(Received for publication, September 11, 1996)
From the Centro di Endocrinologia e Oncologia
Sperimentale del Consiglio Nazionale delle Ricerche, Dipartimento di
Biologia e Patologia Cellulare e Molecolare, University of Naples
"Federico II", 80131 Naples, Italy and the ¶ Servizio di
Spettrometria di Massa del Consiglio Nazionale delle Ricerche,
80131 Naples, Italy
A fragment of bovine thyroglobulin encompassing residues 1218-1591 was prepared by limited proteolysis with thermolysin and continuous-elution polyacrylamide gel electrophoresis in SDS. The reduced and carboxymethylated peptide was digested with endoproteinase Asp-N and fractionated by reverse-phase high performance liquid chromatography. The fractions were analyzed by electrospray and fast atom bombardment mass spectrometry in combination with Edman degradation. The post-translational modifications of all seven tyrosyl residues of the fragment were characterized at an unprecedented level of definition. The analysis revealed the formation of: 1) monoiodotyrosine from tyrosine 1234; 2) monoiodotyrosine, diiodotyrosine, triiodothyronine (T3), and tetraiodothyronine (thyroxine, T4) from tyrosine 1291; and 3) monoiodotyrosine, diiodotyrosine, and dehydroalanine from tyrosine 1375. Iodothyronine formation from tyrosine 1291 accounted for 10% of total T4 of thyroglobulin (0.30 mol of T4/mol of 660-kDa thyroglobulin), and 8% of total T3 (0.08 mol of T3/mol of thyroglobulin). This is the first documentation of the hormonogenic nature of tyrosine 1291 of bovine thyroglobulin, as thyroxine formation at a corresponding site was so far reported only in rabbit, guinea pig, and turtle thyroglobulin. This is also the first direct identification of tyrosine 1375 of bovine thyroglobulin as a donor residue. It is suggested that tyrosyl residues 1291 and 1375 may support together the function of an independent hormonogenic domain in the mid-portion of the polypeptide chain of thyroglobulin.
Thyroglobulin (Tg),1 a homodimeric
glycoprotein with a molecular mass of 660 kDa, is the site of the
biosynthesis of 3,5,3-triiodothyronine (T3) and
3,5,3
,5
-tetraiodothyronine (thyroxine, T4) (reviewed in
Ref. 1). T3 and T4 are synthesized via the
iodination and coupling of a small subset of tyrosyl residues within
the polypeptide chains of Tg. The coupling reaction takes place by the
transfer of an iodophenyl group from a donor 3-monoiodotyrosine or
3,5-diiodotyrosine to an acceptor 3,5-diiodotyrosine. This causes the
formation of T3 or T4, respectively, at the
acceptor site and dehydroalanine at the donor site (2, 3). Both
reactions are catalyzed by thyroid peroxidase. Different tyrosyl
residues have different reactivities toward iodine, so that iodination
proceeds in a sequential order, which is controlled by the native
structure of Tg (4, 5). Early iodinated tyrosyl residues are
preferentially involved in iodothyronine synthesis (6); the coupling of
iodotyrosines, in turn, has stringent steric requirements (7). In fact,
out of 72 tyrosyl residues per bovine Tg monomer, only 15 are iodinated and a maximum of 6-8 of them undergo coupling to form T3
and T4 (8, 9).
So far, four major hormonogenic tyrosines have been identified, by the isolation and sequencing of hormone-rich peptides from Tgs of various animal species and comparison of their sequences with the cDNA-deduced sequences of bovine (10) and human Tg (11). Tyr-5 was the most favored site for T4 formation in most species studied, including humans (12), calf (13), sheep, hog (14), rabbit (15), and guinea pig (16). In hog (17), rabbit (15), guinea pig (16), and human Tg subjected in vitro to low-level iodination (18), Tyr-2553 (human Tg numbering) was the second most efficient T4-forming residue, whereas Tyr-2746 was a site of preferential synthesis of T3 (15, 16, 18, 19). Another T4-forming site found in rabbit and guinea pig Tg corresponded to human Tyr-1290: in those species this site was third in ranking order of hormonogenic efficiency and its function was greatly enhanced by TSH (15, 16). Nevertheless, so far it has received little attention in the bovine and human species. Tyrosines reported as possible donor sites include Tyr-5, -926, -986 or -1008, -1375 (20), -2469 and/or -2522 of bovine Tg (21), and Tyr-130 of human Tg (22).
