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*
Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands;
National Center for Computer-Aided Chemistry and Bioinformatics (CAOS/CAMM), Catholic University Nijmegen, Nijmegen, The Netherlands; and
Department of Medical Biochemistry, Sylvius Laboratory, Leiden University Medical Center, Leiden, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alternatively, progress has been made recently in the use of synthetic peptide libraries to analyze the peptide specificity of T cell clones (8, 9, 10, 11). The synthetic epitopes arising from synthetic peptide library screening are not natural epitopes, but mimics of natural epitopes. These mimicry epitopes may subsequently be used for the identification of natural Ags.
For Ag definition by database searching with mimicry epitope sequence information, a certain degree of sequence similarity between the mimicry epitope and the natural epitope is required. It has been reported that T cell clones can be activated upon stimulation by ligands that hardly share any sequence homology (12, 13, 14). This might impair database searches based on mimicry epitopes. Here we show that the natural Ag can be unambiguously identified using a synthetic library approach for three unrelated CD4+ T cell clones.
We describe a complete protocol for library screening, search pattern definition, and database searches that led to the identification of T cell Ags. DR3-restricted CD4+ T cell clones were used to screen two sublibraries with slightly different DR3-binding submotifs (15, 16). Two clones, MT1 (17) and MT2 (18), are Mycobacterium tuberculosis reactive. The third clone, HG (N. C. Schloot, O, M. C. Batstra, G. Duinkerken, R. R. de Vries, T. Dyrberg, A. Chaudhuri, P. O. Behan, and B. O. Roep, unpublished observation), recognizes human 65-kDa glutamic acid decarboxylase (GAD65),3 a major autoantigen in insulin-dependent diabetes mellitus (19).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synthetic peptides were synthesized on an Abimed 422 multiple peptide synthesizer (Abimed Analyes-Technik, Langenfeld, Germany) using fluorenylmethoxycarbonyl (F-moc)-protected amino acids and TentagelS-AC resins (Rapp, Tübingen, Germany) as described (10). The purity of the peptides was determined by reversed-phase HPLC, and the integrity of the peptides was determined by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry on a Lasermat mass spectrometer (Finnigan-MAT; Hemel Hempstead, U.K.).
Synthetic peptide library design and synthesis
Hybrid TentaGelH-AM resin (particle size, 90 µm; loading, 100
pmol/bead, 16 pmol acid stabile attached, 84 pmol acid labile attached)
(Rapp) (20) was used to synthesize two random one-bead/one-peptide
14-mer peptide libraries containing two different DR3-binding motifs
(15, 16). The hybrid resin allows for a convergent library screening
using the acid cleavable part of the peptide material attached to the
resin (20), combined with highly efficient peptide identification by
Edman sequencing, using the noncleavable part of the peptide material
attached to the resin. The rationale of the library design is the
same as described before (10). The design of the two libraries with two
different DR3-binding motifs is summarized by the following
synthesis schemes: library 1,
XXX(L,I,M,V,A,Y,F)XXDXXXXXXX-GABA; and library 2,
XXX(L,I,M,V,A,Y,F)XX(N,E,Q,S,T)X(K,R,H)XXXXX-GABA, with X
being one of 19 L-amino acids (all natural amino acids except C, which
was omitted for synthetic reasons), and GABA being 
-aminobutyric
acid. Each library was synthesized using chemistry as described above,
following a mix and split protocol (21, 22), yielding two
one-bead/one-peptide libraries, each with a complexity of 4 x
106.
Synthetic peptide library screening and bead sequencing
Convergent peptide cleavage and screening was performed as described (20). Shortly, libraries were divided into pools of 20,000 beads. Part of the peptide material was released from the beads for testing in a T cell proliferation assay (first round of screening). Beads of active pools were subdivided to pools of 70 beads. Again, peptides were released partially for the second round of screening. For the third round of screening beads were divided in limiting dilution. The remaining acid labile-attached peptide was released and tested. Cleavage conditions for the hybrid resins (20) differ from those described for TentaGelS-AM (10). Peptide sequences were determined by manual application of single beads (still containing sequenceable amounts of peptide; 10–15 pmol) to a cartridge and subsequent sequencing using a Hewlett Packard (Palo Alto, CA) G1005A protein sequencer.
