| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
kan Hedstrand,
Jan Haavik,
Jaakko Perheentupa,
Corrado Betterle,
Jan Gustafsson,
Eystein Husebye,
Fredrik Rorsman and
Olle Kämpe
Departments of Medical Sciences (O.E., H.H., F.R., O.K.) and Women’s and Children’s Health (J.G.), University Hospital, Uppsala University, SE-751 85 Uppsala, Sweden; Department of Biochemistry and Molecular Biology (J.H.) and Division of Endocrinology, Institute of Medicine (E.H.), University of Bergen, NO-5021 Bergen, Norway; the Hospital for Children and Adolescents (J.P.), University of Helsinki, FIN-00014 Helsinki, Finland; and Institute of Semeiotica Medica (C.B.), Clinical Immunology and Allergy, University of Padova, IT-35128 Padua, Italy
Address correspondence and requests for reprints to: Olov Ekwall, M.D., Department of Medical Sciences, University Hospital, Uppsala University, SE-751 85 Uppsala, Sweden. E-mail: olov.ekwall{at}medsci.uu.se
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
A clone encoding PAH was used for in vitro transcription/translation, followed by immunoprecipitation with sera from 94 APS I patients and 70 healthy controls. Of the APS I patients, 25% had PAH antibodies, and no reactivity was detected in the controls. No association with the main clinical components of APS I was found with PAH antibodies. Altogether, 59 sera from the 94 APS I patients reacted with at least one of TPH, TH, or PAH, whereas 35 showed no reactivity. Nineteen of the sera contained antibodies towards all enzymes, 12 to TPH only and 12 to TH only. No sera showed antibodies that reacted to only PAH. An immunocompetition assay demonstrated that the reactivity against PAH represents a cross-reactivity with TPH, whereas antibodies against TPH and TH are directed towards epitopes unique for the two enzymes.
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The family of tetrahydrobiopterin-dependent hydroxylases consists of
the three highly homologous enzymes: tryptophan hydroxylase (TPH),
tyrosine hydroxylase (TH), and phenylalanine hydroxylase (PAH). The
enzymes have an overall linear homology of about 35%; and in the
catalytic domain, a homology of 70% is seen (Fig. 1
) (15). All three enzymes catalyze the
hydroxylation of amino acids and depend on a tetrahydropteridine as a
cofactor. They all have central roles in the biosynthesis of the
neurotransmitters serotonin and dopamine (Fig. 2). TPH catalyzes the hydroxylation
of tryptophan into 5-OH tryptophan and is the rate-limiting enzyme in
the synthesis of serotonin. It is a 230-kDa tetramer consisting of
identical subunits, each with a molecular mass of 58 kDa, mainly
expressed in serotonergic cells in the central nervous system and the
intestine (16). TH consists of four 55- to 59-kDa subunits, as four
isoforms (TH 1–4) attributable to alternative splicing, forming
tetramers with molecular masses of 204–217 kDa. It is the
rate-limiting enzyme in the biosynthesis of catecholamines, where it
converts tyrosine into L-dopa, and it is mainly expressed
in the adrenal medulla and catecholaminergic neurons throughout the
body. PAH is found in an equilibrium between tetrameric and a dimeric
forms composed of 50-kDa subunits. It is mainly expressed in the liver,
where it catalyzes the conversion of phenylalanine into tyrosine.
Mutations in PAH are the most common defect responsible for
phenylketonuria.
|
|
In this study, we set out to investigate whether PAH is an autoantigen in APS I and to identify whether cross-reactivity exists between antibodies to these three, highly homologous, enzymes.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Sera were obtained from 10 Swedish, 9 Norwegian, 62 Finnish, and 13 Italian patients with APS I. The clinical characteristics of the patients have been described previously (4, 10, 11, 12, 19). Seventy healthy Swedish blood donors served as controls.
Clones
TPH and TH were cloned from cDNA libraries derived from human small intestine and scalp. A clone coding for human PAH was obtained from the American Type Culture Collection (Manassas, VA; ATCC no. 61605). The clones were subcloned into the pSP64 poly A vector (Promega Corp., Madison, WI), as described elsewhere (11, 12), to permit and optimize in vitro transcription and translation (ITT).