The main goal of this work was to establish whether Tyr-1291 of bovine Tg is also a site of T4 formation. To this purpose, a preparation of bovine Tg containing 1.05% iodine by mass was subjected to limited proteolysis with thermolysin and the products were separated by preparative SDS-PAGE. A thorough mass spectrometric analysis of a peptide spanning residues 1218-1591, together with an analysis of its iodine and iodoamino acid content, were performed. Post-translational modifications of three out of seven tyrosyl residues were documented at an unprecedented level of definition: in particular, we report the first direct evidence of the entire spectrum of modifications typical of a hormonogenic acceptor and a hormonogenic donor site at residues 1291 and 1375, respectively, of bovine Tg.
Thermolysin from Bacillus
thermo-proteolyticus rokko (EC 3.4.24.4) and
L-1-tosylamide-2-phenylethylchloromethyl-treated bovine
pancreatic trypsin (EC 3.4.21.4), dithiothreitol, iodoacetic acid,
glycerol, thioglycerol, 3-iodo-L-tyrosine (MIT),
3,5-diiodo-L-tyrosine (DIT), 3,5,3-triiodothyronine
(T3), 3,5,3
,5
-tetraiodothyronine (thyroxine,
T4) were from Sigma Chimica (Milan,
Italy); endoproteinase Asp-N from Pseudomonas fragi (EC
3.4.24.33) and endoproteinase Lys-C from Lysobacter
enzymogenes (EC 3.4.21.50) were from Boehringer Mannheim Italia
(Milan, Italy). Aminopeptidase M from porcine kidney (EC 3.4.11.2) and
Pronase from Streptomyces griseus were from Calbiochem (San
Diego, CA). Phenylisothiocyanate and EDTA were from Fluka Chimica
(Milan, Italy). AcrylAide cross-linker and GelBond PAG film were from
FMC BioProducts (Rockland, ME), other products for electrophoresis were
from Bio-Rad Laboratories (Milan, Italy). Extracti-gel resin and
bicinchoninic acid Protein Assay Reagent were from Pierce (Rockford,
IL). HPLC grade solvents were obtained from Carlo Erba (Milan, Italy).
The Vydac C-18 column (250 × 4.6 mm, 5 µm) was from The
Separation Group (Hesperia, CA) and the Brownlee C-8 column (250 × 4.6 mm, 5 µm) from Applied Biosystems (Santa Clara, CA); PD-10
Sephadex G-25 cartridges and Sephacryl S-300 HR were from Pharmacia
Biotech (Uppsala, Sweden).
Bovine Tg was prepared from fresh bovine thyroids from the local abbattoir. The tissue was finely minced with scissors and Tg extracted briefly on ice in 0.1 M sodium phosphate, pH 7.2, and purified by fractional precipitation with 1.4-1.8 M ammonium sulfate, 50 mM Tris/HCl, pH 7.2, and gel filtration on Sephacryl S-300 HR in 130 mM NaCl, 50 mM Tris/HCl, pH 7.2, at 4 °C.
Limited Proteolysis of TgLimited proteolysis of Tg with
thermolysin was carried out as described previously (23). Tg at the
concentration of 1 mg/ml in 130 mM NaCl, 50 mM
Tris/HCl, pH 8.0, was incubated with thermolysin at the
enzyme/substrate ratio of 1/1000 or 1/100 (w/w) at 30 °C for the
time indicated. The digestion was stopped by adding EDTA to a final
concentration of 10 mM and concentrated SDS-PAGE sample buffer to a concentration of 10 mM Tris/HCl, pH 6.8, 1%
SDS, 5% -mercaptoethanol, 1.36 M glycerol, 0.0025%
bromphenol blue, and by heating the samples in a boiling water bath for
1.5 min.
