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Thyroid Division (N.T., M.J.B., J.W.H., P.R.L.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; Department of Internal Medicine III (E.K., T.J.V.), Erasmus University Medical School, 3000 DR Rotterdam, The Netherlands
Address all correspondence and requests for reprints to: Theo J. Visser, Ph.D., Department of Internal Medicine III, Erasmus University Medical School, PO Box 1738, Room Bd 234, 3000 DR Rotterdam, The Netherlands.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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rT3 >> T4 > T3 for dID1, and
rT3 >> T2S T3S >
T4 T3 for hID1. Whereas ORD of
T4 and of T4, T3, and
T3S showed relatively little variation between the
different d/hID1 constructs, large differences were found for the ORD
of rT3 and T2S. Both reactions were favored by
the presence of the amino acids G, E and, in particular, F, present in
hID1 at positions 45, 46, and 65, instead of the dID1 residues N, G,
and L, respectively. However, although ORD of rT3 was not
affected by the presence (hID1) or absence (dID1) of the TGMTR(48–52)
sequence, the ORD of T2S was markedly inhibited by the
presence of this sequence. Therefore, we have identified structural
elements in ID1 that have substrate-specific impacts on deiodination.
Our results suggest the specific interaction of the mono-substituted
inner ring of the substrates rT3 and T2S but
not the disubstituted inner ring of T3, T3S, or
T4, with the aromatic ring of F65 in ID1, perhaps by
- interactions. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three membrane-bound iodothyronine deiodinases (IDs) are involved with the stepwise deiodination of T4 (1, 2, 3). ID1 is located mainly in liver, kidney, and thyroid; although it has both ORD and IRD activity, it is especially important for the production of plasma T3 by ORD of T4. ID2 is present predominantly in brain, pituitary and brown adipose tissue; it has only ORD activity and is essential for the local production of intracellular T3 from T4 in these tissues. ID3 is found especially in brain, fetal tissues, and placenta; it has only IRD activity and is probably important for inactivation of T4 and of locally generated and plasma-derived T3. All IDs have recently been characterized as homologous approximately 30-kDa selenocysteine (Sec)-containing enzymes (6, 7, 8, 9, 10, 11, 12, 13, 14). Although Sec is likely to be the catalytic center in all these isoenzymes, the potent, presumably Sec-targeted ID1 inhibitors 6-propyl-2-thiouracil, iodoacetate, and aurothioglucose do not or only weakly inhibit ID2 or ID3 (1, 2, 3).
In addition to its dual catalytic activity, other remarkable features of ID1 in most species are the high preference for rT3 as (ORD) substrate as well as the very efficient deiodination of different sulfated iodothyronines (1, 2, 3, 4, 5). IRD of T3 sulfate (T3S) by human ID1 (hID1) and rat ID1 (rID1) is approximately 40 times faster than IRD of nonsulfated T3. IRD of T4 by rID1 is facilitated approximately 200-fold following its sulfation, whereas ORD of T4S is undetectable (4, 5). The already optimal ORD of rT3 is not influenced by sulfation, but ORD of T2 sulfate (T2S) by rID1 is approximately 50 times more efficient than ORD of nonsulfated T2 (4, 5). Previous studies have indicated that dog ID1 (dID1) behaves differently from the enzyme of other species, because of its relatively slow ORD of rT3 (8, 15, 16). The major difference between the primary structures of dID1 vs. rID1 and hID1 is the deletion of the TGMTR(48–52)2 sequence (7, 8). However, mutational analysis of ID1 cDNA transfected into HEK293 cells demonstrated that it is not this 5-amino acid deletion but predominantly the F65L substitution, which explains the slow ORD of rT3 by dID1 vs. hID1 and rID1 (8).
Whereas these results identified F65 as an important residue for rT3 ORD, they did not establish whether or not differences between dID1 and hID1 or rID1 extended to ORD of other monosubstituted inner-ring substrates such as T2S. Furthermore, it was unclear how the differences identified affected IRD of the other favored substrate T3S. Information on these issues would allow conclusions as to whether the F65L and the other differences were substrate-specific or altered ID1 function in general. We undertook the present study to compare differences in substrate specificity between dID1 and hID1 by analysis of the ORD and/or IRD of an extensive panel of iodothyronine derivatives by the native enzymes in liver microsomes and by wild-type, mutant, and chimeric d/hID1 constructs expressed in HEK293 cells. The results show that the effects of the differences between dDI and hID1 are highly substrate-specific, pointing to important differences in binding contacts in ID1 for substrates that will undergo preferential ORD from those undergoing IRD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2000
Ci/mmol) was obtained from Amersham (Amersham, UK);
[3,5-125I]T3 (35 Ci/mmol) was obtained
courtesy of Dr. Claus Horst and Rudy Thoma (Henning Berlin GmbH);
[3',5'-125I]rT3 and
3,[3'-125I]T2 (2000 Ci/mmol) were prepared
by radioiodination of T2 and 3-iodothyronine, respectively,
as described previously (17); 125I-labeled and unlabeled
T3S and T2S were prepared by sulfation of
labeled and unlabeled T3 and T2, respectively,
using chlorosulfonic acid as described previously (18);
125I-labeled and unlabeled
N-bromoacetyl-T3 (BrAcT3) were
synthesized from N-bromoacetylchloride and
[125I]T3 or T3 as described
previously (8, 16); dithiothreitol (DTT) was obtained from Sigma (St.
