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(Received for publication, May 5,
1995; and in revised form, September 14, 1995) From the
Thyroxine-binding globulin (TBG) is the main transport protein
for thyroxine (T
Heat stability of proteins has been a subject of intensive
research for several decades. The introduction of genetic engineering
techniques (1, 2) and the need for heat-resistant
proteins for research (3) , food biotechnology(4) , and
other industrial processes (5) has stimulated further research
in this area(6, 7, 8) . Early claims of
universally applicable mechanisms to predict heat stability of proteins
have been followed by reports that deny the existence of general
strategies to improve protein stability(9, 10) .
However, the stabilizing effects of several alterations of protein
structures have been well established. These include an increase in the
hydrophobicity of the protein
core(11, 12, 13, 14) , improved
packing density(15) , interaction of Current knowledge about stabilizing factors stems mostly from the
analysis of wild-type proteins and their less stable variants. Only a
few proteins with significant increases in thermal stability have been
described. These include the well characterized
barnase(23, 24) , Thyroxine-binding
globulin (TBG) ( So far, six
partial and three complete TBG deficiency variants have been analyzed
at the gene level(35, 36) . All partial deficiency
variants have different degrees of heat lability associated with one or
two nucleotide substitutions resulting in amino acid replacements. A
causal relationship of the mutation and impaired function has been
established for three of these TBG variants(37, 38) .
Screening of serum samples has revealed several more heat-labile, but
only one heat-resistant, thyroxine-binding globulin
variant(39) . This unique serum sample belonged to an
African-American man from a Chicago family that had no stigmata of
thyroid dysfunction. Except for resistance to heat and acid
denaturation, the TBG of the propositus (TBG-CH) had normal
thyroxine-binding kinetics, a microheterogeneous isoelectric focusing
pattern and immunological properties identical to the normal TBG
(TBG-N) and was present in serum in normal concentration(39) . TBG belongs to the serine protease inhibitor (serpin) superfamily, a
heterogeneous group of more than 60 proteins, including
We now present
the amino acid sequence of TBG-CH deduced by gene sequencing; confirm
by in vitro expression that the single amino acid substitution
is sufficient to impart heat resistance to the molecule and provide an
explanation for the increase in thermal stability of TBG-CH by
structure modeling.
In vitro transcription and cell-free translation
were performed as described previously(37) .
Figure 1:
Schematic representation of the TBG-CH
gene and strategy of sequencing. Exons are depicted by boxes with black areas indicating coding regions, and introns
are represented by lines. The translation initiation (ATG) and termination (TAG) codons, the two
alternative polyadenylation sites (poly-A(64) ), and
the position of the TBG-CH mutation with the resulting amino acid
substitution are indicated. Arrows show the regions and
directions of sequencing.
Figure 2:
Sections of sequencing gels showing the
mutation in the TBG-CH gene compared with TBG-N. Replacement of the
normal adenine 2767 with a thymine in codon 309 results in substitution
of the normal tyrosine (Tyr, TAT) with phenylalanine (Phe,
TTT) in TBG-CH.
Figure 3:
SDS-PAGE analysis of TBG variants
synthesized in reticulocyte lysate. Synthetic RNAs of TBG-N and TBG-CH
were translated in reticulocyte lysate in the absence (-CMM) and presence (+CMM) of canine
microsomal membranes. The [
Figure 4:
SDS-PAGE analysis of TBG variants
expressed in Xenopus oocytes. Oocytes injected with TBG-N and
TBG-CH sRNAs were incubated with [
Figure 5:
Scatchard analysis of T
Figure 6:
Heat denaturation of TBGs expressed in Xenopus oocytes. TBG secreted into the oocyte incubation
medium was heated at 60 °C, and aliquots were removed at the
indicated time intervals for the determination of residual T
The enhancement of protein stability by rational design is
one of the great goals of protein engineering. General principles are
still not available because of the complex interactions and the strong
positional and context dependence of the effect of a particular amino
acid substitution on the stability of a specific protein(8) .
