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Original Studies |
FIRS Laboratories, RSR Limited (S.C., J.S., L.P., J.F.S., H.T., V.P., S.R., M.P., B.R.S., J.F.), Llanishen, Cardiff, Wales, United Kingdom CF4 5DU; Istituto di Semeiotica Medica, University of Padua (C.B., M.V.), Padua, Italy; and Department of Medicine, University of Wales College of Medicine (S.C., J.S., H.T., B.R.S., J.F.), Heath Park, Cardiff, Wales, United Kingdom CF4 4XN
Address all correspondence and requests for reprints to: J. Furmaniak, FIRS Laboratories, RSR Limited, Parc Ty Glas, Llanishen, Cardiff, Wales, United Kingdom CF4 5DU.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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light chains. The affinities of four of the
antibodies were in the range 2.0 x 108
M-1 to 7.0 x 108
M-1, and the affinity of the other was
2.3 x 107 M-1. 21-OH MAbs
did not cross-react with 17
-hydroxylase (17-OH) or P450 side
chain cleavage enzyme. Studies using a series of 21-OH fragments
allowed the identification of short stretches of amino acids (AA) that
were involved in forming the MAb binding sites. AA 391–405, defined as
epitope region (ER) 1, were found to be important for binding of
M21-OH1 and M21-OH2, AA 406–411 (ER2) were important for M21-OH3 and
M21-OH4 binding, and AA 335–339 (ER3) for M21-OH5 binding. In
addition, MAb Fab or F(ab')2 fragments were used to study
21-OH AAb epitopes in competition experiments. These investigations
demonstrated that 21-OH AAbs recognize similar epitopes to the MAbs,
with ER2 and ER3 being part of two distinct major epitopes, and ER 1
being part of a minor epitope. Mixtures of M21-OH antibody Fab or
F(ab')2 fragments caused almost complete inhibition
(80%–95%) of AAb binding in 24 out of 25 sera, and in the case of
the remaining serum, the effect was marked but incomplete (67%
inhibition). There were no major differences between the binding
characteristics of AAbs from patients with different forms of
autoimmune adrenal disease. All five 21-OH MAbs reacted with human
adrenal tissue in an immunofluorescence test, but only M21-OH1 and
M21-OH2 reacted with bovine adrenal tissue in these experiments. None
of the MAbs reacted with human ovarian tissue in an immunofluorescence
test. Overall, these studies indicate that 21-OH AAbs bind to at least
three different epitopes in the C-terminal part of 21-OH, and two of
these epitopes appear to be human 21-OH specific. |
Introduction |
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Previous studies have shown that AAb binding sites on 21-OH are in the main part conformational and are formed by the central and C-terminal parts of the protein (10, 11). Furthermore, a close relationship between the parts of the sequence important for 21-OH enzyme activity and 21-OH AAb binding has been demonstrated in studies on the direct effect of 21-OH AAb on enzyme activity in vitro, and in binding studies using 21-OH containing amino acid (AA) mutations associated with reduced 21-OH enzyme activity in nonclassical adrenal hyperplasia (11, 12, 13). Although 21-OH AAb heterogeneity was evident in these studies, there were no clear differences between 21-OH AAb binding characteristics in patients with different forms of autoimmune adrenal disease, i.e. isolated AD, AD in the context of autoimmune polyglandular syndrome (APS) types I and II, and 21-OH AAb-positive patients without overt adrenal failure (14). To study the AAb binding epitopes on 21-OH in more detail, a panel of mouse monoclonal antibodies (MAbs) to human recombinant 21-OH was produced, and their interaction with 21-OH examined by Western blotting, immunofluorescence test (IFT), and immunoprecipitation assay (IPA). Fab or F(ab')2 preparations were isolated from the 21-OH MAbs and used in binding inhibition studies. These studies allowed the identification of three different short AA sequences on 21-OH that appear to be important for parts of the AAb binding sites.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The full-length 21-OH gene (1–1482 bp) and a modified 21-OH gene sequence with an internal deletion (504–1137 bp) (10) were inserted into the bacterial expression vector, pGEX-2T (Pharmacia Biotech, St. Albans, UK) (pGEX/21-OH1 and pGEX/21-OH7, respectively), for the expression of the 21-OH fusion protein with glutathione-S-transferase (GST) (15).
