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*
Department of Molecular and Cellular Biology, and
Microchemistry Facility, Harvard University, Cambridge, MA 02138;
Dana Farber Cancer Institute, Harvard Medical School, Boston, MA 02115; and
§
Department of Immunology, The Weizmann Institute of Science, Rehovot, Israel
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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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Because Cop 1 is a mixture of random polypeptides, it may contain different sequences that bind to different HLA proteins; in this case only a fraction out of the whole mixture would be an "active component." Alternatively, the whole mixture may be competent, i.e., all polypeptides binding to any HLA-DR molecule. In view of the crystallographic analysis of several HLA-DR molecules complexed with the antigenic peptides (17, 18, 19), as well as the binding motifs of natural MHC-associated ligands that were elucidated by acid-extraction and sequencing (20, 21, 22, 23, 24, 25), it has been intriguing to find out whether all four amino acids that compose Cop 1 are involved in its binding in the groove of HLA-DR molecules.
The study here was undertaken to attempt to identify these active components present in the bound Cop 1 and to determine their binding motifs. To isolate the bound fraction of Cop 1 with no interference from endogenous peptides, recombinant "empty" HLA-DR1, -DR2, and -DR4 molecules produced in insect cells were employed. Because the average length of the Cop 1 polypeptides used is 75–80 amino acids, the epitopes lying in the groove of HLA-DR molecules are likely to be found internally in the polypeptide chains with protruding ends, making direct analysis of the bound amino acid sequences by microchemical methods very complicated. To access these regions, N-terminal peptidase treatment of the protruding ends of Cop 1 polypeptides was employed. This approach proved to be useful in trimming of N-terminal ends of peptides that protrude out of class II MHC proteins, while protecting epitopes bound to the groove from proteolysis (26, 27). Various characteristics relating to the fraction bound and its motifs, including amino acid composition, HPLC profiles, and pool sequencing, together with the immunological recognition of these fractions, are presented here.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Soluble HLA-DR1, -DR2, and -DR4 molecules were expressed in Drosophila S2 cells as described (18, 28, 29). Cells were grown in roller bottles in ExCell 401 medium (JRH Biosciences, Lenexa, KS) supplemented with 0–5% fetal bovine serum (Sigma Chemicals, St. Louis, MO) at 26°C. Cells were harvested 4–5 days after induction by 1 mM CuSO4. Immunoaffinity purification of HLA-DR1, -DR2, and -DR4 molecules was performed as reported previously (18). Briefly, supernatant from harvested cells was sequentially passed through protein A, protein G, and protein A-LB3.1 columns, followed by elution of the bound HLA-DR with 50 mM 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS) (pH 11.5) and neutralized with 200 mM phosphate (pH 6.0). Proteins were concentrated on a Centriprep 10 membrane (Amicon, Beverly, MA).
Antigens and Antibodies
Cop 1 is a synthetic random copolymer prepared by polymerization
of the N-carboxyanhydrides of L-tyrosine,
-benzyl-L-glutamate, L-alanine, and
,N-trifluoroacetyl-L-lysine (1) (the end
product is a mixture of acetate salts of random polypeptides). Cop 1,
batch 52596, in the molar ratio of 1 Y:1.5 E:4.3 A:3.3 K, with an
average m.w. of 8150, was obtained from Teva Pharmaceutical
Industries (Petach Tikva, Israel). Rabbit anti-Cop 1 polyclonal Abs
(IgG fraction, biotin-labeled) were also from Teva Pharmaceutical
Industries.
Treatment of HLA-DR-Cop 1 complexes with aminopeptidase I
Cop 1 (1 mM) was incubated with recombinant water-soluble
"empty" HLA-DR1, -DR2, or -DR4 molecules (100 µM) in PBS for
40 h at 37°C. Aminopeptidase I (2 units) (Sigma Chemicals) was
added for the last 18 h of incubation. Samples were then
spin-concentrated to a final volume of 
100 µl using
Centricon 10 ultrafiltration devices (Beverly, MA). Bound Cop 1
was eluted from HLA-DR by addition of acetic acid (10%) and incubated
at 70°C for 15 min, followed by ultrafiltration and vacuum
concentration in a SpeedVac (Savant), as described previously (12).