Analytical SDS-PAGE in reducing conditions of the digestion products was performed according to Laemmli (24) on 4-16% total acrylamide gradient gels polymerized on GelBond PAG plastic backing (FMC BioProducts). The gels were stained with 0.1% Coomassie Brilliant Blue R-250 in 25% 2-propanol (v/v), 10% acetic acid (v/v), destained in 25% methanol (v/v), 10% acetic acid, soaked in 0.7 M glycerol, and air dried. The main peptides produced were identified on the basis of their mobility, according to detailed characterization of their NH2-terminal peptide sequences provided in a previous study (23).
Purification of Peptide b6TLBovine Tg was
digested for 80 min with thermolysin at the enzyme/substrate ratio of
1/100 (w/w) and the digestion stopped as described above. The fragments
were precipitated in chloroform/methanol (25), redissolved in SDS-PAGE
sample buffer, and separated by preparative continuous-elution
SDS-PAGE, using an electrophoresis chamber Bio-Rad model 491. A
discontinuous gel with an annular cross-section was prepared according
to Laemmli (24) in a cylindrical assembly having a diameter of 3.5 cm,
whose center was occupied by a cooling core having a diameter of 1.5 cm. The 50-ml separating gel contained 12% total acrylamide and was
6.3 cm high; it was topped with a 10-ml 1.3-cm stacking gel containing
3.75% total acrylamide. The products of digestion of 25 mg of Tg with
thermolysin were loaded onto a single gel. The electrode buffer
contained 0.025 M Tris, 0.19 M glycine, 0.1%
SDS, pH 8.2. The apparatus was designed so that, as soon as the
migrating bands reached the lower extremity of the gel, they were
conveyed by a stream of electrode buffer, aspirated by a peristaltic
pump from a reservoir, to a fraction collector. Electrophoresis was
carried at 20 mA. Collection was started as soon as the tracking dye
began to exit from the gel (in 16 h); 4 fractions per hour were
collected, with the pump flow rate set at 20 ml/h, for 24 h. The
fractions were analyzed by SDS-PAGE. Those of interest were pooled and
the pool was concentrated by lyophilization, freed from Tris/HCl, and
glycine by filtration through PD-10 Sephadex G-25 cartridges (Pharmacia Biotech) in distilled water, and from SDS by filtration through Extracti-gel resin (Pierce) (1 ml of resin every 50 ml of the original
pool) in distilled water. The sample was finally lyophilized and stored
at 20 °C.
Purified peptide b6TL was dissolved in 300 µl of 0.3 M Tris/HCl, pH 8.0, containing 6 M guanidine/HCl, 1 mM EDTA, and treated with dithiothreitol (10/1 molar excess with respect to cysteinyl residues) at 37 °C for 2 h. The reduced peptide was carboxymethylated by reaction with a 5/1 molar excess of iodoacetic acid, with respect to total -SH groups, at pH 8.0 at room temperature for 30 min in the dark. The sample was freed from low molecular weight compounds by filtration through a PD-10 G-25 column in 50 mM ammonium bicarbonate, pH 8.5, and lyophilized.
Enzymatic DigestsThe reduced and carboxymethylated peptide b6TL was hydrolyzed with endoproteinase Asp-N at the enzyme/substrate ratio of 1/100 (w/w) in 50 mM ammonium bicarbonate, 10% (v/v) acetonitrile, pH 8.5, at 37 °C for 18 h. Hydrolyses of HPLC-purified peptides with trypsin and endoproteinase Lys-C were carried out in 50 mM ammonium bicarbonate, pH 8.5, at 37 °C, using an enzyme/substrate ratio of 1/50 (w/w), for 4 and 20 h, respectively. All the reactions were immediately followed by lyophilization.
Separation of Peptides Obtained by Hydrolysis with Endoproteinase Asp-NThe peptides obtained by hydrolysis of 0.5 mg of peptide b6TL with endoproteinase Asp-N were fractionated by HPLC with a Vydac C-18 column (250 × 4.6 mm, 5 µm) equilibrated in 0.1% (v/v) trifluoroacetic acid in water (solvent A), containing 4% of 0.07% trifluoroacetic acid in acetonitrile (solvent B). After 5 min at 4% of solvent B, elution was performed by a two-step linear gradient of solvent B percentage from 4 to 25% over 25 min, and from 25 to 60% over the following 45 min. The flow rate was 1 ml/min.