ITT and immunoprecipitation
The pSP64 polyA-clones were transcribed and translated in vitro using the TNT SP6 coupled reticulocyte lysate system (Promega Corp.). The sizes of the radioactively-labeled products were determined on an SDS-PAGE minigel (Bio-Rad Laboratories, Inc., Richmond, CA). Immunoprecipitation was performed (20), and the results were expressed as an index [(cpm sample - cpm negative control)/(cpm positive control - cpm negative control) x 100]. Samples were run in triplicate. APS I patients, known to have high titers of TPH, TH, or PAH antibodies, were used as positive controls; and a blood donor, as a negative control. The upper normal limit of the Ab index was set to the mean value for the blood donors plus 4 SDs. For TPH, the cut-off value was 12; for TH 27 and for PAH, it was set at 30.
Immunocompetition assay
Five sera from APS I patients with reactivity towards different combinations of the three antigens were identified. We selected sera reacting with all three enzymes, with both TPH and TH, with both TPH and PAH, and with TPH or TH alone. No serum reacted with the combination of TH and PAH or with PAH solely. The five sera were tested for the immunoreactivity towards the antigens/antigen it reacted with, in a dilution series ranging from 1:20–1:40,000. The highest serum dilution for each combination of antigen and serum that gave a clearly positive immunoreactivity index was determined. These serum dilutions, ranging from 1:40–1:640, were used in the following experiments.
Radioactive and nonradioactive TPH, TH, and PAH were produced in parallel by ITT. Because the three proteins, with approximately the same molecular weight, differ in their methionine content (with TPH having 6 methionines—TH 4, and PAH 3) 1 U of labeled or unlabeled antigen was determined to be the amount of protein representing 40,000; 27,000; and 20,000 cpm of TPH, TH, and PAH, respectively. In 96-well microtiter plates, patient serum was allowed to react with 1 U of labeled antigen together with none, 1, 2, 4, 8, 16, or 32 U of unlabeled TPH, TH, or PAH; and the reactivity was measured as described earlier (11). The experiments were repeated twice.
Statistical analysis
Fisher’s exact test was used to compare the frequencies of reactivity to TPH, TH, and PAH in the sera from patients with the different clinical components of APS I.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The efficiencies of the ITT reactions varied among the different enzymes. For TPH, 6–7% of the total [35S]-methionine was incorporated in a representative experiment, whereas the incorporation rates for TH (3%) and PAH (2%) were lower. This was approximately as expected, based on their relative content of methionines (see above). Analysis on SDS-PAGE gels showed bands with the expected sizes for all enzymes.
Immunoprecipitation with sera from 94 APS I patients
Altogether, 25 out of 94 APS 1 sera (27%) had a positive index to
PAH. The frequency of PAH reactivity varied between APS 1 sera from the
different countries, where 17 of 62 Finnish sera (27%), 5 of 10
Swedish sera (50%), 0 of 9 Norwegian sera (0%), and 4 of 13 Italian
sera (31%) were positive to PAH. As previously reported, 47 of 94 sera
(50%) had a positive index to TPH and 41 of 94 (44%) to TH (Fig. 3) (11, 12). No reactivity against any of
the 3 enzymes was found in sera from 70 healthy blood donors. The
occurrence of TPH antibodies in serum samples from these 94 APS 1
patients was significantly associated with intestinal dysfunction
(P < 0.0001), and antibodies against TH were
associated with alopecia areata (P = 0.02) (11, 12).
Using Fisher’s exact test, no significant correlations were found
between the presence of PAH-Abs and major clinical characteristics of
APS I (Table 1).
|
|
).
|
Immunoreactivities against different combinations of serum,
labeled antigen, and unlabeled antigen were plotted; and clear patterns
could be seen where unlabeled antigen, in rising concentration, either
competed with the labeled antigen or did not affect the reactivity. As
seen in Fig. 5, the reactivity against
TPH in sera (patients 1 and 4) with reactivity against both TPH
and PAH can be eliminated or markedly reduced by adding unlabeled PAH.
In the same manner, reactivity against PAH, in all sera reacting with
PAH, is eliminated by adding unlabeled TPH but not by adding unlabeled
TH. In patients (patients 2 and 3) with reactivity against TPH only or
against both TPH and TH, the reactivity against TPH is not altered by
adding unlabeled PAH. TH reactivity in all sera with TH antibodies is
unaffected by the presence of unlabeled TPH or PAH. As a control, in
each experiment, reactivity against all three antigens is eliminated
when adding the same unlabeled antigen.