The full-length 21-OH gene was also cloned into pYES2.0 (Invitrogen,
Leek, The Netherlands) (pYES2/21-OH1) downstream of the T7 promoter as
previously described (2). Various in-frame deletions and
truncations of the 21-OH gene were carried out using different
restriction enzyme sites, and the modified genes were cloned into
pYES3, a derivation of pYES2, as described previously (10). In
addition, construct p21-OH14 was produced using PpumI
restriction enzyme (Table 1
and Fig. 1).
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and Fig. 1
). These constructs were
sequenced by the dideoxy termination method using Sequenase Version 2.0
kit according to the manufacturer’s instructions (United States
Biochemical, supplied by Amersham International plc, Little Chalfont,
UK) using the M13 (-40) sequencing primer.
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Sera were obtained from 21-OH AAb-positive patients (n =
25) with APS type I (n = 5), APS type II (n = 5), isolated AD
(n = 5), 21-OH AAb-positive patients with normal adrenal function
(potential AD; n = 5), and 21-OH AAb-positive patients with
impaired adrenal function (subclinical AD; n = 5) (Table 3). Out of five patients with potential
AD, three had Graves’ disease, one had Hashimoto’s thyroiditis, and
one had insulin-dependent diabetes mellitus and premature ovarian
failure (POF). Among five patients with subclinical AD, one did not
have evidence of other autoimmune disease, two had Hashimoto’s
throiditis, one had Hashimoto’s thyroiditis and POF, and one had
Graves’ disease. Disease diagnosis was based on clinical,
immunological, and biochemical grounds (patients whose sera were used
in this study have been described in detail previously, see Refs. 7, 17). Pooled positive serum prepared from ten 21-OH AAb-positive
patients with AD, with 21-OH AAb levels ranging from 7–22 U/mL (7),
was used in some experiments. Pooled serum from 20 healthy blood donors
was used as a 21-OH AAb-negative control. All 20 sera were negative for
21-OH AAb in the 125I-labeled 21-OH AAb assay (7).
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Eight-week-old female BALB/C mice were immunized with 50 µg 21-OH1-GST per mouse. In the case of 21-OH7-GST, the recombinant fusion protein was treated with thrombin (Pharmacia Biotech, following manufacturer’s instruction) to remove GST, and 50 µg cleaved 21-OH7 protein per mouse was used for immunization. Four days after the final iv boost the mice spleen cells were fused with the myeloma cell line X63-Ag8.653 and cloned as described previously (18) and ascites produced. 21-OH binding by the mouse antibodies was assessed using 35S-labeled (4) or 125I-labeled (7) 21-OH in combination with antimouse IgG-agarose (Sigma, Poole, UK) or solid-phase protein A (RSR Ltd., Cardiff, UK).
Scatchard analysis (19) of the 125I-labeled 21-OH/21-OH MAb interaction was carried out as described previously (20) using unlabeled purified human recombinant 21-OH expressed in yeast (7).
Isolation of Fab or F(ab')2 from 21-OH MAbs
21-OH MAb IgG was purified from ascites fluid by affinity chromatography on Prosep-A (Bioprocessing Ltd., Consett, UK) according to the manufacturer’s instructions. MAb subclass and light chain type was determined using a commercial kit (Life Technologies, Paisley, UK).
The purified IgG preparations were treated with either pepsin (Sigma) at an enzyme/protein ratio of 1:10 or mercuripapain (Sigma) at an enzyme/protein ratio of 1:10 and passed through a Prosep-A column to remove any intact IgG or Fc fragment from the Fab or F(ab')2 respectively (21).
Control IgG and Fab preparations were obtained from a TSH receptor mouse MAb (22).