Electrospray Mass Spectrometry (ES/MS)ES mass spectra of the peptides produced by hydrolysis of peptide b6TL with endoproteinase Asp-N were recorded with a PLATFORM mass spectrometer (Fisons, Manchester, United Kingdom) equipped with an electrospray ion source. Samples from the HPLC separation (10 µl, 50 pmol) were injected into the ion source at a flow rate of 10 µl/min; the spectra were scanned from 2000 to 400 at the speed of 10 s/scan. Mass calibration was carried out using the multiple charged ions from a separate introduction of horse heart myoglobin (average molecular mass 16, 950.5 Da). The quantitative analysis was performed by integration of the multiple charged ions of the single species. Molecular masses are reported as average values.
Fast Atom Bombardment Mass Spectrometry (FAB/MS)FAB mass spectra were recorded with a VG Analytical ZAB-2SE double-focusing mass spectrometer fitted with a VG caesium gun operating at 25 kV. Samples (0.1 nmol) were dissolved in 5% acetic acid and loaded onto a glycerol-coated probe tip; thioglycerol was added to the matrix just before introducing the probe into the ion source. The amplification of the electric signal was reduced during the magnet scan, according to the intensity of the mass signals observed on the oscilloscope. The values correspond to the monoisotopic masses of the protonated molecular ions of the peptides and are reported as integer numbers.
Peptide RecognitionThe mass signals recorded in the spectra were associated with the corresponding peptides, on the basis of the expected molecular masses, using a computer program (26). Edman degradation steps were performed on HPLC-purified peptides, and were followed by the mass spectrometric analysis of the truncated peptides, in order to confirm the assignments, as already described (27).
Analytical TechniquesIodine determinations were performed as described (28). The concentration of Tg was estimated by the absorbance at 280 nm, using a percentual extinction coefficient of 10.5 (29). The concentration of peptide samples was assayed using a bicinchoninic acid Protein Assay Reagent (Pierce) and bovine Tg as the standard. For the analysis of iodoamino acids, triplicate samples were hydrolyzed by a modification of a method already described (30): 0.4-mg aliquots of Tg and purified peptide b6TL were incubated at 37 °C with Pronase at the enzyme/substrate weight ratio of 1/1 in 0.5 ml of 0.1 M Tris/HCl, 50 mM 2-mercapto-1-methylimidazole, pH 8.0, to which 10 µl of toluene were added; after 24 h, aminopeptidase M at the enzyme/substrate ratio of 1/10 was added and digestion prolonged for another 24 h at 37 °C. Iodoamino acids were separated by reverse-phase HPLC in a Kontron HPLC equipped with a Brownlee C-8 column (250 × 4.6 mm, 5 µm), as already described (31). Iodoamino acid peaks were identified by comparison with iodoamino acid standards: the contents of iodoamino acids were calculated from the iodine contents of the respective peaks. Since 2-mercapto-1-methylimidazole co-eluted with MIT and interfered in the iodine assay, its contribution was determined in triplicate samples of bovine serum albumin which were subjected to the identical treatment.
A detailed analysis of the products of the limited
proteolysis of bovine Tg with thermolysin has been reported (23).
Typical time courses and a flow-diagram of the proteolysis at pH 8.0 at 30 °C are shown in panels A and B,
respectively, of Fig. 1. The proteolytic peptides
corresponded exactly to those which were previously observed and
characterized by amino-terminal sequencing (23). Therefore, in the
present work the proteolytic peptides were identified according to
their electrophoretic mobilities, on the basis of the data already
reported (23).
For the preparation of peptide b6TL, five 25-mg aliquots of a bovine Tg containing 1.05% iodine by mass were hydrolyzed with thermolysin at the enzyme/substrate ratio of 1/100 at pH 8.0 at 30 °C for 80 min. The fragments were separated by preparative continuous-elution SDS-PAGE, concentrated, further purified, and lyophilized as described under "Experimental Procedures." The analysis by SDS-PAGE of the fractions of a typical preparation is shown in panels C and D of Fig. 1. In the end, 2.2 mg of pure peptide were obtained (Fig. 1, panel E). Because peptide b6TL represented 10% of the peptides detected by densitometry of the gel (Fig. 1, panel A) (23) and these were 80% of the starting protein material, the yield of the purification procedure was 22%.