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The possible roles of TPH and TH as autoantigens in the pathogenesis of
APS is supported by their tissue distributions and correlations of the
antibodies with different components of the syndrome. Antibodies to
TPH, which is highly expressed in the gut, are coupled with
gastrointestinal dysfunction; and antibodies to TH, expressed in hair
follicles, are correlated with alopecia areata. It is difficult to
explain the significance of PAH antibodies, because only a weak
(P = 0.065), not statistically significant,
correlation is found with autoimmune hepatitis, one of the disease
components in APS I. Autoimmune hepatitis, a feared component of APS I,
has earlier been shown to be strongly correlated with the presence of
autoantibodies against the enzymes CYP1A2 and
aromatic-L-amino acid decarboxylase (10, 21).
Although the connection between antibodies to PAH and autoimmune
hepatitis fails to reach statistical significance, it is interesting
that PAH is expressed in liver and kidney. Figure 4
shows that some
sera have unique immunoreactivity against either TPH or TH, whereas no
serum reacts with PAH only. This suggests that the reactivity towards
TPH and TH, at least in some cases, results from different
immunizations and that the reactivity towards PAH mainly reflects a
cross-reactivity with TPH or TH. Sera with antibodies against both TPH
and TH may contain either antibodies that recognize both enzymes
separately, or cross-reacting antibodies. It has previously been shown
that monoclonal antibodies that react with all three enzymes can be
generated in mice (22).
In immunocompetition assays performed using sera that react against TPH and/or PAH, the reactivity is reduced by adding nonradioactive TPH and/or PAH, but not TH. In contrast, reactivity against TH cannot be altered by adding nonradioactive TPH or PAH, nor can adding nonlabeled PAH or TH alter the reactivity against TPH in serum with solely TPH reactivity. This suggests that a shared epitope between TPH and PAH is responsible for all the reactivity against PAH. Antibodies against TH, or TPH in sera not reacting with PAH, are directed towards epitopes unique for these antigens. This observation, in combination with the fact that no patient serum shows reactivity with solely PAH, whereas 12 patients have unique reactivity towards either TPH or TH, strengthens the conclusion that the reactivity against PAH reflects cross-reactivity with TPH. Although we can not definitely exclude the possibility of antibodies cross-reacting with TPH and TH, this work implies that there are three subsets of autoantibodies reacting with biopterin-dependent hydroxylases in APS I patients. One subset reacts with epitopes unique for TPH, a second with epitopes unique for TH, and a third with epitopes shared by TPH and PAH.
Further investigation of the polyclonality of the antibodies is
required. The pattern seen in Fig. 5, for patient no. 1, where the
addition of unlabeled PAH cannot totally eliminate the reactivity
towards TPH, whereas unlabeled TPH totally eliminates PAH reactivity,
suggests that a number of epitopes exist, some of them unique for TPH
and others shared by TPH and PAH.
For TPH and TH, we have seen a marked reduction in enzymatic activity in the presence of patient serum, with TPH or TH autoantibodies, in a 1:100 dilution (11, 12). When measuring PAH activity in the presence of PAH-Ab-positive patient serum in a 1:100 dilution, using the same methodology as with TPH and TH, no decrease in enzymatic activity can be seen (data not shown). This can be interpreted either as if the epitopes responsible for cross-reactivity between TPH and PAH are not involved in the regulation of PAH activity, or as if the cross-reacting antibodies have a weaker affinity for PAH than for the corresponding epitopes on TPH. The lack of ability in PAH-Ab-positive sera to reduce PAH activity strengthens the conclusion that PAH reactivity only reflects a cross-reactivity with TPH, because the capability to reduce the activity of the antigen is a characteristic shared by many autoantibodies directed towards enzymes (10, 23).
In Fig. 3, where the reactivities against TPH, TH, and PAH in sera from
94 APS I patients and 70 blood donors are plotted, a difference in the
immunoreactivity pattern among the 3 enzymes can be seen. For TPH, the
patients can easily be divided into 1 group of clearly positive sera
with high Ab-indexes, and 1 group of sera without TPH antibodies. For
TH, and especially PAH, it is harder to divide the group into strictly
positive or negative sera. This pattern may reflect the nature of
antigen-antibody binding for the 3 enzymes. TPH-Ab-positive sera are
distinctly positive and give a marked effect on enzymatic activity,
even in dilutions of 1:1000, indicating a strong binding with epitopes
capable of regulating enzymatic activity. Sera reacting with TH show a
wider distribution in the immunoreactivity plot and are not as
inhibitory as TPH-Ab-positive sera. This may indicate a more
heterogeneous antibody population with varying affinity and binding
site. For PAH, it is hard to distinguish positive from negative sera,
and no effect on PAH activity by PAH-Ab positive sera can be seen (data
not shown), which may illustrate that weak, cross-reactive epitopes are
involved.