The properties of natural proteins often depend on just one amino acid
at a specific site, as shown by the many deleterious point mutations in
proteins such as Heat resistance
has been described in a few natural variants and some engineered
proteins. However, the gain of thermal stability in most of these
variants is offset by a loss of function, i.e. reduced enzyme
activity. Most TBG variants identified to date have unaltered or
decreased heat stability. The altered properties of some of these
variants can be explained by the changes in their primary structure, i.e. loss of a negative charge (TBG-S(48) ) or
creation of a new site for N-linked glycosylation
(TBG-Gary(56) ). In other variants (TBG-Montreal(45) ,
TBG-CD5(57) , TBG-Quebec(58) , TBG-San
Diego(59) , and TBG-PDJ(60) ), alterations of the
primary structure are more subtle, and functional studies are required
to understand the effect of the amino acid replacement on the structure
of the protein(37, 38, 61) . All
heat-sensitive TBG variants have also defects in their T The
variant TBG-CH is unique in its pronounced heat resistance with
preservation of normal T The choice of the Xenopus oocyte system to analyze the properties of TBG-CH was
based on the previous use of this translation system to characterize
transport and secretion abnormalities of mutant forms of AT (62, 63) and TBG (TBG-Montreal, TBG-CD5, TBG-Gary, and
TBG-CDJ). Microinjected Xenopus oocytes have been shown to be
a legitimate surrogate system for the study of inherited TBG variants,
since the biological, physical, and immunological properties of these
variant TBGs from serum of affected individuals were faithfully
reproduced by them(37, 38, 61) . In
vitro translated TBG-CH had the same properties as the respective
serum TBG, confirming that the substitution of the normal tyrosine at
position 309 by phenylalanine is the cause for the increased heat
stability of this variant TBG. The residue corresponding to tyrosine
309 of TBG-N (tyrosine = Y, Fig. 7) corresponds
to a highly conserved phenylalanine (phenylalanine = F,
at position 312 in AT, Fig. 7) in the serpin superfamily of
proteins, of which TBG is a member. Comparison with the
crystallographic structure of the archetypical serpin, AT, shows that
this amino acid resides in a deep intramolecular pocket and ties the
Figure 7:
Alignment of the amino acid sequences of
human SERPINs showing the conserved phenylalanine 312 (AT numbering)
and the corresponding tyrosine 309 of TBG-N. Sequence alignment was
performed with the MegAlign utility of Lasergene (DNASTAR) using the
Clustal method with the PAM250 residue weight table. Tyrosine 309 of
TBG-N (Y, boxed in black) corresponds to a highly conserved
phenylalanine (F, in bold) in most of the other
SERPINs. TBG-CH with its phenylalanine for tyrosine substitution is
thus more closely related to the serpins than TBG-N. Abbreviations: TBG, thyroxine-binding globulin; ACT,
The most significant cause for
increased thermal stability of proteins has been found to be an
increase in the hydrophobicity of the protein
core(11, 12, 13, 14) , which can be
further improved by optimizing the internal packing density of the
molecule(15) . These two driving forces also appear to be
responsible for the heat resistance of the unique variant TBG-CH.
Volume 270,
Number 47,
Issue of November 24, 1995 pp. 28234-28238
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) in blood. It shares considerable sequence
homology with 
-antitrypsin (AT) and other members of
the serine proteinase inhibitor (serpin) superfamily of proteins. The
crystallographic structure of AT has been determined and was found to
represent the archetype of the serpins. This model has been used for
structure-function correlations of TBG. Sequence analysis of the
heat-resistant variant TBG-Chicago (TBG-CH) revealed a substitution of
the normal tyrosine 309 with phenylalanine. For further analysis,
vectors containing the coding regions of normal TBG (TBG-N) and TBG-CH
were constructed, transcribed in vitro, and expressed in Xenopus oocytes. Both TBGs were secreted into the culture
medium and could not be distinguished by gel electrophoresis. Scatchard
analysis of T binding to TBG-N and -CH revealed no
significant differences in binding affinity. The rate of heat
denaturation of TBGs was determined by measurement of residual T binding capacity after incubation at 60 °C for various
periods of time. The half-life values of denaturation of TBG-N and -CH
were 7 and 132 min, respectively. The tyrosine 309 to phenylalanine
substitution of TBG-CH involves a highly conserved phenylalanine
residue of the serpins. The respective phenylalanine 312 of AT ties the
-helix hI1 to the molecule, thus stabilizing the tertiary
structure. A substitution with tyrosine would disrupt this interaction.