Louis, MO); and Sephadex LH-20 was obtained from Pharmacia (Woerden,
The Netherlands). [125I]T3,
[125I]T3S, [125I]T2
and [125I]T2S could be used without further
purification, but [125I]rT3 was purified on
Sephadex LH-20 before each experiment (19). Normal adult dog liver tissue was obtained from a German shepherd and a bull terrier courtesy of Dr. Jan A. Mol (University of Utrecht, School of Veterinary Medicine, Utrecht, The Netherlands). Dog liver microsomes were prepared as previously described for the human liver microsomes also used in this study (20). Microsomes were suspended in 100 mM phosphate (pH 7.2), 2 mM EDTA (P100E2), and 1 mM DTT at a protein concentration of approximately 10 mg/ml, aliquoted, snap-frozen in liquid N2, and stored at -80 C until analysis.
Preparation and expression of ID1 constructs
cDNAs coding for wild-type dID1 or hID1 were constructed and
subcloned into the expression vector CDM-8 as previously described (7, 8, 21). Oligonucleotide-directed mutagenesis by PCR amplification using
Pfu polymerase (Stratagene) was performed to derive the mutant
proteins. Nucleotides coding for the human or dog amino acids were
introduced into the same position of the dID1 or hID1 cDNA,
respectively, as described previously (8). Human embryonic kidney
(HEK293) cells were maintained in DMEM + 10% FCS in the absence of
tetracycline as described previously (21). Transfections were performed
by CaPO4-DNA precipitation as described previously (6). Two
days after transfection, cells were harvested and sonicated in 0.25
M sucrose, 200 mM phosphate (pH 6.9), 1
mM EDTA, and 10 mM DTT. The sonicates (10 mg
protein/ml), were aliquoted, snap-frozen in liquid N2, and
stored at -80 C until analysis. Quantitation of the amounts of enzyme
expressed was done by saturation analysis of its affinity-labeling with
BrAc[125I]T3 as described previously (8, 16).
Deiodinase assays
Two different deiodinase assays were done, involving 1)
incubation with unlabeled substrate and measurement of the deiodinated
iodothyronine(s) produced with specific RIAs (20), or 2) incubation
with 125I-labeled substrate and isolation and quantitation
of the 125I- released (21). Assay A was used
to study the ORD of T4 to T3, the IRD of
T4 to rT3 (eventually further converted to
T2) and, in some experiments, the ORD of rT3 to
T2, and the IRD of T3S to T2S. In
general, varying amounts of enzyme protein were incubated in triplicate
for 15–60 min at 37 C with varying concentrations of T4,
T3, rT3, or T3S in 200 µl
P100E2 and 5 or 10 mM DTT.
Incubations were stopped by addition of 5 vol of ice-cold 0.1
M NaOH, and the extracts were analyzed for T3,
rT3 and/or T2 by RIA. T2S produced
from T3S was measured by T2 RIA after acid
hydrolysis (20). Assay B was used for analysis of the ORD of
rT3, the IRD of T3S, and the ORD of
T2S. In general, varying amounts of enzyme protein were
incubated in triplicate for 15–60 min at 37 C with approximately
100,000 cpm [3',5'-125I]rT3,
[3,5-125I]T3S or
[3'-125I]T2S and varying concentrations of
unlabeled substrate in 200 µl P100E2 and 5 or
10 mM DTT. The reactions were stopped by addition of 100
µl 5% BSA at 0 C. Protein-bound iodothyronines were precipitated by
addition of 500 µl 10% trichloroacetic acid, and the radioiodide in
the supernatant was further isolated on Sephadex LH-20 minicolumns as
previously described (21). Enzymatic deiodination was corrected for
nonenzymatic product formation in blank incubations without enzyme.