T cell clones
MT1 (=Rp151-1) is a DR3-restricted CD4+ T cell clone that recognizes the N-terminal part of a 65-kDa heat shock protein of mycobacteria (HSP65(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)) (17). MT2 (=CAAp151-3) (18) is a DR3-restricted CD4+ T cell clone that recognizes protein 85 (85B(55–68)) (23, 24) of M. tuberculosis (28). HG (=PM1#11) is a DR3-restricted CD4+ T cell clone that recognizes human 65-kDa glutamic acid decarboxylase (GAD65(339–352)) (N. C. Schloot et al., unpublished observations).
Proliferation assay
CD4+ T cell proliferation assays for testing library pools and synthetic peptides were performed using 1 x 104 T cells and 5 x 104 irradiated HLA-DR3-matched PBMCs per well in flat-bottom 96-well plates in complete Iscove’s modified Dulbecco’s medium (150 µl) (Life Technologies, Gaithersburg, MD) containing 10% pooled human serum. Phytohemagglutinin (10 µg/ml), purified protein derivative of M. tuberculosis (10 µg/ml), and IL-2 (T cell growth factor, 10% Lymphocult, Biotest Diagnostics, Danville, NJ) were used as positive controls for T cell proliferation. [3H]Thymidine (0.5 µCi in 50 µl RPMI 1640) was added after 72 h, cells were harvested (Micro Cell Harvester, Skatron, Lier, Norway), and activity of the T cell DNA was counted after another 18 h (Model 1205 Betaplate, Liquid Scintillation Counter, LKB Instruments, Gaithersburg, MD). Library pools were tested in a quantity of 7 µl per well, giving final test concentrations of 5 nM for each individual peptide and 0.1% DMSO (v/v).
Database searching with PeptideSearch
The M. tuberculosis SHOTGUN database (TB_shotgun.dbs, December 1997) was retrieved from the Sanger Center (http://www.sanger.ac.uk/Projects/M_Tuberculosis/) by ftp (ftp.sanger.ac.uk). The SHOTGUN database, covering the complete genome of M. tuberculosis (strain H37Rv), was converted by translating all possible open reading frames on both the coding and the noncoding strand into hypothetical protein sequences. An incompletely defined codon was translated to an X (±1% of the database). Pattern searches were performed in the translated TB_shotgun.dbs using PeptideSearch (25).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Two distinct but related sets of peptides have been
described to bind to HLA-DR3 molecules (15, 16). The first set uses a
hydrophobic amino acid at relative position 1 and a D at relative
position 4. The second set consists of peptides that need a third
(basic) anchor at relative position 6 (K, R, or H), probably due to a
weak anchor at position 4 (E, Q, N, S, T). Two 14-mer peptide libraries
were synthesized that reflect these two binding motifs. Both libraries
had a complexity of 4 x 106. The first library
reflects 1912 x 7 x 1 theoretically possible
14-mers. For the second library, this theoretical complexity is
1911 x 7 x 5 x 3. This means that both
libraries are highly incomplete. Identification of mimicry epitopes
from these libraries implies that T cells can recognize a large
number of different 14-mer peptides. Because a mix and split protocol
is used for synthesis, random positions are not biased to certain
synthetically preferable combinations. Pools of each library,
containing 20,000 individual peptides, were screened with two
DR3-restricted M. tuberculosis-reactive T cell clones (MT1
and MT2) and a DR3-restricted human GAD65-reactive T cell clone (HG)
(Table I
). Pools were considered positive
when counts exceeded five times the background counts. For clone MT1,
activity was observed in one pool of library 1. Convergent screening
(10, 20) led to the identification of stimulatory peptide MT1-P1
(NSTVAYDEAMIFAQ) (Table II). For clone
MT2, two active pools of library 1 were obtained, resulting in the
identification of MT2-P1 (NSAIGIDIPVARRD) and MT2-P2
(SHFVGXDIPVSLKH) (Table II). For clone HG, activity was
observed in one pool of library 1 as well as in one pool of library 2,
demonstrating that this clone is able to recognize peptides bearing two
distinct binding motifs. The stimulatory peptides were identified as
SIAMAFDPQIPMAA (HG-P1) and TDSLAFEPKVPRRQ (HG-P2) (Table II).