Analysis of Peptide b6TL by Mass SpectrometryA
50-kDa peptide starting at residue 1291 (peptide b6TL)
(Fig. 1, panels A and B) was reduced and
carboxymethylated, digested with endoproteinase Asp-N, and the digest
was fractionated by reverse-phase HPLC on a Vydac C-18 column (250 × 4.6 mm, 5 µm). The chromatogram is shown in Fig. 2.
All fractions were directly analyzed by ES/MS, and some were
freeze-dried and analyzed also by FAB/MS. The results of the analysis
by ES/MS are reported in Table I. The mass signals in
the spectra were associated with the corresponding peptides along the
sequence of bovine Tg, between residues 1200 and 1630, using a suitable
computer program (26) (Fig. 3). Several cleavage sites
were only partially hydrolyzed during the digestion, which yielded
several overlapping peptides. A few aspecific cleavages occurred at the
amino side of glutamic acid residues. However, the data permitted
verification of the entire amino acid sequence of peptide
b6TL, which was identical to the cDNA-derived sequence
(10). Ala-1591 was identified as the COOH-terminal residue of peptide
b6TL. In fact, two peptides, spanning residues 1567-1591
and 1580-1591, both ended at Ala-1591 and, therefore, were not
expected on the basis of the enzymatic specificity of endoproteinase
Asp-N. Moreover, no peptide was detected whose sequence matched Tg
sequence beyond Ala-1591. The mass spectrometric analysis of the HPLC
fractions of peptide b6TL (Table I) permitted
characterization of its seven tyrosyl residues at positions 1234, 1291, 1375, 1450, 1464, 1484, and 1512, identifying post-translational
modifications of Tyr-1234, Tyr-1291, and Tyr-1375.
|
Tyr-1234 Is Partially Converted to MIT
Two molecular species,
having mass values of 4136.3 ± 0.4 Da and 4262.9 ± 0.4 Da,
were detected by ES/MS in fractions 23 and 24, respectively. The first
value was in perfect agreement with that expected for peptide
1218-1252, produced by endoproteinase Asp-N by aspecific cleavage at
Glu-1253 (4136.7 Da, see Table I); the second value was compatible with
that expected for the same peptide in which an iodine atom had been
added to Tyr-1234 (m = +126). To verify this,
fractions 23 and 24 were digested with trypsin and the products
analyzed by FAB/MS. The spectrum of fraction 23 showed a protonated
molecular ion (MH+) at m/z = 771, corresponding to unmodified peptide 1230-1235, while that of fraction
24 contained a MH+ ion at m/z = 897 (
m = +126), confirming the conversion of Tyr-1234 to
MIT. No other forms of this peptide were found, which restricts the
modifications of Tyr-1234 to the formation of MIT.
Among the
peptides expected from the digestion with endoproteinase Asp-N, peptide
1290-1303 DYSGLLLAFQVFLL, containing a single Tyr residue at position
1291 and having an expected mass value of 1598.8 Da, was absent in the
peptide map. However, mass values corresponding to this peptide having
MIT, DIT, T3, and T4 at position 1291 were
found in HPLC fractions 15 (1724.9 ± 0.1 Da for MIT; 1850.3 ± 0.2 Da for DIT) and 29 (2066.5 ± 0.5 Da for T3;
2192.5 ± 0.6 Da for T4) (Table I and Fig.
4). These assignments were confirmed by submitting the
above fractions to FAB/MS followed by two manual Edman degradation
steps, after which the m/z values of the truncated peptides
were measured again by FAB/MS (Fig. 5). After the first
Edman cycle, all peptides showed a shift of 115 mass units,
corresponding to the loss of Asp-1290. After the second step, all
peptides collapsed to the same m/z value of 1320, due to the
loss of MIT (fraction 15,
m =
289), DIT (fraction
15,
m =
415), T3 (fraction 29,
m =
631), and T4 (fraction 29,
m =
757), respectively, from position 1291. This experiment demonstrated the presence of all the molecular species involved in the pathway of T3 and T4 synthesis
at position 1291, with the exception of unmodified Tyr.