Radioiodide formation from labeled rT3 and T3S
was multiplied by 2 to account for the random labeling and deiodination
of the 3' and 5' positions, and the 3 and 5 positions,
respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, showing that
deiodination of all substrates tested was markedly faster with dog than
with human liver microsomes, except for rT3, which was
deiodinated much faster in human than in dog liver. Figure 1A presents
the total deiodination rate (ORD plus IRD) of T4, and this
is broken down in proportion to the individual deiodination products in
Fig. 1B. Although there is a somewhat greater contribution of the ORD
product T3 with dID1 than with hID1, the most obvious
difference concerns the proportions of rT3 and
T2, being the main products generated by dID1 and hID1,
respectively. These results are in agreement with the rT3
deiodination data (Fig. 1A), indicating that the rT3
produced by IRD of T4 is much more stable in incubations
with dID1 than with hID1.
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shows the Lineweaver-Burk plots of such data with
rT3 (Fig. 2A), T3S (Fig. 2B) or T2S
(Fig. 2C) as the substrate, and Table 1 provides the
Km and Vmax values of the various deiodinations
catalyzed by the dog and human enzymes. Whereas Km values
for deiodination of T4, T3 and T3S
by dID1 are three to four times higher than their Km values
for hID1, the differences are much greater with rT3
(50-fold) and T2S (25-fold) as substrate. The
Vmax values for deiodination of T4,
T3 and T3S are 11–31 times higher with dID1
than with hID1, which is explained by the difference in enzyme content
of the microsomes (16). However, differences in Vmax values
were much smaller with rT3 (1.6-fold) and much higher
(100-fold) with T2S as substrate. The
Vmax/Km ratios were calculated as a measure of
the efficiency by which the different deiodinations are catalyzed by
the same enzyme, demonstrating the marked difference in substrate
specificity between dID1 (T3S > T2S
>> rT3 > T4 T3) and hID1
(rT3 >> T2S T3S >
T4 T3).
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).
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shows the deiodination rates of
rT3, T2S, and T3S by wild-type dID1
(DWT), dID1 mutants (DMs), wild-type hID1 (HWT), and hID1 mutants
(HMs), expressed as percentage of those observed with HWT. The results
clearly demonstrate that the different mutations have very little
effect on the IRD of T3S. As demonstrated previously, ORD
of rT3 by DWT was very low, and insertion of the
TGMTR(48–52) sequence in DM1 brought little improvement. Small
increases in rT3 ORD were observed if the residues N45 and
G46 in DWT were replaced by the human amino acids G and E,
respectively, in DM4, whereas full restoration to HWT activity was
obtained by the additional L65F and Y66F substitutions in DM7. The
relatively slow ORD of T2S by DWT compared with HWT was
even further decreased by insertion of the TGMTR(48–52) sequence in
DM1. Some improvement was observed with the N45G and G46E mutations in
DM4, whereas ORD of T2S was increased to almost HWT
activity by the additional L65F and Y66F mutations in DM7. Replacements
of other residues in DWT by the corresponding amino acids in HWT (Q39R,
H40N, V42L, Y58H, A62I and M68T) had little or no effect on ORD of
either rT3 or T2S (not shown).
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).
Figure 5 shows the Lineweaver-Burk plots of the ORD of
rT3 (Fig. 5A) and that of T2S (Fig. 5B) by DWT,
DM7, HWT, and HM6, and the kinetic parameters (Km,
Vmax and Vmax/Km ratio) for these
reactions and for the IRD of T3S are given in Table 2. Affinity-labeling of the proteins with
BrAc[125I]T3 indicated similar expression
efficiencies for the different constructs (not shown). The results
demonstrate that Km values of rT3 for both DWT
and HM6 are approximately 50 times higher than those for HWT and DM7,
whereas Vmax values are very similar for all these enzymes.
Therefore, Vmax/Km ratios for rT3
ORD are much greater for HWT and DM7 than for DWT and HM6. The
Km value of T2S was lower for HWT than for the
other enzymes, whereas Vmax values were clearly lowest with
HM6. Consequently, the Vmax/Km ratio for
T2S ORD is highest with HWT, intermediate with DWT and DM7,
and lowest with HM6. Both Km and Vmax values of
T3S IRD are higher with DWT and DM7 than with HWT and HM6,
so that the Vmax/Km ratio varied less than
3-fold for the different enzymes.
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shows the levels of the different deiodination
products after incubation of 1 µM T4 with
these enzymes. Although the rate of T4 deiodination was
somewhat higher with DM7 than with the other enzymes, the most
remarkable difference was that rT3 accumulated in
incubations with DWT and HM6, whereas it was further deiodinated to
T2 in incubations with DM7 and HWT.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An important question regarding the regulation of thyroid hormone bioactivity concerns the structural elements in ID1 that are related to the ORD and IRD activities of the enzyme. The fact that ID1 catalyzes both ORD and IRD, even with the same substrate (e.g. T4), suggests different modalities for substrate binding so that either the iodines of the inner ring or those of the outer ring are in close proximity of the presumed catalyic center of the enzyme, i.e. the Sec residue (3). This may involve two distinct binding sites for ORD and IRD substrates or a single site to which substrates bind with a considerable degree of freedom or in different orientations. Here we report the first detailed investigation of the relationship between the structure of ID1 and its capacity to catalyze the ORD and IRD of different substrates.