PAGE and Western blotting
Preparations of recombinant 21-OH expressed in yeast or in Escherichia coli (7) were run on 9% gels in the presence of SDS and 10 mmol/L dithiothreitol (23) and blotted onto nitrocellulose. The Western blotting procedure was carried out according to the method described by Birk and Koepsell (24), and the membranes reacted with diluted MAb followed by antimouse Ig horseradish peroxidase conjugate and enhanced chemiluminescence reagents according to the manufacturer’s instructions (Amersham International plc.). A mouse MAb to glutamic acid decarboxylase 65 was used as control (25).
Immunofluorescence studies
The reactivity of 21-OH MAbs was also tested by a classical indirect immunofluorescence technique using thin cryosections of human and bovine adrenal tissue and biotin conjugate followed by avidin fluoresceine isothiocyanate-conjugated antimouse IgG (Sigma). Reactivity of 21-OH MAbs was also tested using cryostat sections of normal human ovary tissue by the same method. MAb IgGs were tested in serial dilutions (from 1 mg/mL to 1 ng/mL) until reaching the end point. A mouse MAb to the TSH receptor was used as a control (22).
Reactivity of 21-OH MAbs with other steroidogenic enzymes
35S-Labeled 17-OH and 35S-labeled
P450 side chain cleavage enzyme (P450scc) were prepared in the
in vitro transcription/translation (TnT) system (Promega,
Southampton, UK) as described previously (17). The
[35S]17-OH and [35S]P450scc proteins
were then used in IPAs (17) to test the reactivity of the 21-OH MAbs.
Rabbit antibody to 17-OH and P450scc were used as controls (17).
Analysis of reactivity of 21-OH MAb with modified 21-OH proteins
Modified 35S-labeled 21-OH proteins containing
various deletions and truncations (Tables 1 and 2 and Fig. 1) were
produced in the in vitro TnT system and were used in IPAs to
assess 21-OH MAb binding as described previously (4).
Effect of Fab or F(ab')2 preparations on binding of intact monoclonal 21-OH IgG and 21-OH AAbs to 125I-labeled 21-OH
Further analysis of the epitopes recognized by the 21-OH MAbs and by 21-OH AAb was carried out using a technique that exploits the ability of protein A to bind to intact antibody complexed with 125I-labeled antigen but not to Fab or F(ab')2 bound to labeled antigen (26).
Fab or F(ab')2 preparations, at various dilutions, were incubated for 7 h at 4 C with 125I-labeled 21-OH (30,000 cpm/50 µL). Intact 21-OH antibodies (either 50 µL 21-OH MAb IgG or 50 µL patient sera appropriately diluted in assay buffer) were then added and incubation continued for 18 h at 4 C. 125I-Labeled 21-OH bound to either intact 21-OH MAb or 21-OH AAb was separated by the addition of solid-phase protein A.
Computer analysis of epitope region (ER) AA sequences
The complete complementary DNA sequences of human and bovine 21-OH (27, 28) were translated into AA sequences using the computer software DNASIS, version 2.1 (Hitachi Software Engineering America, San Francisco, CA). The AA sequences of the ERs in both human and bovine 21-OH were aligned using the Clustal method.
Statistical methods
Statistical analyses were carried out using SPSS for Windows software. The statistical significance of any difference of the effect of Fab or F(ab')2 preparations on 21-OH AAb binding between different forms of autoimmune adrenal disease patient groups was determined by Mann-Whitney U test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five mouse MAbs to 21-OH were produced; one (M21-OH1) in response
to immunization with 21-OH fragment 21-OH7 and four (M21-OH2, -3, -4,
and -5) in response to immunization with the full-length fusion protein
21-OH1-GST (Table 4). Binding of the MAbs
to 125I-labeled 21-OH was dose dependent, and the antibody
dilution profiles are shown in Fig. 2.
M21-OH1 was IgG2a subclass, and M21-OH2, -3, -4, and -5 were IgG1
subclass. All five antibodies had light chains (Table 4). Scatchard
analysis of the interaction between 125I-labeled 21-OH, and
each 21-OH MAb was carried out and the affinity constants are shown in
Table 4.