HPLC separation and microsequencing
After elution as above, typically 5–10% of the Cop 1 mixtures were fractionated by microbore HPLC using a Zorbax C18 1.0-mm reverse-phase (RP) column (Saratoga, CA) on a Hewlett-Packard 1090 HPLC with 1040 diode array detector. At a flow rate of 54 µl/min, Cop 1 was eluted with a gradient of 0.055% trifluoroacetic acid in acetonitrile (0% at 0–10 min, 33% at 73 min, and 60% at 105 min). Strategies for peak selection, RP separation, and Edman microsequencing have been described previously (22, 30). Pooled fractions were submitted to automated Edman degradation on a Hewlett-Packard G1005A protein sequencer using the manufacturer’s Routine 3.5 analytical method.
PAGE
SDS-PAGE was conducted with the NOVEX (San Diego, CA) minicell electrophoresis system. Separation gel was 10% in acrylamide and stacking gel was 5%. HLA-DR1-Cop 1 complexes were run under nonreducing conditions for 1 h at 200 V, stained with Coomassie Brilliant blue, fixed for 3 h in 10% methanol/10% acetic acid, and dried on Cellophane paper (Bio-Rad, Richmond, CA) at 25°C.
Ab binding assay
The cross-reactivity between Cop 1 and its fractions was detected by direct ELISA assay using biotinylated anti-Cop 1 polyclonal Abs. Cop 1 or fractions were diluted to 0.4 and 2.0 µg/ml and plated in duplicate on a 96-well microtiter immunoassay plates (PRO-BIND, Falcon, Lincoln Park, NJ) (100 µl per well). All incubations were for 1 h at 37°C and washes were three times with Tris-buffered saline (TBS)/0.05% Tween-20 (TBS = 137 mM sodium chloride, 25 mM Tris (pH 8.0), 2.7 mM potassium chloride). The wells were then blocked with TBS/3% BSA, followed by addition of biotinylated anti-Cop 1 Abs (1:5000, 100 µl per well). Ab-ligand complexes were detected using streptavidin-conjugated alkaline phosphatase (1:3000, Bio-Rad) and p-nitrophenyl phosphate in triethanolamine buffer (Bio-Rad). The absorbance at 410 nm was monitored by a microplate reader (Dynatech MR4000).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Quantitation and amino acid analysis of Cop 1 bound to HLA-DR1,
-DR2, and -DR4 molecules.
Cop 1 was incubated with water-soluble HLA-DR1, -DR2, or -DR4 molecules
at the molar ratio of 1:1 for 40 h at 37°C. These recombinant
"empty" HLA-DR molecules are usually stably assembled in the
presence of exogenously added peptide Ag. However, Cop 1 can substitute
for peptides in promoting stabilization and with no interference from
endogenous peptides (15). Unbound Cop 1 was separated from bound Cop 1
by Centricon ultrafiltration. Bound Cop 1 was then extracted from the
HLA-DR by acid treatment (22) and subjected to amino acid analysis. At
least 95% of added Cop 1 was bound to isolated HLA-DR1 and -DR4, and
80% was bound to HLA-DR2 proteins (Table I
). Cop 1 eluted from HLA-DR1, -DR2, and
-DR4 molecules showed ratios of Y:E:A:K similar to unseparated Cop 1
(Table I). These results indicate that the bound fraction of Cop 1
reflects the amino acid composition of the whole mixture and suggest
that there is little or no preferential binding of Cop 1 components to
different HLA-DR proteins. These data were supported by a different set
of experiments in which Cop 1 was incubated with an excess of HLA-DR1,
-DR2, and -DR4 molecules that had been purified from human homozygous
EBV-transformed B cell lines, and then passed through a size-exclusion
column. Nearly all of the Cop 1 was found in the fractions
corresponding to the high m.w. complexes with each of the HLA-DR
molecules (data not shown), with <10% in each case at the lower m.w.
position of Cop 1, also indicating that most of the copolymer binds to
these molecules.
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) using an acetonitrile gradient, as
described in Materials and Methods. In contrast to typical
profiles of single peptides or peptide pools (22, 31, 32, 33, 34), untreated
Cop 1 showed a very broad peak with several smaller peaks, which spread
between 40- and 75-min elution time (Fig. 1A). This
elution profile is characteristic of a mixture of random polypeptides
and resembles HPLC separations of other batches of Cop 1 (unpublished
data). Similar profiles were obtained when Cop 1 was eluted from
HLA-DR1 (Fig. 1B), -DR2 (Fig. 1C), or -DR4 (Fig. 1D) molecules, suggesting that the bound fraction is similar
to the whole Cop 1 mixture in its chemical properties.
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,
A–D), which corresponds to the initially higher molar ratio
of A in Cop 1 (1). There was no sequence specificity or preferential
positioning of any amino acid of Cop 1, indicating that the bound
fraction is also random, similar to the entire Cop 1. The yields of
other amino acids resulting from endogenous peptides of HLA-DR
molecules were minor (data not shown). It should be noted that these
data represent sequencing of the first 20–25 amino acids from the N
termini of bound Cop 1 polypeptides, which most likely protrude from
the groove of HLA-DR molecules, and therefore do not reflect the actual
binding motif(s).
