0001-2815
Tissue Antigens 1999: 53: 227–236Printed in Denmark . All rights reserved
227
J. Yague The South Amerindian allotype HLA-B*3909M. Ramos has the largest known similarity in peptideJ. VazquezA. Marina specificity and common natural ligands withJ.P. AlbarJ.A. Lopez de Castro HLA-B27
Abstract: HLA-B*3909 has only been found among South Amerindians,and presumably arose locally in these populations. It differs from B*3901 bya single Tyr to Ser change at position 99. To analyze the influence of thispolymorphism on peptide specificity, pool sequence analysis and sequencingof multiple individual ligands from B*3901 and B*3909 were carried out.Both allotypes bind peptides with Arg2 or His2 and nonpolar C-terminalresidues. However, whereas His2 is the predominant B*3901 motif, a ma-jority of the B*3909-bound peptides have Arg2. In addition, B*3909 bindspeptides with Pro2, and also shows an increased preference for Pro3. Inspite of their differences, both subtypes bind overlapping peptide repertoires,as indicated by the identification of several identical ligands from theirrespective peptide pools. B*3909 is significantly more similar in its peptidespecificity to HLA-B27 than B*3901. This is due to the increased preferenceof B*3909 for Arg2 and to low suitability of HLA-B27 for His2. The simi-larity between HLA-B27 and B*3909 was confirmed by identification of anatural ligand common to both allotypes. In addition, multiple HLA-B27ligands bound efficiently B*3909 in vitro. The results indicate that, amongthe HLA class I allotypes of known peptide specificity, B*3909 is the mostsimilar in its peptide binding properties to HLA-B27, which is absent inSouth Amerindians. This may have implications for the susceptibility ofindividuals in these populations to spondyloarthropathies.
Indigenous populations in South America exhibit limited repertoires
of HLA class I alleles. However, the B-locus appears to have sub-
stantially diversified after the ancestor population settled on the
South American continent, giving rise to many HLA-B alleles not
found elsewhere, whose origin is explained mainly by microconver-
sion events involving ancestor alleles frequent in these populations
(1–5). Two lines of evidence suggest that the HLA-B repertoires of
South American Indians have evolved as an adaptive response to
the local environment. First, the size of the HLA-B repertoire in
unmixed South American populations is not significantly different
from those in North America, but the latter populations show little
Authors’ affiliations:
J. Yague1,M. Ramos1,J. Vazquez1,A. Marina1,J.P. Albar2,J.A. Lopez de Castro1
1Centro de BiologıaMolecular Severo Ochoa,Consejo Superior deInvestigaciones Cientıficas(CSIC) and UniversidadAutonoma de Madrid,Facultad de Ciencias,Cantoblanco, Madrid,Spain,
2Immunology and OncologyDepartment, Pharmacia-CSIC, Centro Nacional deBiotecnologıa, Cantoblanco,Madrid, Spain
Correspondence to:Dr. Jose A. Lopez de CastroCentro de Biologıa Molecular
Severo OchoaUniversidad Autonoma de
MadridFacultad de CienciasCantoblanco28049 MadridSpainFax: π34 91 397 8087
Yague et al : Peptide specificity of B*3909
evidence for class I alleles of recent origin. This suggests that allelic
turnover is taking place in South America, so that new B-locus alleles
progressively replace new ones (3, 5). Second, the new allotypes
usually differ from their ancestors by amino acid changes in the pep-
tide-binding site, and therefore must have distinct peptide binding
and presenting properties (3–5). It is likely that the number of founder
HLA-B alleles in South America was very small. Thus, many of the
new variants derive from a single founder allele and constitute sets of
subtypes with the same serologic specificity. For instance, at least 14
HLA-B35 and 8 HLA-B39 alleles have only been found in South and
Central America (5). Thus, to understand the nature of the adaptive
response of the HLA-B repertoire it is essential to determine how the
newly acquired polymorphism affects peptide specificity.
