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Autoimmune Disease Unit, Cedars-Sinai Research Institute, and University of California School of Medicine, Los Angeles, CA 90048
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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15 biotins/molecule protein) of lysine
residues on the surface of purified, native TPO resulted in loss of
multiple tryptic cleavage sites, as determined by analysis of tryptic
polypeptide fragments on reverse-phase HPLC. TPO was then complexed
with a monoclonal human autoantibody Fab (TR1.9) before biotinylation.
After dissociation from TR1.9, TPO was recovered by gel filtration. A
trypsin site, previously observed to be lost after TPO biotinylation,
was restored when biotinylation was performed on the TPO-TR1.9 complex.
The epitope-protected lysine (K) was present in a 30-aa TPO fragment
that, by N-terminal sequencing, was found to be K713. Altered
recognition by TR1.9 of a TPO-myeloperoxidase chimeric molecule
involving this region supported the epitope protection data. In
conclusion, we provide the first identification of an amino acid
residue (K713) comprising part of an epitope within the TPO
immunodominant region. This focal residue localizes the facet on the
large, highly complex TPO molecule that contains the immunodominant
region and provides the basis for rational guided mutagenesis studies
to more fully characterize this region. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TPO is very large (107 kDa) and, based on the known three-dimensional structure of closely related myeloperoxidase (8), is a highly complex, convoluted, globular molecule that may be likened to bowl of spaghetti. It is, therefore, not surprising that identification of conformational, possibly discontinuous, TPO autoantibody epitopes (9, 10, 11) on the native Ag has not been possible using synthetic peptides. Some polypeptide fragments of TPO are recognized by TPO autoantibodies in patients’ sera (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25). However, the wide spectrum and large size of these fragments as well as the fact that (in the native structure) polypeptides can traverse the molecule and contribute to widely separated facets on the surface of the protein have not led to deduction of the site of the immunodominant region. Moreover, polyclonal patient serum interaction with polypeptides may reflect the minor component of TPO autoantibodies that recognize denatured, linear determinants. Other approaches have involved competition between mouse mAbs (26) or rabbit antisera (27) for human autoantibody binding to TPO. However, potential steric hindrance by IgG molecules, which are even larger than TPO, does not allow definitive localization of an epitopic region.
Identification of the TPO autoantibody immunodominant region could be attained by determination of the three-dimensional structure of crystals of TPO-autoantibody complexes. However, although the crystal structure of one TPO autoantibody Fab has been reported (28), this goal, or indeed determining the structure of uncomplexed TPO (29, 30), has to date not been achieved. In this context, definitive identification of even a single amino acid within the TPO immunodominant region would provide vital information by indicating which facet of TPO contains this region, and this individual residue could then be the landmark for future guided mutagenesis studies based on the known structure of myeloperoxidase (MPO) (8). In the present study we used an epitopic footprinting approach to identify for the first time a specific TPO amino acid residue (Lys713) in the autoantibody immunodominant region.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant human TPO, expressed in Chinese hamster ovary (CHO)
cells as an 848-aa, secreted protein (31), was affinity
purified as described previously (32). TR1.9, one of four
recombinant human TPO autoantibodies (expressed as a Fab) that define
the immunodominant determinants on TPO (7), was affinity
purified as described previously (33). This Fab was cloned
by screening a combinatorial Ig gene library constructed from
thyroid-infiltrating B lymphocytes. TPO-TR1.9 complexes were prepared
by incubating (in a typical preparation) 2 mg of TPO with 6 mg of TR1.9
in 1.5 ml of 10 mM Tris, pH 7.0, for 2 h at room temperature.
Complexing efficacy (20 µg aliquot) was analyzed by fast protein
liquid chromatography (Pharmacia LKB, Piscataway, NJ) using a Sephacryl
S-200 HiPrep 16/60 column equilibrated with PBS. Subsequently, 1 mg of
uncomplexed TPO and the remaining TPO-TR1.9 complex were separately
dialyzed against 50 mM NaHCO3, pH 8.5, and then
incubated with EZ-Link Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL) at a
biotin to protein molar ratio of 100:1 for 2 h at room
temperature. Free biotin was removed by five cycles of dilution and
concentration in 10 mM Tris, pH 7.0, using a Centricon YM-30
(Millipore, Bedford, MA). The biotin-TPO molar ratio was determined by
the 2-(4'-hydroxyazobenzene)-benzoic acid (HABA) method according to
the protocol of the manufacturer (Pierce).