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) showed that all the fractions were
similarly recognized by anti-Cop 1 Abs, suggesting that these
fractions share similar epitopes with each other and with Cop 1.
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Treatment of Cop 1 bound to HLA-DR1, -DR2, or -DR4 molecules with
aminopeptidase I.
To determine the actual binding motifs, Cop 1 was incubated with HLA-DR
molecules at the molar ratio of 10 Cop 1:1 HLA-DR in PBS for 40 h
at 37°C. Aminopeptidase I, a metalloprotein isolated from
Streptomyces griseus (35), was added for the last
18 h of incubation, to remove N-terminal ends of Cop 1
polypeptides, protruding from the groove of the HLA-DR molecules, as
well as to digest the unbound Cop 1. The resulting Cop 1-HLA-DR
complexes were analyzed by SDS-PAGE. As shown in Fig. 4, Cop 1-DR1 complexes were resistant to
SDS-induced dissociation, forming higher m.w. complexes with HLA-DR1
ß heterodimers that were observed as numerous bands on the
polyacrylamide gel above the molecular mass protein standard of 50 kDa
(lane 5), and as described previously (15). In
the presence of aminopeptidase I (a 33-kDa protein appearing as a thin
band below the molecular mass protein standard of 35 kDa, lanes
2, 4, and 6), all the unbound Cop 1 (a
smear in the lower part of the gel, lanes 1 and
5) was completely digested (lanes 2
and 6), whereas Cop 1-DR1 complexes were protected
(lane 6). It should be noted that upon
incubation of aminopeptidase I with HLA-DR1 alone a complex was formed,
represented by a band below the 50-kDa molecular mass protein standard
(lane 4) probably caused by binding of some
aminopeptidase I-derived digestion products to HLA-DR1. Similar
treatment with aminopeptidase I was applied to Cop 1 bound to HLA-DR2
and -DR4 molecules. Bound Cop 1 remaining after aminopeptidase I
treatment was eluted from HLA-DR by acid extraction, as described in
Materials and Methods.
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) using an acetonitrile gradient, as
described in Materials and Methods. In contrast to a very
broad peak corresponding to untreated Cop 1 (Figs. 2
A and
5A), proteolytic Cop 1 fragments eluted from HLA-DR1 (Fig. 5C), -DR2 (Fig. 5E) or -DR4 (Fig. 5G)
showed profiles similar to peptide pools isolated from purified human
HLA-DR molecules (31, 32, 33). On the other hand, almost no peptides were
eluted from "empty" HLA-DR1 (Fig. 5D), -DR2 (Fig. 5F), or -DR4 (Fig. 5H) molecules, or from a total
Cop 1 digestion with no HLA-DR added (Fig. 5B). Peaks for
further analysis were selected in the region where the untreated Cop 1
was eluted (Figs. 2A and 5A), between 40 and
75 min elution time. For each HLA-DR molecule, only peaks corresponding
to Cop 1 peptides, which did not overlap those eluted from that same
HLA-DR molecule with no Cop 1, were pooled for sequencing.
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A and 6A), significantly
higher levels of E were found at the first and second cycles, of K at
the second and third cycles, and of Y (presumably at P1 of the bound
peptide) at the third to fifth cycle, for peptides bound to HLA-DR1
(Fig. 6C), -DR2 (Fig. 6E), or -DR4 (Fig. 6G). This spread of residues
over two or three positions in the pool sequencing data is probably
caused by the ragged N termini of the Cop 1 components after
aminopeptidase treatment, similarly to other treated naturally
processed class II MHC ligands (25). Position 3 was assumed to
correspond to P1 because in the structure of the HA 306–318/HLA-DR1
complex P-2 is at the flush end of the groove and P1 is amino acid 3,
i.e., Y308, in a deep pocket (17). For HLA-DR2, both Y and A levels
were increased at cycle 3 (Fig. 6E). However, HLA-DR2
(DRB1*1501) has a P1 pocket lined by ß86 Val, which could not
accommodate Y but can accommodate A (18, 32, 33). Although Y can be
accommodated at the P4 pocket, no enrichment of Y at this position
(cycles 6, 7) was observed (Fig. 6E). No sequence
specificity or preferential positioning was observed for anchor
positions following P1 (P4, P6, or P9 of HLA-DR1 or -DR4; P4, P7, of
DR2b molecules) (Fig. 6, C, E, and G).