Further investigations of the immunoreactivity against epitopes of the
three enzymes could include epitope mapping of the enzymes by
construction of chimeric proteins. In this case, the three-dimensional
structures for TH and PAH are known, and the structure for TPH is
possible to predict (Fig. 1) (24, 25, 26). Once the TPH structure is
definitely solved, we will have a unique possibility to design a study
that will be able to take both conformational and linear epitopes into
account (27). By looking at the linear homologies, regions where the
sequences differ can be identified. The three-dimensional
structures can tell us if these regions are displayed on the surface of
the proteins as possible epitopes. The region of interest can be
genetically changed for the corresponding region from one of the other
enzymes, and the resulting chimeric enzyme expressed in
vitro, followed by immunoprecipitation.
The identification of TPH, TH, and PAH as autoantigens in APS I is surprising and puzzling. Even if the reactivity seen against PAH only illustrates a cross-reactivity with TPH, the fact remains that APS I patients have the ability to produce antibodies against several enzymes of central importance in the biosynthesis of different neurotransmitters. From earlier studies, we know that glutamic acid decarboxylase and aromatic-L-amino acid decarboxylase are autoantigens in APS I, as well as enzymes of great importance in the synthesis of GABA, serotonin, and dopamine (8, 9). We postulate that these enzymes have shared immunogenic properties or that these proteins play a role in the pathogenesis of APS I.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received March 4, 2000.
Revised May 3, 2000.
Accepted May 15, 2000.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  N. G. Gavalas, E. H. Kemp, K. J. E. Krohn, E. M. Brown, P. F. Watson, and A. P. Weetman The Calcium-Sensing Receptor Is a Target of Autoantibodies in Patients with Autoimmune Polyendocrine Syndrome Type 1 J. Clin. Endocrinol. Metab., June 1, 2007; 92(6): 2107 - 2114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Candeloro, C. B. Voltattorni, R. Perniola, M. Bertoldi, C. Betterle, M. Mannelli, R. Giordano, A. De Bellis, C. Tiberti, S. Laureti, et al. Mapping of Human Autoantibody Epitopes on Aromatic L-Amino Acid Decarboxylase J. Clin. Endocrinol. Metab., March 1, 2007; 92(3): 1096 - 1105. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. S. B. Wolff, M. M. Erichsen, A. Meager, N. F. Magitta, A. G. Myhre, J. Bollerslev, K. J. Fougner, K. Lima, P. M. Knappskog, and E. S. Husebye Autoimmune Polyendocrine Syndrome Type 1 in Norway: Phenotypic Variation, Autoantibodies, and Novel Mutations in the Autoimmune Regulator Gene J. Clin. Endocrinol. Metab., February 1, 2007; 92(2): 595 - 603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Skoldberg, F. Rorsman, J. Perheentupa, M. Landin-Olsson, E. S. Husebye, J. Gustafsson, and O. Kampe Analysis of Antibody Reactivity against Cysteine Sulfinic Acid Decarboxylase, A Pyridoxal Phosphate-Dependent Enzyme, in Endocrine Autoimmune Disease J. Clin. Endocrinol. Metab., April 1, 2004; 89(4): 1636 - 1640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Betterle, C. Dal Pra, F. Mantero, and R. Zanchetta Autoimmune Adrenal Insufficiency and Autoimmune Polyendocrine Syndromes: Autoantibodies, Autoantigens, and Their Applicability in Diagnosis and Disease Prediction Endocr. Rev., June 1, 2002; 23(3): 327 - 364. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Halonen, P. Eskelin, A.-G. Myhre, J. Perheentupa, E. S. Husebye, O. Kampe, F. Rorsman, L. Peltonen, I. Ulmanen, and J. Partanen AIRE Mutations and Human Leukocyte Antigen Genotypes as Determinants of the Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy Phenotype J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2568 - 2574. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
), TH
(•) or PAH (
), and the immunoreactivity is measured. The y-axis
shows the immunoreactivity, expressed as a percentage of the cpm value
measured for 1 U of labeled antigen. The x-axis represents 0, 1, 2, 4,
8, 16, or 32 U of nonlabeled antigen.