The stimulatory capacities of the identified peptides were compared
with their natural counterparts in a T cell proliferation assay (Fig. 1). Retrospectively, it was shown that
suboptimal concentrations of 5 nM (which equals the individual peptide
concentrations during library screening) are indeed stimulatory for all
mimicry epitopes.
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Standard homology database searches with either the complete sequences of MT2-P1 and HG-P2 or part of the sequences of these mimicry epitopes did not lead to identification of the natural epitopes for clone MT2 and HG within the 100 best-matching hits out of either the M. tuberculosis database or a human abstract of the Swiss Prot database. Therefore, a detailed analysis was performed on MT2-P1 and HG-P2 to define amino acid requirements for stimulation of clones MT2 and HG.
Omission mixture analysis (MT2)
Although identification of the natural epitopes for clones MT2 and HG using mimicry epitopes MT2-P1 and HG-2 was performed similarly, only the procedure for clone MT2 is described in detail.
We chose to focus on relative positions 1–7 of MT2-P1
(IGIDIPV), because the similarity in this part of the peptide
with MT2-P2 suggested an important role for HLA binding and TCR
interaction. To determine the absolute importance for T cell
stimulation of the amino acids in this part of the sequence, an
omission mixture analysis was performed. In an omission mixture for
relative position 1, a mixture of 18 peptides was synthesized and
tested for T cell stimulation (consensus sequence
NSAO1GIDIPVARRD, where O1 means all 20 natural
L-amino acids except I, which was omitted because it is present at
relative position 1 in MT2-P1, and C, which was omitted for synthetic
reasons). The omission mixtures for relative position 2 and 5 induced
no proliferative response (Table III).
Therefore, it could be concluded that, in the context of the MT2-P1
sequence, a G at relative position 2 and an I at relative position 5
are essential for proliferation of clone MT2.
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Amino acids L, I, M, V, A, G, W, Y, and F have been
described as anchors for relative position 1 for HLA-DR3 (16). To
confirm the expectation that in MT2-P1 the I at relative
position 1 can only be substituted by this set of amino acids
without disturbing stimulatory capacity, an omission mixture was
synthesized: NSAX1GIDIPVARRD (X1 = all
natural L-amino acids, except C and the expected anchor residues). This
mixture of 10 peptides was not able to induce a significant
proliferation of clone MT2 (Table IV).
The same omission analysis was performed for the other anchor
positions, relative positions 4 and 6. Anchor amino acids for relative
positions 4 and 6, which have been described in literature (15, 16),
and the amino acids present at positions 4 and 6 of MT2-P1 were taken
into account. As interdependence of those anchor positions is expected
(15), relative position 4 was studied in combination with a random
relative position 6, and vice versa:
NSAIGIX2IX3VARRD (X2 = all natural
L-amino acids, except D, N, S, T, and C; X3 = all natural
L-amino acids, except C) and NSAIGIX4IX5VARRD
(X4 = all natural L-amino acids, except C; X5 =
all natural L-amino acids, except P, H, R, K, and C). Both the omission
mixtures for relative position 4 and relative position 6 were inactive
in proliferation assays (Table IV).
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G at relative position 2 and I at relative position 5 were
essential for proliferation of clone MT2. The remaining potential TCR
contact residues within the 7-mer core sequence of MT2-P1 are at
relative position 3 and 7. For these positions, all substitution
peptides (except C substitutions for synthetic reasons) were
synthesized and tested for T cell stimulation (Fig. 2). For relative position 3, L, R, S, T,
N, D, W, and Y are allowed amino acids. For relative position 7, I, T,
and K were shown to be allowed.
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Based on the omission and substitution studies as described
(Tables III and IV and Fig. 2
), a search pattern was constructed for
PeptideSearch. This resulted in a pattern at (relative) position 1
= (L,I,M,V,A,G,W,Y,F), position 2 = G, position 3 =
(I,L,R,S,T, N,D,W,Y), position 4 = (D,N,S,T), position 5 = I,
position 6 = (P,K,R,H), and position 7 = (V,T,I,K). The
translated M. tuberculosis SHOTGUN database was searched
with this pattern, yielding 19 hits (Table V). The natural epitope M.
tuberculosis 85B(55–68), SPSMGRDIKVQFQS (number 9), was indeed
part of the list. In addition to peptide 9, peptides 2, 15, and 16 were
also recognized by clone MT2. Peptide 2 is derived from M.