In all the samples the levels of A were higher than E, Y, and K, which
corresponds to the initially higher molar ratio of A in Cop 1 (1). The
yields of Y, E, A, and K (Fig. 6, D, F, and H),
as well as of other amino acids (data not shown), resulting from
endogenous peptides of HLA-DR molecules or from a complete Cop 1
digestion by aminopeptidase I (Fig. 6B), were minor.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). In the case of HLA-DR1 and -DR4, which have a P1
pocket that is lined by residue ß86Gly, this tyrosine residue would
serve as a primary anchor (17, 18). In the case of DR2, which has
ß86Val, the tyrosne residue is presumably too large for the P1
pocket, but can be accommodated in the P4 pocket; in MBP 85–99, Phe92
is accommodated in the P4 pocket (33). The high fraction of Cop 1 that
binds suggests that nearly every polypeptide within this mixture with
an average size of 75–80 amino acids includes a 13-amino acid stretch
that is accommodated within the groove. The N-terminal sequence analysis of the first 20 amino acid residues of bound Cop 1 most likely represents material that protrude from the grooves of the HLA-DR molecules. Determination of the actual peptide sequence(s) from Cop 1 found within the binding grooves of HLA-DR proteins, has been an important goal. To directly access these amino acids, digestion of the protruding ends of Cop 1 polypeptides with an N-terminal peptidase was employed. This approach was proven to be useful in trimming of N-terminal ends of peptides that protrude out of class II MHC proteins, while protecting epitopes bound to the groove from proteolysis (26, 27).
Detailed peptide motifs for class II MHC molecules were elucidated by
pool sequencing of natural MHC-associated ligands (25). A similar
approach was employed here for Cop 1, a mixture of random polypeptides
of various lengths. Because of the nature of its synthesis, isolation
of individual components has been technically impossible (1).
Regardless of the HLA-DR molecule, Y was found at the first anchor
position (the third residue in the sequence analysis), followed by A in
the subsequent pockets (Fig. 6). These data are in line with the
peptide-binding motifs of HLA-DR1 (DRB1*0101) (17, 21, 22, 39, 40) or
-DR4 (DRB1*0401) (18, 22, 41, 42, 43) molecules. For HLA-DR2 (DRB1*1501),
however, no aromatic residue was found in the first P1 anchor, whereas
the second (P4) could have Y (19, 32, 33). Anomalously, however, in the
Cop 1 bound to HLA-DR2 Y as well as A was also apparently enriched at
P1, but not at P4. The efficacy of Cop 1 might be improved by
substituting V for Y in the copolymer, since V89 is found in MBP 85-99
in the P1 pocket. Substitution of F for Y might be even better, since
it would fit tightly into the P1 pocket as well as fitting into the P4
pocket. The increase in E at P-2 and K at P-1 is not fully understood.
The enrichment of K near the N termini of naturally processed peptides
bound to HLA-DR1 was observed previously and mistakenly interpreted as
an anchor residue (21). These residues may contribute to the stable
interactions of Cop 1 with the HLA-DR molecules and the TCR, similarly
to residue K at P-1 of HA 306–318 peptide complexed with HLA-DR1 (17),
or Y at P-1 and possibly Q at P-2 of CII 1168–1180 complexed with
HLA-DR4 (18). Also, removal of P at P-2 and K at P-1 from HA 306–318
peptide resulted in a lower binding to some HLA-DR1 and -DR4 alleles
(44). Peptide flanking residues that lie outside the MHC anchor
positions at the C-terminal end were shown previously to influence
immunogenicity (45); no comparable information is available at the
N-terminal end. However, P-2 and P-1 in the crystal structures of the
HA 306–318/HLA-DR1 and CII 1168–1180/HLA-DR4 complexes point upwards
and most likely contact the TCR (17, 18). Moreover, peptide analogues
of HA 306–318 with amino acid substitutions at position 307K (P-1)
anergized HA 306–318-specific DR1-restricted human T cell clones (46).
Binding motifs of Cop 1 are summarized in Table II.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jack L. Strominger, Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, MA 02138. E-mail address:
3 Abbreviations used in this paper: Cop 1, copolymer 1; CII, collagen type II; HA, influenza virus hemagglutinin; MBP, myelin basic protein; MS, multiple sclerosis; RA, rheumatoid arthritis; EAE, experimental allergic encephalomyelitis; RP, reverse phase.
Received for publication July 14, 1998. Accepted for publication January 19, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß heterodimers in the absence of antigenic peptide. Cell 68:465.[Medline]
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