In agreement with previous reports (8, 15, 16), we have shown that, in
contrast to hID1 and rID1, rT3 is not the preferred
substrate for native dID1 in liver microsomes as well as the wild-type
recombinant enzyme expressed in HEK293 cells. Based on the
Vmax/Km ratios determined with liver
microsomes, the substrate specificity of native dID1 is
T3S > T2S >> rT3 >
T4 T3, compared with rT3 >>
T2S T4 T3 for native
hID1 (Table 1). Consistent results were obtained when the kinetic
parameters for the deiodination of rT3, T2S and
T3S by the recombinant enzymes were determined (Table 2).
Comparison of the Vmax values determined with the native
and recombinant enzymes indicate higher levels of expression of dID1
than of hID1 in liver microsomes, in agreement with previous findings
(16). Km values of the different substrates for the native
and recombinant enzymes are similar, taking into account the different
concentrations of DTT, the second substrate in these ping-pong type
reactions (1, 2, 3), used in these experiments.
Recent analyses show that the human ID1 enzyme is a type 1 membrane
protein with a single transmembrane domain exiting the endoplasmic
reticulum at approximately residue 36 (24). This places the catalytic
domain of the enzyme in the cytoplasmic compartment. Further, a
previous study by some of us demonstrated that the relatively poor ORD
of rT3 by dID1, compared with hID1 and rID1, was related to
differences in the sequence of amino acids 36–70 of the proteins (8).
Surprisingly, this was not the result of the deletion of the
TGMTR(48–52) sequence in dID1 but rather due to the replacements of
the amino acids Gly at position 45 by Asn, Glu at position 46 by Gly,
and Phe at position 65 by Leu (8). The latter substitution has clearly
the largest contribution to the difference in rT3 ORD
potential between dID1 and hID1 (and rID1). In agreement with previous
findings (8), our results indicate that the deiodination of
T4 is relatively independent of these structural
differences between dID1 and hID1. However, the composition of the
deiodination products accumulating in incubations of T4
with native, wild-type and mutated d/hID1 preparations (Figs. 1B and 6)
differs substantially, depending on the rate of further deiodination of
rT3 to T2 by the different enzymes. In
addition, we found relatively little variation in the IRD of
T3S by the various d/hID1 constructs, as determined at a
single substrate concentration (Fig. 4). This was associated with
limited, parallel variation in the Vmax and Km
values, and thus relatively constant Vmax/Km
ratios for the IRD of T3S by these enzymes (Table 2).
The effects of the various mutations of the dID1 and hID1 structures on
the ORD of T2S in part paralleled those on the ORD of
rT3. Thus, the above-mentioned substitutions at positions
45, 46, and 65 affected the ORD of both substrates similarly. However,
in contrast to rT3, the deiodination of T2S
depended on the presence of the TGMTR(48–52) sequence, in that
insertion of this sequence into the dID1 structure (DM1) was associated
with a marked inhibition of the ORD of T2S (Fig. 4). The
specific effect of the TGMTR(48–52) sequence on the ORD of
T2S may be due to interference with the interaction of the
sulfate group of the substrate with a basic residue in the enzyme, but
this remains speculation. It is remarkable that the ORD of
rT3 and T2S, both substrates with one iodine
substituent on the inner ring, are both favored by the presence of Phe
at position 65 in the deiodinase. However, the deiodination of the
other substrates tested, all with two iodine substituents on the inner
ring, is independent of the occupation of position 65 by Phe or Leu.
This suggests a specific interaction of the inner ring of
rT3 and T2S with Phe65, perhaps due to -
interaction of these aromatic rings.
Taken together, our results suggest that none of the amino acids involved in the 36–70 residue portion of ID1, located just external to the ER membrane, are critical either to the binding of T3S or to its deiodination. Thus, the changes at position 65 have specific influences on the binding of the inner ring-monosubstituted iodothyronines and are not likely to reflect alterations in later steps of the deiodination process. The fact that deletion of a 5-amino acid sequence in the center of a region so critical to substrate binding is not only not deleterious to rT3 ORD or T3S IRD, but even enhances ORD of T2S, suggests that these residues do not play a critical structural role in ID1 function. All of these observations will be relevant to the interpretation of the three-dimensional structure of this bifunctional enzyme when this is resolved by crystallographic studies.
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Acknowledgments |
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Footnotes |
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2 Amino acid numbering is based on the human (and
rat) ID1 sequence.
Received July 15, 1996.
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