Dissociation of the TPO-TR1.9 complex and purification of TPO
Nonbiotinylated TPO, biotinylated TPO (TPO-Bio), and the biotinylated TPO-TR1.9 complex were each incubated for 1 h at 80 C in 6 M guanidine hydrochloride (Sigma, St. Louis, MO) and 700 µM 2-ME before fast protein liquid chromatography on a Sephacryl S-200 column equilibrated with 4 M guanidine hydrochloride. The peaks of nonbiotinylated TPO, TPO-Bio, and TPO recovered from the biotinylated TPO-TR1.9 complex (termed TPO-epitope-protected-Bio (TPO-ep-Bio)) were collected, dialyzed against 10 mM Tris, pH 7.0, and concentrated to 1 mg/ml using a Centricon YM-30. Aliquots of the three different purified TPO preparations were electrophoresed under denaturing and reducing conditions on 10% SDS-polyacrylamide gels (Bio-Rad, Hercules, CA) followed by staining with Coomassie blue.
Trypsin digestion of TPO
TPO, TPO-Bio, and TPO-ep-Bio were treated (18 h at 37 C) with sequencing grade trypsin (Roche, Indianapolis, IN) at an enzyme/substrate ratio of 1/50 (w/w) in digestion buffer (100 mM Tris-HCl, pH 8.5) with 1% acetonitrile. The trypsin digestion profiles of TPO, TPO-Bio, and TPO-ep-Bio were examined by reverse-phase HPLC (see below). The presence of biotin on individual tryptic fragments was assessed by adsorption on streptavidin. Aliquots of digested TPO (20 µg) were incubated (1 h at room temperature) with 200 µl of streptavidin agarose (Pierce) prewashed twice with PBS. After microcentrifugation, the supernatants were subjected to reverse-phase HPLC, and the tryptic fragment profiles were compared with those of the same material not adsorbed with streptavidin.
Reverse-phase HPLC, amino acid composition, and sequencing
For qualitative analyses, 20 µg of trypsin-digested TPO
supplemented with trifluoroacetic acid (0.1% final concentration) was
applied to a C18 reverse-phase 3.9- x 150-mm
column (Waters, Milford, MA). Unless indicated otherwise, tryptic
fragments were eluted with a linear gradient of 0–100% acetonitrile
and 0.1% trifluoroacetic acid for 60 min at 1 ml/min. Eluted peptides
were monitored by absorbance at 214 nm. Digestion of each of the three
TPO preparations (TPO, TPO-Bio, and TPO-ep-Bio) was repeated at least
three times followed by reanalysis by reverse-phase HPLC. After
identification of a peak of interest (see Results), larger
amounts (1–2 mg) of TPO digests were subjected to reverse-phase HPLC
under identical conditions. The peptide peak was collected in 1.8 ml in
a 2-ml polypropylene vial (Corning, Cambridge, MA). The purity of the
peak was confirmed by reinjection of 100 µl of collected material.
Where indicated, the eluted peak was treated (1 h at 80 C) with 700 mM
2-ME followed by reinjection and recovery of peptides separated by
disruption of disulfide bonds. Amino acid composition (400 pmol of
peptide) and N-terminal amino acid sequencing analysis (40 pmol of
peptide) were determined by the Molecular Structure Facility,
University of California-Davis School of Medicine.
Flow cytometry for autoantibody recognition of TPO
CHO cells stably expressing wild-type TPO (34) or a
chimeric TPO-MPO molecule (G2) (35) were propagated in
Ham’s F-12 medium supplemented with 10% FCS, penicillin (100 U/ml),
gentamicin (50 µg/ml), and amphotericin B (2.5 µg/ml). Cells were
released by light trypsinization and incubated (1 h at 4 C) with the
indicated concentrations of purified, recombinant human monoclonal
autoantibodies, TR1.9 and SP1.5, expressed as Fab (6, 7).