HLA-B39 has an added interest because it has a B pocket similar
to the ankylosing spondylitis-associated HLA-B27 antigen. HLA-B27
is absent from unmixed South Amerindian populations (6), but
B*3901, which is found in South America and elsewhere, presents
peptides with Arg2 or His2 (7), and binds in vitro a subset of the natu-
ral HLA-B27 ligands (8). Some HLA-B39 subtypes found outside
South America have amino acid changes that affect the B pocket and,
by altering its side chain specificity, totally change the repertoire of
peptides bound to these subtypes. This is the case with B*3902 (7),
also found in South America (B*39022) (5), and B*3910 (9).
B*3909 was originally found in a Warao Indian from the Orinoco
Delta in Venezuela (10), and subsequently in a Xavante Indian from
Brazil (4). More recently it was found at a high frequency among
the Yucpa of Venezuela (39), but has not been found outside South
America. It differs from the B*3901 subtype by a single Tyr to Ser
change at position 99. By its location, this change could affect the
specificity of pockets B and D, which bind residues 2 and 3, respec-
tively (10). In this study, we have determined the peptide motifs of
B*3909, sequenced multiple novel B*3901 ligands, and tested the
binding of HLA-B27-bound peptides to B*3909. The results show
that the peptide specificity of B*3909 is more similar to HLA-B27
than B*3901, identify an individual ligand that is naturally pre-
sented by both B*3909 and HLA-B27, and provide evidence for a
substantial overlap between the HLA-B27- and B*3909-bound pep-
tide repertoires.
Material and methods
DNA, transfectant cell lines and antibodies
HLA-B*3901 genomic DNA cloned into pBluescript (a kind gift of
Dr. M. Takiguchi, University of Tokyo, Tokyo, Japan) plus pSV2neo
and a full-length cDNA clone with the coding sequence of B*3909
228 Tissue Antigens 1999: 53: 227–236
cloned into RSV5neo (10) were separately transfected into the HLA
class I defective HMy2.C1R cells by electroporation as previously
described (11). C1R transfectants were cultured in Dulbecco’s modi-
fied Eagle medium (DMEM) supplemented with 7,5% heat-inacti-
vated fetal bovine serum (FBS) (both from Gibco, Paisley, UK). The
same B*3909 construct was also co-transfected, together with the
human b2-microglobulin gene, into the transporter associated with
antigen processing (TAP)-deficient murine RMA-S cells as follows:
1.5¿107 cells were electroporated on ice with 15 mg of each con-
struct, at 250V and 960 mF. Cells were kept on ice for 10 min both
before and after the electroporation, and were then plated and cul-
tured under selection as previously described (12). The B*2705-
RMA-S transfectant cells used in this study have been described
elsewhere (13). RMA-S transfectants were grown in RPMI 1640 me-
dium containing 25 mM HEPES buffer and 5% heat-inactivated FBS
(Gibco).
The W6/32 mAb, specific for an HLA-A, B, C monomorphic de-
terminant (14), was purified by affinity chromatography with Hi-
Trap protein G, and coupled to CNBr-activated Sepharose 4B (both
from Pharmacia, Uppsala, Sweden) following standard procedures.
The ME1 mAb, specific for HLA-B27πB7πB22 (15), was used as
supernatant of the hybridoma cell culture.
Flow microfluorometry (FMF) analysis
Expression of HLA-B39 or HLA-B27 was measured by FMF using 50
ml of a solution containing 45 mg/ml of W6/32 in PBS, or 50 ml of
culture supernatant of ME-1 respectively, as previously described
(12).
Purification of HLA-B39-bound peptides
About 1010 cells were grown at 37æC in 2,000 ml roller flasks, in
DMEM pH 7.4, containing 7,5% heat-inactivated FBS, 100 mg/ml
Streptomycin sulfate and 100 units/ml Penicillin G (all from Gibco).
B*3901 and B*3909 were purified by affinity chromatography of
the cell lysates obtained in the presence of protease inhibitors, and
their peptide pools were obtained by acid extraction with TFA and
fractionated by high-performance liquid chromatography (HPLC) as
previously described (12, 16).
Edman sequencing
Peptide pools and some individual HPLC fractions were subjected
to Edman degradation, as previously described (16). Residue assign-
ments in the pool sequence experiments were done as described
elsewhere (17).