Accordingly, stabilization of the TBG molecule by replacement of
tyrosine with phenylalanine in position 309 causes the increased heat
stability of TBG-CH.
-helix dipoles with
charged residues(8) , disulfide bond
formation(16, 17, 18) , N-linked
glycosylation(19) , and the assembly into quaternary
structures(10) . It has also been shown that the combination of
stabilizing mutations leads to a cumulative effect on protein
stability(1, 9, 20, 21, 22) . -repressor
protein(15, 25) , subtilisin(26) , kanamycin
nucleotidyltransferase(20) , and bacteriophage T4
lysozyme(20, 27, 28) . However, the gain of
thermal stability in most of these variants is offset by a loss of
function, i.e. reduced enzyme activity.
)is the main transport protein for thyroxine
(T) and triiodothyronine in human serum(29) . It is
synthesized in the liver and secreted into the blood stream as a 54-kDa
glycoprotein(30) . The primary structure of human TBG and the
organization of the TBG gene have been
described(31, 32) . The mature protein contains 395
amino acids in a single polypeptide chain and oligosaccharides attached
to four of the five potential N-linked glycosylation sites (33) . Familial TBG defects follow a X-linked inheritance
pattern, consistent with the presence of a single TBG gene on the long
arm of the X chromosome(34, 35) .-antitrypsin (AT), -antichymotrypsin,
and corticosteroid-binding globulin, among
others(40, 41, 42) . The crystallographic
structure of AT has been determined (43) and was found to
represent the archetype of the serpins(40) . Since attempts to
crystallize TBG have failed(44) , the AT model was used for
structure-function correlations of the TBG molecule.
Reagents
Restriction endonucleases, DNA
modifying enzymes, and size markers were from New England Biolabs, Life
Technologies, Inc., and Boehringer Mannheim. Oligonucleotide primers
used for amplification and sequencing have been described
previously(45) . The expression vector pSELECT, the altered
sites mutagenesis kit, the Gemini-II in vitro transcription
kit, recombinant RNasin, rabbit reticulocyte lysate, and canine
microsomal membranes were obtained from Promega Biotech. L-Methionine used for the cell-free synthesis of unlabeled TBG
was from Sigma. The cap analog m
G(5`)ppp(5`)G was from
Pharmacia Biotech Inc. Taq DNA polymerase was from
Perkin-Elmer. -S-dATP (specific activity > 800
Ci/mmol) for DNA sequencing and
C-methylated protein size
markers were from Amersham Corp. L-[S]Methionine (specific activity >
1200 Ci/mmol) for metabolic labeling of proteins and
[
I]T
(specific activity, 1500
µCi/µg) for binding analysis were from DuPont NEN. All other
reagents were of analytical grade.Bacteria
Escherichia coli strains P2392,
LE293, JM109, BMH7118, and DH5 were maintained in Luria-Bertani
broth or plates(46) . For maintenance of strains carrying
plasmids, tetracycline (60 µg/ml) or ampicillin (50-100
µg/ml) were added.Construction of a Genomic DNA Library Containing the
TBG-CH Gene
Leukocyte DNA was prepared from blood (47) of a subject known to expess both TBG-CH and TBG-Slow
(TBG-S)(39) . 20 µg of this DNA were partially digested
with EcoRI and run on a 0.8% SeaPlaque (FMC, San Diego, CA)
agarose gel in TAE buffer(46) . DNA from the 9-23
kilobase pair region was excised, isolated by gel extraction, and
ligated into EMBL4-EcoRI-arms (Stratagene, La Jolla, CA). The
resulting -vector library was packaged with the Gigapack Gold in vitro packaging extract (Stratagene) and transfected into E. coli P2392 and grown on LB plates. Plaques were lifted with
BA 85 nitrocellulose filters (Schleicher & Schuell, Dassel,
Germany). Plaque hybridization, further propagation, rehybridization,
and large scale preparation of positive clones was performed
essentially as described previously(46) , using a
P-labeled TBG-cDNA probe synthesized with a nick
translation kit from Amersham labeled with
[
-P]dCTP (specific activity > 3000
Ci/mmol, DuPont NEN) and purified with G-50 quick spin columns
(Boehringer Mannheim, Mannheim, Germany). Clones were screened by
allele-specific amplification(48) , and those that did not have
the TBG-S genotype were assumed to have the TBG-CH genotype.