Tyr-1375 Is a Hormonogenic Donor Residue
The chromatogram of
Fig. 2 contained four peaks (13, 19, 26, and 27), whose analysis by
ES/MS revealed mass signals related to peptide 1366-1381, containing
one Tyr residue at position 1375 (Table I and Fig. 4). The mass value
of 1709.4 ± 0.2 Da, in fraction 19, corresponded to peptide
1366-1381 DVEEALAGKYLAGRFA, with unmodified Tyr-1375. The mass value
of 1615.8 ± 0.2 (m =
94), in fraction 13, could be accounted for by a form of peptide 1366-1381 in which Tyr-1375 had been converted to dehydroalanine. The mass values of
1836.3 ± 1.0, in fraction 26, and 1961.6 ± 0.1, in fraction 27, were compatible with the addition of one and two iodine atoms to
Tyr-1375, respectively. These identifications were confirmed by
incubating the four fractions with endoproteinase Lys-C, to cleave
peptide 1366-1381 into peptides 1366-1374 and 1375-1381. When
analyzed by FAB/MS (Fig. 6), the four digests had in
common the MH+ ion at m/z = 931 predicted
for peptide 1366-1374 DVEEALAGK, whereas they differed in the
MH+ ions corresponding to peptide 1375-1381. In fact, the
spectrum of fraction 19 showed the MH+ ion at
m/z = 797 predicted for the unmodified peptide
1375-1381 YLAGRFA, while the spectra of fractions 26, 27, and 13 showed MH+ ions at m/z = 923, 1049, and
703, corresponding to peptide 1375-1381, in which Tyr-1375 had been
converted to MIT, DIT, and DHA, respectively. The analysis by FAB/MS,
after one step of Edman degradation (Fig. 6), showed the expected mass
shifts, by which all four peptides moved to m/z = 634, due to the loss of Tyr (fraction 19,
m =
163), MIT
(fraction 26,
m =
289), DIT (fraction 27,
m =
415), and DHA (fraction 13,
m =
69). These data proved that Tyr-1375 is an
iodophenyl donor residue. Mass signals corresponding to various forms
of peptide 1355-1393, in which Tyr-1375 was present as such or had
been modified to MIT, DIT, and DHA were detected also in fraction 16 (Table I).
Asn-1346 Is Not Glycosylated
The sole putative site of N-linked glycosylation of peptide b6TL, corresponding to Asn-1346 (within the consensus sequence Asn-Ile-Thr) (10), was unmodified. In fact, peptides 1330-1354 (fraction 18) and 1336-1354 (fraction 10) had mass values typical of the non-glycosylated species (Table I), and no evidence was found of glycosylated forms of the above peptides.
Efficiency of Tyr-1375 as a T4- and T3-forming SiteThe data of Table II indicate that the iodine content of peptide b6TL (1.11% by mass) exceeded slightly the average iodine content of the parent bovine Tg (1.05% by mass). Thus, the fraction of total Tg iodine contained in 2 mol of peptide b6TL/mol of Tg dimer (0.16) was only slightly higher than the fraction of Tg mass that they accounted for (0.15). In particular, 13% of total iodine in peptide b6TL was found in T4 and 3% in T3, as opposed to 21 and 5%, respectively, in bovine Tg. Tyr-1291 contributed 10% of the T4 and 8% of the T3 content of Tg. The relative amounts of iodine incorporated into iodothyronines and iodotyrosines were 1 versus 5 in peptide b6TL, and 1 versus 3 in Tg. On the basis of the moles of iodoamino acids formed per mole of Tg, the overall extent of modification of Tyr-1234, -1291, and -1375 appeared to be quite large, considering that the other 4 Tyr residues were unmodified (see Table I). Because 0.4 mol of iodothyronines were formed per mole of 660-kDa Tg (i.e. per 2 mol of Tyr-1291), the efficiency of hormone formation at this site, at this level of Tg iodination, was 20%. The 2.6 mol of DIT per mole of Tg in peptide b6TL accounted for another 65% of the combined 4 mol of Tyr-1291 and -1375 per mole of Tg dimer, considering that the modification of Tyr-1234 was restricted to formation of MIT. Out of 2.5 mol of MIT per mole of Tg found in peptide b6TL, more than 0.5 mol had to be formed in correspondence of Tyr-1291 and -1375, and less than 2.0 by the iodination of the 2 mol of Tyr-1234 per mole of Tg dimer, as the ES/MS spectrum of peak 23 revealed the presence of some unmodified Tyr-1234 (see Table I). This makes it probable that the amount of DHA formed at Tyr-1375 was of the same order of magnitude of the amount of iodothyronines formed at Tyr-1291 and leaves room for only a small amount of unmodified Tyr at positions 1291 and 1375. In fact, no unmodified Tyr-1291 was found in the mass spectra.