Yague et al : Peptide specificity of B*3909
Synthetic peptides
Peptides were synthesized in an AMS 422 Multiple Peptide Syn-
thesizer (Abimed, Langelfeld, Germany) using the Fmoc chemis-
try, and purified by reverse-phase HPLC. Their correct compo-
sition was determined by amino acid analysis and mass spec-
trometry as previously described (12). Peptides were stored at 4æC
as stock solutions in water. One peptide (AHDETEFYL) was dis-
solved in aqueous ammonia (0.56% v/v) due to its low solubility
in water.
Mass spectrometry analysis and sequencing
The peptide composition of individual HPLC fractions was analyzed
by matrix-assisted desorption-ionization time of flight (MALDI-
TOF) mass spectrometry using a calibrated Biflex instrument
(Brucker-Franzen Analytik, Bremen, Germany) as previously de-
scribed (18). Peptide sequencing by quadrupole ion trap mass spec-
trometry was carried out using an LCQ instrument (Finnigan MAT,
San Jose, CA, USA). Only one peptide (SRDKTIIMW eluted from
B*3901) was sequenced using the microspray interface as described
by Paradela et al. (18). All other sequences were obtained using the
‘‘nanospray’’ interface originally described by Wilm and Mann (19)
as detailed elsewhere (9, 20). Sequence assignments were usually
confirmed by re-fragmenting (MS3) some ‘‘daughter ions’’ coming
from the parental one (20). Most of the sequence assignments were
further confirmed by reproducing the MS/MS spectrum with the
corresponding synthetic peptides.
Epitope stabilization assay
The quantitative epitope stabilization assay used to assess peptide
binding to HLA in vitro has been described elsewhere (21). Briefly,
RMA-S transfectants were incubated at 26æC for 24 h. They were
then incubated 1 h at 26æC with 10ª4 to 10ª9 M peptides without
FBS, transferred to 37æC and collected for FMF analysis with the
W6/32 (for HLA-B39) or ME1 mAb (for HLA-B27) after 4 h. 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. Relative binding of
multiple peptides was assessed as follows. First, the C50 of a refer-
ence peptide was calculated. Second, the concentration of each other
peptide required to obtain the fluorescence value at the C50 of the
reference peptide was found by interpolation. This was designated
as EC50, which is equal to the C50 for the reference peptide. Relative
binding was expressed as the ratio between the EC50 values of the
peptides compared. Peptides with EC50Æ5 mM were considered to
229Tissue Antigens 1999: 53: 227–236
bind with high affinity; EC50 values between 5 mM and 50 mM were
considered to reflect intermediate affinity; EC50 between 50 mM and
100 mM indicated low affinity; EC50.100 mM was considered to
reflect lack of binding.
Results
HLA-B39-bound peptide sequencing: B*3909 presents a
natural HLA-B27 ligand
The differences in peptide specificity between B*3909 and B*3901
were determined by pool sequencing of B*3909-bound peptides (Fig.
1), and by sequencing 10 individual ligands from B*3901 (1 heptamer,
1 octamer, 7 nonamers, and 1 decamer) (Fig. 2A) and 12 ligands from
B*3909 (5 octamers and 7 nonamers) (Fig. 2B). The GRYATAA pep-
tide is, to our knowledge, the first heptamer sequenced from a major
histocompatibility complex (MHC) class I-bound peptide pool. The
large majority of these peptides were sequenced by electrospray
quadrupole ion trap mass spectrometry. An example is illustrated in
Fig. 3. The MS3 capability of the LCQ instument was used frequently
to confirm the assignments based on the MS/MS spectra, and to re-
solve ambiguities resulting from incomplete fragmentation of the
peptide by MS/MS. A detailed example of the use of MS3 for sequenc-
ing a B*3901 ligand was previously reported (20).
Three identical peptides were sequenced from both B*3901 and
B*3909 (Fig. 2), indicating that both subtypes bind overlapping pep-
tide repertoires in vivo. One of these peptides (EHAGVISVL) was
also previously sequenced from the B*3801-bound peptide pool (7).
Among the peptides sequenced from B*3909, SRDKTIIMW (Fig.
3) was previously reported from both B*2702 (22) and B*2701 (23).
This result demonstrates that B*3909 binds in vivo a natural ligand
of at least two HLA-B27 subtypes.