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In Western blotting analysis, all five MAbs bound specifically to full-length 55-kDa 21-OH whether expressed in yeast or in E.coli (data not shown). M21-OH1–5 reacted with recombinant 21-OH expressed in yeast up to a dilution of 0.1 µg/mL, whereas with recombinant 21-OH expressed in E.coli up to a dilution of 1 µg/mL. None of the 21-OH MAbs reacted with nonrecombinant bacterial or yeast proteins (including control strains) in Western blotting analysis at these dilutions.
In addition, the reactivity of the 21-OH MAbs with 17-OH and P450scc
was tested by IPA. None of the 21-OH MAb IgGs reacted with either
[35S]17-OH or [35S]P450scc. In
particular, M21-OH1–5 bound from 1.3–2.0% of
[35S]17-OH compared with 1.4% binding of control IgG
(TSH receptor MAb), and this contrasted with
[35S]17-OH binding of 58% to a specific 17-OH
rabbit antibody (diluted 1:100). In the case of
[35S]P450scc, binding to the 21-OH MAbs did not exceed
2.8% compared with 1.9% binding of control IgG and 45% binding to a
specific P450scc rabbit antibody (diluted 1:100).
Immunofluorescence studies
When tested by immunofluorescence, IgGs from M21-OH1–5 reacted
with human adrenal tissue, and all showed the positive staining
patterns (Fig. 3)
typical for adrenal cortex antibody (ACA). M21-OH1 was positive up to a
dilution of 1 ng/mL, M21-OH2 up to a dilution of 1 µg/mL, M21-OH3 up
to a dilution of 100 ng/mL, M21-OH4 up to a dilution of 1 µg/mL, and
M21-OH5 up to a dilution of 1 µg/mL. M21-OH1 and M21-OH2 showed
positive staining when bovine adrenal tissue was used in IFT, whereas
M21-OH3, -4, and -5 were negative in IFT based on bovine adrenal tissue
(Table 4). All five MAbs were negative in IFT on human ovarian tissue.
Control mouse IgG (from a TSH receptor MAb) was negative in
immunofluorescence tests on human adrenal tissue, bovine adrenal
tissue, and human ovarian tissue.
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In the competition studies we originally planned to use bivalent
F(ab')2 preparations of the MAb. However, preliminary
studies using peptic digestion showed that IgGs from M21-OH2, -3, and
-4 were highly sensitive to pepsin, and all three MAbs were rapidly
degraded without the formation of significant amounts of
F(ab')2 fragments. Consequently, F(ab')2
fragments were isolated from M21-OH1 and M21-OH5, whereas Fab fragments
prepared by papain digestion were obtained from M21-OH3 and the control
TSH receptor MAb. These Fab and F(ab')2 preparations were
used in binding inhibition studies with all five MAbs. Binding of
intact M21-OH1 IgG to 125I-labeled 21-OH was inhibited by
the M21-OH1 F(ab')2, whereas Fab from M21-OH3,
F(ab')2 from M21-OH5, and Fab from the control IgG had
little or no effect (Fig. 4a). Binding of
intact M21-OH2 IgG to 125I-labeled 21-OH was also inhibited
by M21-OH1 F(ab')2, but not by M21-OH3 Fab, M21-OH5
F(ab')2 and the control Fab (Fig. 4b). Binding of intact
M21-OH3 IgG to 125I-labeled 21-OH was inhibited by M21-OH3
Fab. In contrast, M21-OH1 F(ab')2, M21-OH5
F(ab')2, and the control Fab had little or no effect (Fig. 4c). Binding of intact M21-OH4 IgG to 125I-labeled 21-OH
was inhibited by M21-OH3 Fab, but not by M21-OH1 F(ab')2,
M21-OH5 F(ab')2, and the control Fab (Fig 4d). Binding of
intact M21-OH5 IgG to 125I-labeled 21-OH was inhibited by
M21-OH5 F(ab')2, whereas M21-OH1 F(ab')2,
M21-OH3 Fab, and the control Fab had little or no effect (Fig. 4e).