Sequence Analysis
A -vector containing the
TBG-CH gene was digested with BamHI, and the resulting
5.5-kilobase pair insert was subcloned into a pGEM-7Zf+ vector.
The coding regions and exon-intron junctions of the TBG gene in this
vector were sequenced by the dideoxynucleotide termination method (49) using the Sequenase-2 kit from U. S. Biochemical Corp.
Construction of Vectors
A vector containing the
full-length cDNA of TBG-N had been constructed before(37) . The
TBG insert of this vector was excised with BamHI, separated by
agarose gel electrophoresis, isolated with miniprep filters (Millipore,
Bedford, MA), and subcloned into the pSELECT expression vector by
standard techniques(46) . This construct was designated pSpT-N.
Site-directed mutagenesis with the oligonucleotide
5`-TTCAGCATGCCTTTTCTGAAAATGC-3` (the underlined T is substituted for
the normal A) was performed to obtain a vector coding for TBG-CH.
Briefly, single-stranded DNA was generated from the vector pSpT-N and
incubated with the mutagenesis oligonucleotide and with a repair
oligonucleotide to reinstitute ampicillin resistance of the pSELECT
vector. The complete second strand was synthesized with DNA-polymerase
and -ligase, and the resulting reaction products were used to transform
the DNA repair-deficient BMH7118 strain of E. coli. Mutated
vectors were selected by supplementing the medium with ampicillin.
After preparation of vector DNA, a second round of transformation in
JM109 E. coli was performed to obtain pure vector
preparations. Individual clones for both the TBG-N and TBG-CH
constructs were tested by restriction endonuclease analysis for proper
orientation of the coding region relative to the T7 RNA polymerase
promoter and verified to contain the complete and proper nucleotide
sequence by sequencing alkali denatured double-stranded vector DNA as
template.Preparation of Oocytes and RNA Injection
Ovaries
were removed from mature Xenopus laevis (Nasco) (50) and suspended in OR-IIa medium (83 mM NaCl, 2.5
mM KCl, 1 mM MgCl
, 1 mM
NaHPO, and 5 mM HEPES, pH
7.6)(51) . After manual dissection of the follicles, oocytes
were dissociated from the surrounding connective tissue by incubation
in OR-IIa containing 0.2% collagenase type IA (Sigma) for 2 h with
shaking at room temperature. The liberated oocytes were then rinsed
extensively in OR-IIb medium (OR-IIa with 1 mM CaCl and 100 µg/ml gentamycin), and stage VI oocytes (52) were separated and kept up to 3 days in OR-IIb with daily
medium changes. After injection with 100 nl of sRNA (0.5
µg/µl), oocytes were kept on ice for 1 h and then for 2-6
h at 19 °C in OR-IIb. Intact oocytes (>95%) were transferred to
24-well plates (Costar) and kept in OR-IIc (OR-IIb with 1 mM sodium pyruvate), 5 µl/oocyte, at 19 °C for up to 4 days,
with daily exchange of medium. Typically, 100 oocytes were injected
with each sRNA preparation. Control oocytes were either injected with
water or noninjected, with identical results. In some experiments, the
oocytes were metabolically labeled by addition of 250 µCi of
[S]methionine/500 µl of medium.