Main anchor residues: Arg2 is a predominant B*3909
peptide motif
The main peptidic anchor positions (P) for B*3901 and B*3909, as
in most HLA-B proteins, are P2 and the C-terminal one (PW). Like
B*3901, B*3909 allowed for both Arg and His at P2. However, Arg2
was largely predominant upon pool sequencing: the molar ratio of
pth-Arg relative to pth-His at cycle 2 was 4 for B*3909 (Fig. 1), and
only 2 for B*3901 in a control experiment (data not shown). In
addition, of the 13 B*3901 ligands sequenced, including 10 in this
study and 3 from a previous one (7), 5 (38%) have Arg2 and 8 (62%)
have His2 (Fig. 2A). Among B*3909 ligands (Fig. 2B), 9 (75%) have
Arg2, and only 2 (17%) have His2. These results indicate that
Yague et al : Peptide specificity of B*3909
Fig. 1. Pool sequence analysis of B*3909-bound peptides. Num- positions 2 to 4) or 50% (in positions 5 to 9) over either of the previousbers indicate picomole yields of each pth-amino acid at each cycle of Edman two cycles and their absolute yield was .5% of the total amount of pth-degradation. Residues were assigned as previously described (17). Briefly, amino acids at that cycle. Residues within each group in each cycle area residue was assigned at a given position when its absolute yield in that ordered according to their absolute yields, but this does not necessarily re-cycle was increased at least 50% over either of the two previous cycles, flect their relative frequency in that position. Pool sequencing was donebut not if it decreased compared to the previous one. Assigned residues three times from independent preparations, with similar results. One repre-(underlined) were considered strong when they increased at least 300% (in sentative experiment is shown.
B*3909 polymorphism modulates the relative allowance for Arg and
His at P2, towards increasing preference for Arg2.
Pro2 was also observed in B*3909 upon pool sequencing, where
its pth increased about 400% over the previous cycle (Fig. 1), but
was not reported for B*3901 (7). In our control experiment of
B*3901-bound peptides, pth-Pro at cycle 2 was increased relative to
cycle 1 below 50%, which is the adopted criterion for motif assign-
ments by pool sequencing (data not shown). Thus, our result indi-
cates that preference for Pro2 is increased in B*3909 relative to
B*3901. In addition, one ligand with Pro2 was sequenced from
B*3909 (Fig. 2B).
Only Leu and other aliphatic C-terminal residues were previously
reported for B*3901 (7). We have now identified a B*3901 ligand
with C-terminal Phe, indicating that aromatic PW residues are also
allowed in this subtype. However, a great majority of the B*3901
ligands sequenced in this study had C-terminal Leu (Fig. 2A). Both
Leu and Phe were predominant among B*3909-bound peptides by
pool sequencing (Fig. 1). Among the individual B*3909 ligands se-
quenced, most had C-terminal Leu, one had Met, one had Trp, and
230 Tissue Antigens 1999: 53: 227–236
none had Phe. Thus, B*3909 and B*3901 have very similar C-ter-
minal motifs, consisting predominantly of Leu and, secondarily,
other aliphatic and aromatic residues, but not Tyr.
Secondary anchor motifs: distinct preference of B*3909 for
Pro3
Both B*3901 and B*3909 accommodate a wide variety of residues
at P1. Large aliphatic residues, Asn and Gln were rare or absent
among the sequenced ligands from both subtypes. The only notice-
able difference was an increased frequency of Ser plus Thr residues
in B*3901 (54%) relative to B*3909 (25%).
At position 3, both subtypes accommodated also chemically di-
verse residues. However, a main difference was the higher frequency
of Pro3 in B*3909. This residue was not previously reported for
B*3901 (7), nor was it found among the B*3901 ligands sequenced
in this study (Fig. 2A). In contrast, Pro3 accounted for 18.5% of the
total pth-amino acids detected at cycle 3 upon pool sequencing of
B*3909 peptides (Fig. 1). This value was only 1.7% in our control
Yague et al : Peptide specificity of B*3909
pool sequencing from B*3901 (data not shown). In addition, Pro3
was found in 4 (33%) of the individual B*3909 ligands sequenced
(Fig. 2B). Thus, preference for Pro3 is a feature distinguishing
B*3909 from B*3901. Asp3 was found more frequently among
B*3909 ligands, but it was similarly increased in B*3909 and
B*3901 upon pool sequencing (Fig. 1 and data not shown).