This data is summarized in Table 5.
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Binding of 21-OH AAb in pooled patient sera to
125I-labeled 21-OH was not inhibited by M21-OH1
F(ab')2, but M21-OH3 Fab and M21-OH5 F(ab')2
had clear effects (Fig. 4f). A combination of the three Fab or
F(ab')2 preparations inhibited 21-OH binding by AAb almost
completely (Fig. 4f).
Fab or F(ab')2 preparations from M21-OH1, -3, and -5 were
also used in inhibition studies with individual 21-OH AAb-positive
sera. The results of these studies are shown in Table 3 and are
expressed as a percent inhibition of 125I-labeled 21-OH
binding relative to binding of AAb in the absence of Fab or
F(ab')2 preparations.
In the majority of patients the binding of AAb to
125I-labeled 21-OH was not or only partially inhibited by
M21-OH1 F(ab')2 with a mean inhibition of 15% ± 12.7
(mean ± SD, n = 25; range 0–50%). However in
the case of sera from three patients (one in the APS type II group and
two in the AD group) M21-OH1 F(ab')2 caused inhibition of
AAb binding of 50%, 30%, and 47%, respectively (Table 3).
In contrast to M21-OH1 F(ab')2, M21-OH3 Fab resulted in
clear inhibition of AAb binding to 125I-labeled 21-OH in
all 25 patient sera studied with a mean inhibition of 54% ± 14.5
(mean ± SD, n = 25; range 27–75%) (Table 3).
M21-OH5 F(ab')2 also showed clear inhibition of AAb binding
to 125I-labeled 21-OH in all 25 patient sera with a mean
inhibition of 76% ± 12.4 (mean ± SD, n = 25;
range 35–92%) (Table 3).
A combination of the three Fab or F(ab')2 preparations
resulted in essentially complete inhibition of AAb binding to
125I-labeled 21-OH in 24 out of 25 patients with a mean
inhibition of 89.5% ± 4.6 (mean ± SD, n = 24;
range 80–95%). However, in the case of 1 patient in the AD group, the
combination of three Fab and F(ab')2 preparations resulted
in only 67% inhibition (Table 3).
There were no statistical differences in the effect of each Fab or F(ab')2 fragment on AAb binding between the different patient groups (P > 0.05). However when a combination of all the three Fab or F(ab')2 preparations was used, there were small but statistically significant differences between the inhibition of AAb binding of the APS type II group and the potential AD group (P = 0.01), the AD group and the potential AD group (P = 0.01), and the subclinical AD group and the potential AD group (P = 0.01); there were no statistically significant differences between the other groups (P > 0.05).
21-OH MAb epitope analysis
In the IPA based on 35S-labeled 21-OH, all five MAbs
reacted with full-length 21-OH and with 21-OH truncated at either AA
448 or 418 (from constructs p21-OH1, p21-OH2, and p21-OH3; Table 1). In
addition, M21-OH5 reacted with 21-OH truncated at AA 381 (from
construct p21-OH4) and with 21-OH with the internal deletion 382–414
AAs (from construct p21-OH12; Table 1). None of the 21-OH MAbs bound to
21-OH truncated at either AA 335 or AA 282 (from constructs p21-OH14
and p21-OH5, respectively; Table 1).
Further studies were carried out using modified 21-OH proteins, with
smaller stretches of AAs deleted. All the 21-OH MAbs reacted with 21-OH
with the AAs 494–412 deleted (from constructs pND21-OH2 and pND21-OH3;
Table 2). M21-OH1, -2, and -5 reacted with 21-OH with AAs 494–406
deleted (from construct pND21-OH4; Table 2), whereas M21-OH3 and -4 did
not react with this modified protein (Table 2). M21-OH1–4 did not
react with modified 21-OH proteins truncated at AA 391, 360, 340, or
335 (from constructs pND21-OH5–8, respectively; Table 2). In contrast,
M21-OH5 bound to 21-OH proteins truncated at AAs 391, 360, or 340 but
not to 21-OH truncated at AA 335 (Table 2).