Extraction and Analysis of TBG Synthesized in
Oocytes
The medium of microinjected oocytes was removed,
supplemented with 1 mM phenylmethylsulfonyl fluoride, and
stored at -20 °C until further use.SDS-PAGE
Products of cell-free translation or
synthesized in Xenopus oocytes were analyzed by the method of
Laemmli (53) , using polyacrylamide gels at 10% T and 2.7% C.
Stacking gels were 3.75% T, 2.7% C. Gels were dried and
autoradiographed at -90 °C on X-AR5 film (Eastman Kodak Co.)
with an intensifying screen.Measurement of T
Parameters of TBinding to
TBG binding to TBG were
measured by a method previously described in detail(54) .
Briefly, TBG preparations were incubated with
[I]T
in the presence of increasing
amounts of unlabeled T. TBG-bound T was
separated from free T with anion exchange resin beads
(Mallinckrodt), and the protein-bound I activity was
determined. The affinity constants (K
) of TBG
preparations were determined by the method of Scatchard(55) .Heat Denaturation
The rate of heat denaturation of
TBG was determined by measurement of the residual T binding
capacity of TBG in samples incubated at 60 ± 0.1 °C for
various periods of time. Data were expressed as a percentage of the
T binding capacity of TBG-N before heat exposure.
Half-lives (T) of denaturation were calculated
from the slope of the linear regression semi logarithmic plots.
Sequence Analysis of the TBG-CH Gene
A genomic
-EMBL4 library was prepared from EcoRI-digested leukocyte
DNA obtained from an heterozygous member of the TBG-CH family
expressing both TBG-CH and TBG-S. The latter can be identified by its
slower electrophoretic mobility, its cathodal shift on isoelectric
focusing, and the substitution of the normal aspartate with asparagine
due to a mutation in codon 171(48) . Screening of the EMBL4
library with a TBG-cDNA probe and subsequent analysis by
allele-specific amplification specific for TBG-S identified clones that
contained the TBG-CH allele, which is contained in a single 14-kilobase
pair EcoRI DNA fragment. For further analysis, a 5.5-kilobase
pair BamHI fragment of the TBG-CH gene containing the entire
coding region was subcloned and sequenced. Fig. 1shows the
structure of the TBG gene and the strategy of sequencing. Analysis of
the TBG-CH gene revealed a single base substitution replacing the
normal adenine 2767 with thymine, resulting in the substitution of the
normal tyrosine 309 with phenylalanine (Fig. 2).
Synthesis of Normal and Mutant TBG in Reticulocyte
Lysate
An expression vector for TBG-N was constructed by
subcloning a TBG-cDNA into the pSELECT phagemid. A vector containing
the mutation specific for TBG-CH was then constructed by site-directed
mutagenesis. The linearized vectors were transcribed in vitro to obtain synthetic mRNAs (sRNAs). The products of cell-free
translation of TBG-N and TBG-CH sRNAs in reticulocyte lysate were
indistinguishable on SDS-PAGE. Both variants had a major band at 44
kDa, and several new bands of higher molecular weight after signal
peptide processing and core glycosylation (Fig. 3).
S]methionine-labeled
reaction products were submitted to SDS-PAGE and autoradiographed. Both
types of TBG were synthesized with equal efficiency and had identical
patterns of nonglycosylated and glycosylated forms. The lane labeled MWM contained
C-labeled molecular weight
markers.
Expression of Normal and Mutant TBG sRNAs in Xenopus
Oocytes
Oocytes were removed from X. laevis, culled,
injected with TBG sRNAs, and incubated in medium with
[S]methionine. The TBGs synthesized and secreted
into the medium were then submitted to SDS-PAGE. No significant
differences were observed between TBG-N and TBG-CH (Fig. 4).
Both showed a microheterogeneous 60-kDa product that was secreted in
similar amounts. The TBGs from microinjected Xenopus oocytes
were also submitted to isoelectric focusing. Again, no differences were
found between TBG-N and TBG-CH (data not shown), as shown previously
for the respective serum TBGs(39) .