Both subtypes were similar in their relaxed selectivity at P4, P5,
and P6. At P7, no Ala was reported from B*3901 (7), and was found
in only the heptamer from this subtype. In contrast, Ala7 was in 3
(25%) of the B*3909 ligands, which is compatible with an increased
preference for Ala7 in this subtype. Finally, no significant differ-
ences between B*3901 and B*3909 were observed at P8 among pep-
tides longer than octamers.
Peptide binding in vitro: the relationship between B*3909
and B*2705
The similarities between some of the peptide motifs of B*3909,
namely Arg2 and nonpolar C-terminal residues, with those of HLA-
Fig. 2. Amino acid sequence of individual peptides from (A) the trap mass spectrometry, except ARDETEFYL, which was sequenced byB*3901-bound and (B) the B*3909-bound peptide pools. The HPLC Edman degradation. The SRDKTIIMW (see Fig. 3) was sequenced by bothfraction number from which the sequence was obtained, the protein data- methods. Edman sequencing was also used in some cases to confirm as-base match of the sequence, and the molecular mass of the sequenced spe- signments from mass spectrometry, especially those concerning the two N-cies, as determined by quadrupole ion trap mass spectrometry, are indi- terminal peptide residues. Residues with the same molecular mass (I/L andcated. Peptides labelled with asterisk (*) are those that were sequenced from Q/K) were assigned, whenever possible, on the basis of unambiguousboth subtypes. Most of the peptides were sequenced by quadrupole ion matching with database sequences.
231Tissue Antigens 1999: 53: 227–236
B27, and the shared SRDKTIIMW ligand, indicate that both allo-
types have overlapping peptide specificities. To gain further insight
into the extent of this overlap, two issues were addressed by testing
natural HLA-B27 and -B39 ligands, and analogs in which Arg and
His were interchanged at P2, for binding to both B*2705 and
B*3909. The first issue was the relative suitability of Arg2 and His2
for each allotype. Four peptides were tested: a B*3909 ligand with
Arg2 (ARDETEFYL), its analog with His2, a B*3901 ligand with
His2 (YHTDTTVKF), and its analog with Arg2. As shown in Fig.
4A, the two peptides with Arg2 bound to B*2705, whereas those
with His2 did not bind. These results indicate that His2 is not a
suitable residue for B*2705. In contrast, both Arg and His2 were
nearly equivalent for binding to B*3909 (Fig. 4B): both the B*3909
ligand and its His2 analog bound efficiently (C50: 7 mM), whereas
both the B*3901 ligand and its Arg2 analog bound very weakly.
The second issue was whether natural HLA-B27 ligands with
Arg2 would bind to B*3909. To address this, 13 natural HLA-B27
ligands and one B*3909 ligand were tested for binding to B*3909
(Fig. 5 and Table 1). Among the peptides tested, those with C-ter-
Yague et al : Peptide specificity of B*3909
Fig. 3. (A) Sequencing of the SRDKTIIMW peptide from the of carbon monoxide (28 daltons) from ions of the b series. The b4 ion wasB*3909-bound peptide pool by quadrupole ion trap mass spec- generated with very low yield, but was assigned on the basis of its moretrometry. The MS/MS fragmentation spectrum of ion at mass/charge clear detection with the corresponding synthetic peptide. The sequence of(m/z) 575.2 is shown. The assigned peptide sequence is indicated, detailing this peptide was also obtained by Edman degradation, which confirmed thethe major backbone fragment ions according to the nomenclature of Roep- SR and KT sequence assignments (data not shown). (B) MS/MS frag-storff and Fholman (38). Ions of the a series are produced by neutral loss mentation spectrum of the synthetic SRDKTIIMW peptide.
minal Arg, Lys, Ala or Tyr did not bind or bound weakly. This is
consistent with sequence data showing the absence of these motifs
among natural B*3909 ligands (Figs. 1 and 2B). Four peptides with
C-terminal Leu, Val, or Phe bound to B*3909 as efficiently or better
than the B*3909 ligand tested. Thus, besides the common peptide
bound in vivo to both HLA-B27 and B*3909, these results indicate
that B*3909 binds many of the natural HLA-B27 ligands carrying
Arg2 and aliphatic/aromatic C-terminal residues, thus revealing the
basis for an extensive overlap between the B*3909- and the HLA-
B27-bound peptide repertoires.