Consequently, AAs 391–405, defined as ER1 appeared important for binding of M21-OH1 and M21-OH2. AAs 406–411 (ER2) appeared important for binding of M21-OH3 and M21-OH4, and AAs 335–339 (ER3) appeared important for binding of M21-OH5.
Computer analysis of ER sequences
Computer analysis was carried out on the AA sequences of human and
bovine 21-OH to compare the sequences in the ERs 1, 2, and 3. ER1 (AA
391–405), recognized by M21-OH1 and M21-OH2, was found to be 87%
homologous with two AA changes: arginine at position 400 in human 21-OH
is changed to glutamine in bovine 21-OH and tryptophan at position 405
in human 21-OH is changed to arginine in bovine 21-OH (Fig. 5). ER2 (AA 406–411) recognized by
M21-OH3 and M21-OH4, was found to be 100% homologous. ER3 (AA
335–339) recognized by M21-OH5, was found to be 80% homologous, with
only one AA difference between human and bovine 21-OH: proline at
position 335 in human 21-OH is changed to threonine in bovine 21-OH
(Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-OH or P450scc in immunoprecipitation
assays. Furthermore, the MAbs did not react with steroidogenic enzymes
in human ovarian tissue as judged by an IFT.
Our investigations indicated that the panel of five MAbs were able to
recognize three distinct sections of the 21-OH protein. M21-OH1 and -2
showed the same reactivity to one distinct part of 21-OH, M21-OH3 and
-4 both reacted to another part, and M21-OH5 reacted with yet another
part of the 21-OH protein sequence (Table 1 and Fig. 4). Although the
MAbs recognized different parts of 21-OH, they showed comparable
affinities for the protein (Table 4).
Extension of the competition studies to 21-OH AAbs in a pool of patient sera indicated that the AAbs recognized the same regions on 21-OH as the MAbs. In particular, M21-OH5 F(ab')2 and M21-OH3 Fab preparations markedly inhibited the binding of 21-OH AAbs (in the patient pooled sera) to 125I-labeled 21-OH, suggesting that these two MAbs recognize major autoantigenic epitopes on 21-OH. Further competition experiments were then carried out with 21-OH AAbs in sera from 25 individual patients with different forms of autoimmune adrenal disease. These studies showed that all the sera studied contained 21-OH AAbs that recognized the same sections of 21-OH as M21-OH3 (section 2) and M21-OH5 (section 3). M21-OH1 Fab was less effective than M21-OH3 or -5 in inhibiting the interaction between 21-OH AAbs and labeled 21-OH. A combination of Fab or F(ab')2 preparations from M21-OH1, -3, and -5 inhibited 21-OH AAb binding to 125I-labeled 21-OH essentially completely (>80%) in 24 out of the 25 sera. This suggested that essentially all the 21-OH AAbs in these 24 sera bound to the epitopes on 21-OH, which were dependent on AAs in sections 1, 2, and/or 3 of the 21-OH sequence. The single exceptional serum out of the 25 was from a patient with isolated AD, and in this case, a combination of the three Fab or F(ab')2 preparations only partially inhibited 21-OH AAb binding to 125I-labeled 21-OH (67% inhibition). Further studies with 21-OH protein truncated at AA 282 and labeled with 35S showed that this serum recognized epitope(s) in the N-terminal part of 21-OH (data not shown), which would not be recognized by any of the five MAbs we produced. This observation is in agreement with previous studies on Western blotting of yeast expressed modified 21-OH proteins that showed that 6/14 21-OH AAb-positive sera reacted with an N-terminal epitope between AA 15 and 162 (11). Also, Song et al. (29) reported that about 10% of 21-OH AAb-positive sera react with an epitope in bacterially expressed 21-OH N-terminal fragments (AA 1–272).