Binding of TR1.9 and SP1.5 was detected with PE-conjugated monoclonal
mouse anti-human 
Ab (Caltag, Burlingame, CA). As controls,
cells were incubated with buffer alone or second Ab alone. Flow
cytometry was performed (10,000 events) using a FACScan with CellQuest
software (Becton Dickinson, San Jose, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Purified TPO autoantibody TR1.9 (expressed as a Fab) has
previously been reported to form an immune complex with purified TPO,
as evident by immunoprecipitation of tracer quantities of
[125I]TPO (7) and by gel
filtration of larger quantities of unlabeled proteins
(32). For maximal epitope protection, we complexed TPO
with a 3- to 6-fold molar excess of TR1.9 (e.g., 1 mg of TPO and 3 mg
of TR1.9). Both TPO alone and the TPO-Fab complex were then heavily
biotinylated under native conditions using a biotin/protein molar ratio
of 100/1. The objective of this approach was to biotinylate as many
Lys residues as feasible on the surface of free TPO. However, a Lys
residue(s) on TPO at the Ab-antigen interface would be protected from
biotinylation (Fig. 1
). The extent of
biotinylation of TPO and the TPO-TR1.9 complex was determined by the
HABA reaction to be typically about 15 biotins/molecule of protein.
Before proteolytic digestion, it was then necessary to remove the TR1.9
Fab from the TPO-ep-Bio.
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A). Although not necessary
because of the absence of Fab, TPO-Bio was processed in parallel (Fig. 2B).
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C, lane 6). It should be noted that glycosylated
TPO stains less well with Coomassie blue than does the nonglycosylated
Fab. In addition, the presence of 15 biotin molecules on TPO,
estimated to contribute about 4 kDa, is evident by the slightly
slower mobility of TPO-Bio and TPO-ep-Bio vs nonbiotinylated
TPO included as a control (Fig. 2C, lanes 4 and
6 vs lanes 2 and 5). The presence of
biotin on TPO-Bio and TPO-ep-Bio was also confirmed by their complete
removal on adsorption with streptavidin-agarose (data not shown). Difference between the trypsin fragment profiles of TPO and epitope-protected TPO
In preliminary experiments we determined the optimal conditions
for trypsinization of nonbiotinylated, uncomplexed TPO. Reverse-phase
HPLC on an acetonitrile gradient revealed complete digestion of the
protein with a large spectrum of released fragments (Fig. 3A). The basis for the
epitope-mapping approach used in the present study was that the tryptic
fragment profile was subtly altered following biotinylation of TPO
(Fig. 3B), presumably because biotinylation of Lys (K)
residues on the surface of the native molecule inhibited trypsin action
at these sites. Because Arg (R) residues are not biotinylated, these
sites, in addition to nonbiotinylated K residues, would remain
available to trypsin. High affinity Ab binding to TPO would prevent
biotinylation of a K residue(s) within its epitope. This epitope
protection would, therefore, restore the trypsin site lost when the Ag
was biotinylated in the absence of Ab (Fig. 1).
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B). Thus, this peak was not present in nonbiotinylated TPO
(Fig. 3A) and disappeared again upon biotinylation of the
TPO-Fab complex (Fig. 3C). The same profiles were confirmed
on more than five biotinylations and digestions. In addition,
biotinylation of this peptide was confirmed by it being one of the
peptides removed by adsorption on streptavidin-agarose beads (Fig. 4). Adsorption of multiple tryptic
fragments is to be expected with extensive TPO biotinylation.
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In principle, the polypeptide fragment appearing at 38 min on
reverse-phase HPLC should contain tryptic sites (R or K residues) at
its N and C termini and an internal biotinylated K residue. To confirm
this expectation as well as to identify precisely the amino acids
comprising this polypeptide, we collected the 38 min peak after
injecting a much larger quantity (2 mg) of trypsin-digested TPO-Bio and
using a more shallow acetonitrile gradient to maximize separation from
adjacent peaks. Reinjection of an aliquot of the collected material
confirmed the purity and elution time (38 min) of the peak (Fig. 5A). However, the amino acid
composition of this polypeptide did not correspond to any tryptic
fragment predicted by computer analysis of the TPO amino acid sequence.
The likely reason for these inconclusive data was evident on amino acid
sequencing, which revealed multiple (two or three) N termini. Either
multiple polypeptides were eluting at the identical location or the
peak contained disulfide-linked polypeptides formed upon refolding of
the denatured and reduced TPO.
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B). Only the dominant peaks (1 and 2)
contained peptides, as determined by amino acid composition analysis.