232 Tissue Antigens 1999: 53: 227–236
Discussion
The contribution of HLA-B diversification among South Amerindi-
ans to help this populations against local antigens should be under-
stood through knowledge of how such polymorphism affects pep-
tide specificity. On the basis of structural homology of the side
chain pockets in the peptide binding site between South American
allotypes and other class I molecules for which the peptide motifs
are known, it has been suggested that the repertoire of HLA-B al-
Yague et al : Peptide specificity of B*3909
leles in South America encompasses almost all the supertypic speci-
ficities, that is, the combination of main peptidic anchor motifs at
P2 and PW of the known HLA-B repertoire (4, 5). Although this
analysis provides a general assessment of peptide specificities, it is
insufficient for three reasons. First, the specificity of a given pocket
can sometimes be influenced by polymorphism outside it through
indirect effects (23). Second, polymorphism that affects residues
located at the boundary of a pocket may alter the structure of the
pocket, and its peptide specificity, in such a way that could be
Fig. 4. Epitope stabilization assay showing the binding of theB*3909 ligand ARDETEFYL, its His2 analog, the B*3901 ligandYHTDTTVKF, and its Arg2 analog to (A) B*2705, and (B)B*3909 on RMA-S cells. Data are means of two individual experi-ments.
233Tissue Antigens 1999: 53: 227–236
difficult to detect by sequence comparisons. Third, polymorphism
in pockets other than those involved in P2 and PW binding may
significantly affect peptide specificity. Thus, comparative analyses
of the peptide binding properties of those HLA-B allotypes orig-
inated in South America are required to understand how they con-
tribute to modulating peptide specificity.
The single amino acid difference (Tyr.Ser99) that distinguishes
B*3909 from B*3901 affects a position that is related to at least the
B and D pockets (24), and could, therefore, affect at least the peptide
motifs at P2 and P3. Indeed, among the main differences between
the two subtypes were those observed at these two positions. Most
noteworthy was the increased preference for Arg2 in B*3909, rela-
tive to B*3901. The results with peptide analogs indicate that Arg2
and His2 are similarly suitable for B*3909. In addition, computer
simulation analyses suggest that the effect of the amino acid change
in this subtype is both to improve Arg2 and to disfavor His2 inter-
action, relative to B*3901 (D. Rognan, personal communication). An-
other important difference between B*3909- and B*3901-bound pep-
tides was the increased frequency of Pro3 in the former subtype.
This suggests that the Ser99 change in B*3909 favors Pro3 in the
D pocket. This residue does not appear to significantly affect the
frequency of other P3 residues, relative to B*3901. Residue 99 is
close to the opening rim of the A pocket (24) and might affect P1
preferences. This would be compatible with the lower frequency of
Fig. 5. Epitope stabilization assay showing the binding of theB*3909 ligand ARDETEFYL and three HLA-B27 ligands toB*3909 on RMA-S cells. The EC50 values of these and other naturalHLA-B27 ligands tested in this assay are given in Table 1. Data are meansof two individual experiments.
Yague et al : Peptide specificity of B*3909
Binding of natural HLA-B27 or B*3909 ligands to B*39091
Peptide Ligand of2 EC50 (mM)