In the current study, no major differences were observed in the regions
of 21-OH recognized by 21-OH AAbs in different patient groups. This is
in agreement with our earlier studies (14) on the reactivity of 21-OH
AAbs in sera from different patient groups with full-length and
modified 21-OH, and suggests that in the autoimmune response to 21-OH
in APS types I and II, isolated AD, and ACA/21-OH AAb-positive patients
without overt adrenal failure, the same autoantigenic epitopes are
recognized. In addition, six of the patients studied (three with APS
type I, one with APS type II, one with subclinical AD, and one with
potential AD; Table 3) had POF. 21-OH AAb in sera from patients with
POF showed similar binding characteristics to different regions on
21-OH as 21-OH AAb in sera from patients without POF.
The sections of 21-OH recognized by the mouse MAbs were analyzed in
more detail in studies with 35S-labeled 21-OH proteins with
small segments of AAs removed (Fig. 1 and Table 2). These experiments
indicated that AA 391–405 were important for M21-OH1 and -2 binding,
and these AAs, i.e. AA 391–405, were defined as ER1.
Similarly, AA 406–411, found to be important for M21-OH3 and -4
binding, were defined as ER2, and AA 335–339, found to be important
for M21-OH5 binding, were defined as ER3. In view of the effective
competition between the mouse MAbs and 21-OH AAbs ER1, ER2, and ER3
must also be an important part of the AAb binding sites. ER2 and ER3
appear to be parts of two distinct major AAb epitopes, whereas ER1
appears to be part of a minor epitope.
Further information on ER1–3 was obtained in immunofluorescence studies using human and bovine adrenal tissue sections. All five mouse MAbs reacted with human tissue, but only M21-OH1 and -2 reacted with bovine tissue. This suggested that ER2 and -3 are human specific, whereas ER1 is present on both human and bovine 21-OH. Some of the AA sequence differences between human and bovine 21-OH are consistent with this specificity. In particular, AA 335 in ER3 (AA 335–339) is the hydrophobic proline in human 21-OH and hydrophillic threonine in bovine 21-OH, and this difference is likely to have important implications for folding of ER3. Also, AA 405, immediately adjacent to ER2 (AA 406–411) is the hydrophobic tryptophan in human 21-OH and hydrophillic arginine in bovine 21-OH, and this may be responsible for structural differences between human and bovine 21-OH in ER2. In contrast, the sequence differences between human and bovine 21-OH in ER1 are conservative and should not have a major influence on the structure of ER1. These observations emphasize the importance of using human rather than bovine adrenal tissue sections in immunofluorescence tests for adrenal AAbs. Furthermore, comparison of AA sequence homologies of 21-OH of human (27), porcine (30), and mouse (31) origin tends to suggest that neither porcine nor mouse would be useful substitutes for human adrenal material.
The mapping of autoantigenic epitopes using different MAbs has been used in the case of other autoantigens; for example, thyroid peroxidase, thyroglobulin, and glutamic acid decarboxylase (26, 32, 33). However, this is the first study in the case of 21-OH, and our observations with the MAbs confirm previous reports that 21-OH AAb are heterogenous (10, 11, 13, 29).
Previous studies (10, 11, 12, 13) have suggested that 21-OH AAb binding sites are conformational, and this is consistent with the nature of AAb epitopes in general (5). In the current study we identified three short stretches of 5, 6, and 15 AAs to be involved in 21-OH AAb binding. The mouse MAbs used in these studies were raised to bacterially expressed, denatured 21-OH, and most probably bind to essentially linear epitopes, of which ER1–3 are important parts. In the case of 21-OH AAbs, the AAs contained within ER1–3 are likely to be an essential part of more complex, folded epitopes.
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Acknowledgments |
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Footnotes |
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2 Recipients of RSR fellowships.
Received January 16, 1998.
Revised March 12, 1998.
Accepted March 31, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), M21-OH2 (
), M21-OH3
(
), M21-OH4 (
), and M21-OH5 (
).