Unlike before reduction, amino acid composition and N-terminal
sequencing unequivocally identified peak 2 (Fig. 5B) to
comprise a 30-aa polypeptide (TPO residues 704–733). As predicted, the
polypeptide was flanked by tryptic cleavage sites (N terminus
immediately downstream of R703 and the C terminus at R733). Most
important, to confirm epitope protection, TPO-Bio residues 704–733
should contain a biotinylated K at position 713. Indeed, extended
N-terminal sequencing identified all amino acid residues from 704–714
except for residue 713, which appeared as a strong, unrecognized
signal. The polypeptide eluting at peak 1 could not be clearly
identified because of contamination with the peak 2 polypeptide. Flow cytometric evidence that K713 is part of the TPO autoantibody immunodominant region
TPO residue K713, protected by monoclonal autoantibody TR1.9, is
one of a cluster of amino acids (aa 713–720) replaced with the
homologous MPO residues in a chimeric TPO-MPO molecule (G2)
(35). In this chimera, in which MPO residues SYPRDFVN
replace TPO residues KFPEDFES, there is a K713S substitution in the TPO
molecule. In principle, therefore, TPO-MPO chimera G2 should lack at
least one (K713), and perhaps more, of the amino acid residues in the
TR1.9 epitope (16–20 aa residues for a typical polypeptide epitope).
Human autoantibodies to the immunodominant region do not recognize MPO
(36). We, therefore, used flow cytometry to compare TR1.9
binding to intact CHO cells expressing on their surface either
wild-type TPO or TPO-MPO chimera G2. In addition, we tested binding to
these cell lines of another human monoclonal autoantibody, SP1.5, whose
epitope does not overlap with that of TR1.9 yet still lies within the
autoantibody immunodominant region. With the wild-type TPO, TR1.9
binding closely paralleled that of SP1.5 over a wide range of Ab
dilutions (Fig. 6A). In
contrast, TR1.9 recognized TPO-MPO chimera G2 less well than did SP1.5
(Fig. 6B). These data provide supporting evidence that TPO
residue K713 comprises part of the TR1.9 epitope and, therefore,
identifies an amino acid residue in the TPO immunodominant region.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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).
An important feature of the approach used in the present study is that
biotinylation (in the presence or the absence of Ab) was performed on
the native protein. Hydrophilic K residues are more frequent on the
protein surface and from the extent of TPO biotinylation achieved
(biotin/TPO molar ratio of 15/1), it appears that at least 15 of the
24 K residues are on the surface of TPO. Once biotinylated, maintaining
the native form is no longer necessary; indeed, denaturation
facilitates complete proteolytic digestion.
Other than the use of chimeric TPO-MPO molecules (which failed to identify the immunodominant region) (35, 37), most previous attempts to identify TPO autoantibody epitopes have examined polyclonal autoantibody interactions with polypeptide fragments (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) with the inherent problem of the highly conformational nature of TPO autoantibody epitopes. However, based on the putative three-dimensional model of TPO, an r-pep fragment (TPO aa residues 742–848) (23), although not defining a precise epitope, may lie in the vicinity of K713 located in the present study. The recent use of polyclonal rabbit antisera to TPO peptides in competition studies has located a TPO autoantibody epitope (TPO aa residues 599–617) believed to be part of the immunodominant region (27). However, this epitope lies on the opposite pole of the 100-kDa Ag to K713. Further studies are necessary to understand the relationship between this epitope and K713. Conceivably, each could define the extreme perimeters of the immunodominant region on one face of the molecule; TR1.9 defines only a portion of this region.
It should be emphasized that human mAb are the optimal tools for identifying the TPO immunodominant region by the approach used in the present study. Although some mouse mAb generated to TPO compete for autoantibody binding (26), these mAb cannot be used to identify with certainty an amino acid residue within the autoantibody immunodominant region. Thus, unless the mouse and human mAb epitopes contain the identical amino acids (which is very unlikely), a K residue protected by the mouse mAb may or may not lie within the human autoantibody immunodominant region. However, our present findings do explain some puzzling observations made with a very interesting member of the mouse mAb panel used to study autoantibody binding, mAb 47 (26).
TPO autoantibodies in patients’ sera compete for mouse mAb 47 binding to TPO on ELISA (26). This mAb was the only one in the panel whose epitope could be determined by screening a TPO cDNA fragment library (15). Whether this mAb is directed against the TPO autoantibody immunodominant region has been debated. On the one hand, the weaknesses in this hypothesis were that 1) only very high concentrations of patient serum competed for mAb 47 binding to TPO (26); 2) the possibility existed of steric hindrance by an Ab molecule (150 kDa) even larger than its Ag (100 kDa); 3) human monoclonal autoantibodies (50 kDa Fab) that define the TPO immunodominant region did not compete for mAb 47 binding to TPO (11); 4) it could not be established in quantitative competition studies that the component among the polyclonal serum TPO autoantibodies inhibited by mAb 47 was to the immunodominant region; and 5) replacement of the mAb 47 epitope in a chimeric TPO-MPO molecule did not appear to alter monoclonal autoantibody binding to TPO (35).