ARDETEFYL B*3909 9
RRYQKSTEL B*2705, 01, 03, 04, 06, 10 10
RRIYDLIEL* B*2705, 02, 04 3
RRWLPAGDA B*2705 50
RRFFPYYV B*2705, 02, 03, 04 5
RRFFPYYVY B*2705, 03, 01 nb
RRARSLSAERY* B*2702 60
GRLTKHTKF B*2705, 01, 02, 04, 10 nb
RRFVNVVPTF B*27053, 02 2
RRSKEITVR B*2705 nb
FRYNGLIHR B*2705 nb
RRIKEIVKK B*2705 nb
RRVKEVVKK B*2705 nb
GRIDKPILK B*2705, 10 nb
1 The peptides were tested for binding to B*3909-RMA-S cells using an epitope stabilization assay.
The B*3909 ligand was used as a reference peptide for calculating EC50 values of the HLA-
B27 ligands (see Material and methods); nb: no binding. Peptides labelled with asterisk (*)
are HLA-B27-restricted Epstein-Barr virus (EBV)-derived epitopes. All other ligands come from
endogenous proteins of the cell2 HLA-B27 or -B39 subtypes from whose peptide pools the ligand was sequenced (16, 18, 22,
23, 33–37)3 Our unpublished results
Table 1
small polar residues (SerπThr) at P1 among B*3909 ligands, rela-
tive to B*3901.
Thus, B*3909 polymorphism does not totally change the reper-
toire of bound peptides relative to B*3901, as happened in B*3902
(7) and B*3910 (9). Indeed, three B*3909 ligands were also found in
B*3901. However, the modulatory effect of the B*3909 change
allows for many peptides to bind differentially both subtypes. One
such example was the B*3901 ligand YHTDTTVKF that failed to
bind B*3909 in vitro.
HLA-B39 is associated with the axial type of psoriatic arthritis
(25) and with progression of early disease (26). In addition, B*3901
is associated with HLA-B27-negative ankylosing spondylitis and
pauciarticular juvenile rheumatoid arthritis in Japanese (27). This
has been correlated with the capacity of B*3901 to present peptides
with Arg2. More recently it has been shown this subtype binds
in vitro a subset of the HLA-B27 ligands (8). The peptide binding
properties of B*3909 make it closer than B*3901 to HLA-B27. This
234 Tissue Antigens 1999: 53: 227–236
similarity is based on the increased preference for Arg2, while main-
taining its specificity for nonpolar C-terminal residues. Indeed,
multiple B*2705 ligands bound efficiently B*3909 in vitro and a
common ligand was sequenced from both B*3909 and two subtypes
of HLA-B27 (22, 23). Thus, B*3909 has the greatest similarity with
HLA-B27 in peptide specificity described so far. HLA-B73, which
also binds peptides with Arg2, is significantly different from HLA-
B27 in the C-terminal peptide motifs (28). One previously reported
B*3901 ligand differs from the peptide found in B*3909 and HLA-
B27 only by its lack of a C-terminal Trp residue (7). Since this
peptide was sequenced by Edman degradation, and its molecular
mass, as determined by mass spectrometry, was not reported, there
is a possibility that a C-terminal Trp residue might have gone un-
noticed. Thus, the same B*3909/B27 common ligand might also be
presented by B*3901. Nevertheless, it is also possible that the
B*3901 ligand is indeed different from that found in B*3909. For
instance, we have recently reported that an octamer and its C-ter-
minally extended nonamer are both bound in vivo to B*2705, but
only the nonamer was found in the B*2701-bound peptide repertoire
(18).
HLA-B27 is absent from unmixed South Amerindian populations
(6). Among the HLA class I allotypes in South America of which the
peptide specificity is known, B*3909 is the closest one to HLA-B27.
It has been suggested that HLA-B27 is a highly efficient restriction
element for viral antigens (29), and is associated to slow progression
of AIDS (30–32). Thus, it might be speculated that the similarity of
B*3909 with HLA-B27 in its peptide specificity might have particular
functional significance among South Amerindians. In addition, it
might endow B*3909 individuals with increased susceptibility to
spondyloarthropathy. However, in spite of significant similarities,
B*3909 differs from HLA-B27 in its much greater preference for
acidic and Pro residues at P3. While this property does not necessarily
impair binding to HLA-B27, as demonstrated by the common B*3909/
B27 ligand, which has Asp3, it suggests that the HLA-B27 and
B*3909 peptide repertoires, although overlapping, are significantly
different. A more detailed comparison of the peptide presenting prop-
erties of these two molecules may be worth pursuing.
In conclusion, the peptide specificity of B*3909 shows that an
HLA-B polymorphism of South Amerindian origin makes the new
allotype closer in its peptide presenting specificity to HLA-B27,
which is absent from these populations but widespread among
other human groups.
Yague et al : Peptide specificity of B*3909
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