|
We report a detailed analysis of the post-translational modifications of seven tyrosyl residues comprised in fragment 1218-1591 of bovine thyroglobulin. In particular, we demonstrate the formation of MIT, DIT, T3, and T4 from Tyr-1291, and of MIT, DIT, and DHA from Tyr-1375. Modification of Tyr-1234 was restricted to formation of MIT, while Tyr-1450, -1464, -1484, and -1512 were unmodified.
Mass spectrometry is widely employed for the analysis of post-translational modifications of proteins (32). However, it has been used here for the first time to identify iodinated tyrosyl residues in Tg, and has proved extremely valuable as a source of primary structure data not available from earlier use of Edman degradation. In the past, the identification of hormonogenic sites by the sequencing of hormone-rich peptides of Tg was not always as direct. The only iodotyrosines and iodothyronines directly identified, by the manual method of sequencing with dimethylaminoazobenzeneisothiocyanate (35, 36), were those located at positions 2553, 2567, and 2746 of hog Tg (human Tg numbering) (17, 19), and 5 of human Tg (12, 33, 34). On the other hand, the phenylthiohydantoin-derivatives of iodoamino acids, in iodopeptides subjected to automated sequencing, were generally not identified by comparison with proper standards. The localization of hormonogenic sites in the NH2-terminal peptides of calf (13), sheep and hog Tg (14), in the tryptic peptides of rabbit (15) and guinea pig Tg labeled in vivo with 125I (16), and in Tg from human goiters subjected to low-level iodination in vitro with 125I (18), was based on the monitoring of the contents of 127I or 125I in the automated sequencing cycles, and the determination of the distribution of iodoamino acids. In this regard, although 125I labeling provides an easy way to trace Tg iodopeptides and iodination sites and study hormonal turnover, it is not suited for the study of physiologically iodinated Tg of humans and other large animals.
The identification of donor tyrosyl residues was also indirect in all cases reported so far. In one study of bovine Tg, in which the separation of dehydroalanine-containing peptides exploited the conversion of dehydroalanine to S-(4-aminophenyl)cysteine, the presence of the latter at positions 5, 926, 986 or 1008, and 1375 was inferred from the lack of known phenylthiohydantoin-derivatives in sequencing cycles where tyrosine was expected, and from differences between the actual and expected tyrosine content of the peptides (20). In another study, the labeling of dehydroalanyl residues of bovine Tg with NaB3H4 and their conversion to labeled aspartic acid with Na14CN revealed a small labeled CNBr peptide containing possible donor Tyr-2469 and Tyr-2522, and a larger CNBr peptide, spanning residues 785-1551, possibly harboring other donor residues (21). Finally, the proposal that alanine recovered at position 130 of peptide 1-171 of human Tg derived, in fact, from the conversion of dehydroalanine was largely based on speculation (22).
On the other hand, in the present study, mass spectrometry allowed the direct, unambiguous characterization of the entire spectrum of modifications of every tyrosyl residue within a large fragment of Tg. Not only the identification of T4 and T3 in correspondence of Tyr-1291 of bovine Tg is unprecedented, but also the localization of a donor site at position 1375 cannot be considered merely confirmatory, as it is based, for the first time, on a direct demonstration. By using the combination of limited proteolysis, preparative electrophoresis and mass spectrometry employed here, we project to extend our analysis to other still unsettled aspects of hormonogenesis in Tg, including: 1) the localization of the hormonogenic donor tyrosines of human Tg; 2) the identification of acceptor tyrosines other than tyrosine number 5 in physiologically iodinated human Tg; 3) the resolution of the uncertainties about donor sites at positions 986, 1008 (20), 2469 and 2522 (21) of bovine Tg.
