(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) 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.
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 -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) .
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) , -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.
Thyroxine-binding
globulin (TBG) ()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) .
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
-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.
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 [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.
Figure 4:
SDS-PAGE analysis of TBG variants
expressed in Xenopus oocytes. Oocytes injected with TBG-N and
TBG-CH sRNAs were incubated with [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.
Figure 5:
Scatchard analysis of T 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.
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 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.
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 -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.
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 binding affinity and show increased concentrations of denatured
TBG in serum, compatible with a general defect of the molecule.
The
variant TBG-CH is unique in its pronounced heat resistance with
preservation of normal T 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).
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
-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.
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,
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.
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.