tuberculosis 85C. Peptide 15, which contains an X (due to a DNA
sequencing uncertainty), is derived from either M.
tuberculosis 85A or 85B (X = P). 85A (32 kDa), 85B (30 kDa),
and 85C (30 kDa) are highly homologous proteins, probably originated
from gene duplication (24, 26). They all show mycolyltransferase
activity. Therefore, it is likely that these three proteins are
involved in cell wall assembly (26). Peptide 16 differs at only one
amino acid position from the corresponding 14-mer peptide derived from
M. tuberculosis major protein Ag MPT51 (27)
(SPSMGRDKPVAFLA, difference underlined). This K to I
substitution probably results from a DNA sequencing error present in
the M. tuberculosis SHOTGUN database (a single substitution
of a T for an A can change the codon for K (AAA) into the codon for I
(ATA)). The 14-mer peptide of MPT51 (SPSMGRDKPVAFLA) appeared not to be
able to stimulate clone MT2, which was expected from the search pattern
that was used for searching in the M. tuberculosis SHOTGUN
database (only I was included at relative position 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). This implies that all three clones have a
strict recognition pattern. These data further suggest that recognition
of ligands without any sequence similarity by one T cell clone may not
be a very common phenomenon for epitopes that are recognized in low
nanomolar concentration with a regular binding motif (14, 15, 16). This
implies a probability for Ag definition by database searching using
sequence information from synthetic mimicry epitopes, provided that the
Ag is contained in the database. In two cases, a simple homology search with the core of the sequence of a synthetic mimicry epitope obtained from the library screening led to immediate identification of the natural epitope as part of a large set of heterogeneous database hits. A homology search with the core sequences (relative positions 1–7) of mimicry epitopes (MT1-P1 and HG-P1) identified the corresponding natural Ags within the 100 best-matching database hits (M. tuberculosis database for clone MT1 and a human abstract of the Swiss Prot database for clone HG).
In two other cases (mimicry epitopes MT2-P1 and HG-P2), the size of the
set of hits containing the natural epitope appeared to be too large to
be investigated. Therefore we developed a generally applicable approach
that increases the probability of successful identification of natural
epitopes by pattern searching with a precisely defined pattern (Fig. 3). This approach is based on the
observation that stimulating ligands can be predicted by studying
individual amino acid positions (14). Using a precisely defined pattern
for searching the M. tuberculosis SHOTGUN database, the
natural epitope of clone MT2 (BCG85B(55–68)) was unambiguously
identified. A comparable analysis and subsequently a pattern search in
the nonredundant database was performed for clone HG using mimicry
epitope HG-P2. GAD65 was indeed part of the human fraction of all
pattern hits (data not shown). This indicates that similarity rather
than homology between mimicry epitopes and natural epitopes is required
for natural Ag identification.
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One clone (HG) was able to recognize two similar mimicry epitopes containing two slightly different HLA-DR3-binding motifs (library 1 and library 2 motifs). In this example, our approach succeeded in unambiguous identification of human GAD65 using the sequence information of HG-P2, whereas a homology search with this peptide did not identify the natural Ag. Our approach might further contribute to the search for functional T cell-epitope mimicry in autoimmune disease that is not based on simple-sequence homology.
We conclude that mimicry epitopes identified from synthetic peptide
libraries are highly similar to their natural counterparts. Therefore,
in some cases the natural epitope can be defined by a simple homology
database search, depending on the degree of homology and the size of
the database of interest. Because the success probability of homology
searching is limited, we present a generally applicable protocol based
on similarity, which represents a convenient approach for Ag definition
using sequence information obtained from synthetic mimicry epitopes
(Fig. 3). This approach opens new perspectives for rapid and reliable
Ag definition and represents a feasible alternative for the biochemical
and molecular biology approaches described thus far. Defined Ags can be
used for vaccine design in infectious disease and for the development
of immune intervention strategies in autoimmune disease.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jan W. Drijfhout, Leiden University Medical Center, Department of Immunohematology and Blood Bank, Albinusdreef 2, P.O. Box 9600, 2300 RC Leiden, The Netherlands.
3 Abbreviation used in this paper: GAD65, 65-kDa glutamic acid decarboxylase.
Received for publication April 5, 1998. Accepted for publication June 11, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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