The formation of T4 at a site corresponding to residue 1291 of bovine Tg was already reported in rabbit (15, 16) and guinea pig Tg (16), in which it contributed 17 and 11%, respectively, of Tg's T4. From the data reported in Table II, it appears that also in bovine Tg, Tyr-1291 contributed an appreciable amount of T4, together with a small amount of T3. It also appears that both Tyr-1291 and Tyr-1375 were to a large extent modified, mostly to DIT; however, only in one-fifth of the cases modification proceeded and iodothyronines were formed. On one hand, this probably reflects a high degree of accessibility of both residues. In this regard, the hydrophilicity plot of this region was not particularly informative (not shown); however, it is noteworthy that Tyr-1291 is located 70 residues apart from a cluster of protease-sensitive sites encompassing residues 1142, 1184, and 1218 (23). On the other hand, the prevalence of iodotyrosines at these sites raises interesting questions concerning the factors of steric hindrance that may limit the efficiency of a hormonogenic site, and the structural requirements that must be satisfied for efficient coupling to occur. In rabbit and guinea pig Tg labeled in vivo with 125I, Tyr-1290 (human Tg numbering) contributed greatly to Tg's flexibility in meeting varying demands for hormone formation, as TSH enhanced T4 formation at residue 1290, at the expense of T4 formation at residue number 5, while increasing T3 formation at residue 2746 (16). Under TSH stimulation, the percentage of T4 neo-synthesized at residue 1290 changed from 10 to 14% in rabbit and from 13 to 24% in guinea pig. In guinea pig, tyrosine 1290 was the most active site for new T4 formation even in the presence of basal TSH levels (16). It is possible that also in bovine Tg the formation of T4 at Tyr-1291 increase under TSH stimulation, e.g. as a consequence of iodide shortage. In this regard, it would be interesting to measure the share of Tg's total T4 formed at Tyr-1291 at increasing levels of Tg iodination. On the other hand, in turtle Tg labeled in vivo with 125I, only 5% of T4 and 11% of T3 were newly formed at Tyr-1290 (human Tg numbering) (37), while in human Tg iodinated in vitro with 7.8 atoms of iodine/Tg molecule, only traces of iodothyronines were found at residue 1290 (18). Only further work may establish whether this reflected the low level of Tg iodination, or the low efficiency of Tyr-1290 in human Tg. Interestingly, in human Tg, aspartic acid substitutes for tyrosine at position 1375. In addition, human Tyr-1447 was proposed to be a possible donor site, because it was iodinated early but did not provide inner iodothyronyl rings upon further iodination (18), whereas the corresponding Tyr-1450 of bovine Tg was unmodified in the present study. Although there is no indication that acceptor and donor residues need to be contiguous in the Tg sequence, the apparently low hormonogenic potential of human Tyr-1290 might indicate that, in bovine Tg, T4 formation at Tyr-1291 depends on the presence of donor Tyr-1375, whereas, in human Tg, Tyr-1447 is not as good a donor site.
It was proposed that different hormonogenic sites of Tg evolved independently, and may also function independently from each other and the rest of the Tg molecule (38). Various observations support this hypothesis. Thyroid hormone formation within truncated NH2-terminal Tg fragments, derived from the abortive translation of normal-sized mRNAs, was probably responsible for the correction of hypothyroidism, by iodide supplementation, in a strain of Dutch goats with congenital goiter (39, 40, 41), and for euthyroidism in Afrikander cattle (42, 43, 44). Efficient T4 formation was demonstrated in isolated fragment 1-171 of human Tg (22, 34). Thyroid hormones were also formed upon in vitro iodination of a fragment comprising the 224 COOH-terminal amino acids of rat Tg (45). It would be interesting to test the ability of peptide b6TL, isolated from low-iodine bovine Tg, to sustain T4 (and T3) formation at Tyr-1291 upon peroxidase-catalyzed iodination in vitro. Should T4 be formed, peptide b6TL could represent an interesting model for the study of the minimal structural requirements of the hormonogenic function.