S]methionine.
The labeled TBGs secreted into the medium were submitted to SDS-PAGE
and autoradiographed. Both types of TBG had the same apparent molecular
weight. No significant differences in the efficiency of synthesis and
secretion of TBG-N and TBG-CH were found in four independent
experiments. MWM,
C-labeled molecular weight
markers; ni, noninjected.
Analysis of T
The amount of biologically active TBG synthesized in Xenopus oocytes was examined by measurement of the
TBinding to
TBG binding characteristics. TBGs were synthesized as
described above, but without the addition of
[S]methionine to the medium. Scatchard analysis
of the secreted TBGs revealed no significant differences in T
binding affinity and T binding capacity/oocyte for
TBG-N and TBG-CH (Fig. 5). The amounts of secreted TBG-N and
TBG-CH as quantified directly by SDS-PAGE (Fig. 4) and by
radioimmunoassay (data not shown) were also not significantly
different. These results are in agreement with data obtained from serum
TBG of a subject expressing only the TBG-CH gene(39) .
binding to TBGs expressed in Xenopus oocytes. TBG-N and
TBG-CH were expressed in oocytes. The secreted TBGs were incubated with
[I]T
and increasing amounts of
unlabeled T. No significant differences in T binding affinity (slope) and binding capacity/oocyte (intercept)
were found.
Heat Resistance of TBG-CH
For heat denaturation,
unlabeled TBG-N and TBG-CH were expressed in Xenopus oocytes
as described above. The rate of heat denaturation of the TBGs was
determined by measurement of residual T binding capacity
with a standard resin T binding assay after various
incubation periods. At 60 °C, the half-life of denaturation of
TBG-N was 7 min and that of TBG-CH was 132 min, almost 20 times as long (Fig. 6).
binding activity. Values are expressed as TBG-bound T relative to the basal levels. Note the much slower rate of
denaturation of TBG-CH as compared with
TBG-N.
-antitrypsin, myoglobin, and
hemoglobin. In most cases, the loss of stability is accompanied by a
loss of biological activity. This has been confirmed by systematic
analysis of proteins by site-directed mutagenesis. binding affinity and show increased concentrations of denatured
TBG in serum, compatible with a general defect of the molecule. binding affinity, electrophoretic
mobility, and serum concentration. The isolated increase in stability
of TBG-CH can thus be thought of as a specific effect of the mutant
amino acid on the molecule (Fig. 2).-helix hI1 to the molecule, ()thus stabilizing the
tertiary structure. A substitution with tyrosine with its larger and
hydrophilic side chain would disrupt this interaction. Accordingly,
stabilization of the TBG-CH molecule is most likely due to hydrophobic
interactions of the better fitting side chain of phenylalanine. TBG-CH
with its phenylalanine for tyrosine substitution is thus more closely
related to the serpins than TBG-N.
1-antichymotrypsin; AT, 1-antitrypsin; CBG,
corticosteroid-binding globulin; IPSP, plasma serine protease
inhibitor; ATRP, 1-antitrypsin-related protein; HC2, heparin cofactor II; ANT3, antithrombin-III; PAI1, plasminogen activator inhibitor-1; HS47, 47-kDa
heat shock protein; PAI2, plasminogen activator inhibitor-2; GDN, glia-derived nexin; PC1I, protease C1 inhibitor; ANGT, angiotensinogen.
)-antitrypsin; serpin(s), serine
protease inhibitor(s); T, thyroxine; sRNA, synthetic
messenger RNA; PAGE, polyacrylamide gel electrophoresis.)
We thank Paul Gardner (Howard Hughes Medical
Institute, University of Chicago) for the synthesis of the
oligonucleotide primers, Dr. Robert Huber (Max Planck Institute for
Biochemistry, Martinsried, Germany) for help with the structural data,
and Dr. Graeme Bell (Howard Hughes Medical Institute, University of
Chicago) for helpful discussions of the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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