On the other hand, 1) the linear component of the TPO mAb 47 epitope that could be identified (aa residues 713–721) overlapped almost completely with another linear epitope (C21; residues 710–722) identified using a highly potent patient serum (16); and 2) when used at a 40-fold higher concentration (4 x 10-8 vs 10-9 M) than that in the original study, one of the four recombinant autoantibody Fab that define the immunodominant region weakly inhibited mAb 47 binding to TPO (38). This Fab was TR1.9, whose epitope is now determined to contain TPO amino acid residue K713, a component of the mAb 47/C21 epitope.
Because of the high (44%), relatively uniform homology between TPO and
MPO, the three-dimensional structure of MPO (lacking its pro sequence)
is likely to resemble that of TPO. Indeed, support for this is provided
by the use of this model for guided mutagenesis of TPO with retention
of normal folding and autoantibody recognition (35). Based
on this model, the mAb 47 epitope was mapped to a region on the surface
of TPO (Fig. 7). As mentioned above,
substitution of the TPO residues within this epitope with those for MPO
(TPO-MPO chimera G2) did not abolish binding by the TPO monoclonal
autoantibodies to the immunodominant region, providing evidence against
this linear segment being part of the immunodominant region. However,
identification of K713 as a residue within both the TR1.9 epitope and
the mAb 47 epitope induced us to look for a more subtle change in TR1.9
binding, which was indeed found.
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20 contact amino acids within an epitope, it is clear that
alteration of one, or a few, residues (as in TPO-MPO chimera G2) may
produce only subtle changes in Ab affinity. Our previous inability to
detect gross changes in monoclonal autoantibody binding to TPO-MPO
chimeric molecules points to the limitation of mutagenesis studies and
emphasizes the value of the epitopic protection approach used in the
present study. It is also apparent from the complexity of the
three-dimensional structure of MPO (and hence TPO; Fig. 7) that the
short segment defined by mAb 47 represents only a linear component in a
larger, possibly discontinuous, epitope. For this reason, too, it is
not surprising that the human monoclonal autoantibodies to TPO fail to
recognize 66- to 200-aa polypeptides generated in a TPO cDNA fragment
library (6) (S. McLachlan, J. Guo, L. Farilla, and B.
Rapoport, unpublished observations). In conclusion, using an epitope protection approach, we provide the first identification of an amino acid residue (K713) comprising part of an epitope within the TPO immunodominant region. Localization of this focal residue localizes the facet on the large, highly complex TPO molecule that contains the immunodominant region and provides the basis for rational guided mutagenesis studies to more fully characterize this region.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Basil Rapoport, Autoimmune Disease Unit, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Suite B-131, Los Angeles, CA 90048.
3 Abbreviations used in this paper: TPO, thyroid peroxidase; MPO, myeloperoxidase; TPO-Bio, biotinylated TPO; TPO-ep-Bio, TPO epitope-protected-Bio; CHO, Chinese hamster ovary; HABA, 2-(4'-hydroxyazobenzene)-benzoic acid.
Received for publication September 5, 2000. Accepted for publication October 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor by a recombinant human autoantibody Fab converted to IgE. J. Immunol. Methods 195:81.[Medline]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) is present within
the epitopic site (black heavy line). Trypsin digestion at three sites
generates four fragments, including fragments X and Y that contain the
epitope (all or in part). B, Upon biotinylation of the
protein in the absence of Ab, the K residue within the epitope is
biotinylated, and this tryptic site is eliminated, resulting in the
generation of three (rather than four) tryptic fragments. The X and Y
fragments are lost, but are replaced with a new fragment (Z) containing
the biotinylated K residue. C, The formation of an Ag-Ab
(or Fab) complex before biotinylation blocks the lysine biotinylation
site. Therefore, the lysine in the epitope will again be subject to
trypsin digestion. However, before trypsin digestion it is necessary to
dissociate the Ag-Ab complex and to purify the Ag, although the latter
no longer is required to be native. Once again, trypsin would generate
four fragments, including X and Y, and fragment Z (B)
would be lost. Comparing the digestion profiles of the three antigenic
materials on reverse-phase HPLC, either the newly created fragment Z
(B) or the lost fragments X and/or Y (A)
could be isolated for amino acid composition and sequencing. The lysine
involved in the Ab epitope would, therefore, be identified.
