(Received for publication, September 5, 1995; and in revised form, January 26, 1996)
From the
The interleukin 2 receptor (IL2R) plays a prominent role in the
biology of T cells, B cells, and NK cells during activation. Of the
three chains described, the -chain of the receptor (Tac;
IL2R
; CD25) is the most subject to regulation and is shed from the
surface of activated cells to generate a soluble form in serum and
tissues. Conflicting results have been reported on the native structure
of soluble Tac, suggesting variously a monomer, a dimer, or higher
noncovalent forms, spawning different models for its mechanism of
action. We similarly show a large M
by HPLC
sieving chromatography, suggesting a tetrameric form. However,
stoichiometry-ordered size (SOS) analysis of antibody-antigen complexes
indicated only a single epitope per Tac molecule, compatible with a
monomeric form. This larger M
also conflicted
with prior in vivo data showing rapid filtration of soluble
Tac through the renal glomerulus that was not expected of a larger
complex. Using different solvents, denaturants, and columns in the
chromatography suggested that the elevated M
values were an artifact of solute-column interactions, termed
``ionic exclusion,'' rather than reflecting larger native
structures. Analytical ultracentrifugation using a new type of analysis
specific to glycoproteins demonstrated monomeric masses under all salt
conditions with no tendency to form dimers or higher aggregates.
Finally, circular dichroism spectroscopy showed no salt-dependent
changes to suggest conformational alterations that might correlate with
mobility changes on high pressure liquid chromatography. We conclude
therefore that Tac is monomeric under physiologic conditions.
Assessments of higher molecular weight for the purified soluble protein
by other methods may be explained by the highly acidic nature of the
molecule, which hampers matrix penetration with chromatographic media
and by the high carbohydrate content and low partial specific volumes
that accelerate the molecule in sedimentation media relative to pure
protein standards.
The interleukin 2 receptor is comprised of at least three
cooperating polypeptide chains. The first of these chains, termed Tac,
for T activation antigen, was described by Waldmann and colleagues in
the early 1980s and displayed low affinity binding for IL2. ()A second chain of moderate affinity for IL2 was described.
(See Refs. 1 and 2 for review.) A third chain of the receptor,
(
), was recently cloned by Sugamura and
colleagues(3) . At present, evidence for additional chains is
circumstantial without supporting functional data.
Of these chains,
Tac is the most subject to regulatory events in the cell. Absent in
normal resting lymphocytes, it is strongly up-regulated on activated
cells to collaborate with the and
-chains to form the high
affinity receptor. Furthermore, its down-regulation is an active
process that sheds the molecule from the cell surface into the milieu
and leads to rapid return to resting state following withdrawal of an
activating stimulus. In certain disorders, the accumulation of Tac in vivo can lead to high serum levels and has been used as a
marker of activity in autoimmune and allommune states and of tumor
burden in malignancy. While speculations of a secondary regulatory role
for soluble cytokine receptors have been offered(4) , for
inhibiting cytokine binding (as in genetically engineered
IL1R
(5) ) or for
-chain collaboration (as in
IL6R
(6) ), there are presently no functional data to
support any role for soluble Tac at concentrations encountered in
vivo other than as an intermediate in the down-regulation of
activated cells that shed receptor when activation and proliferation
are no longer needed.
Because of its central importance to T cell
biology, Tac has been extensively studied from clinical, biological,
and molecular perspectives. It is a type I transmembrane glycoprotein
of 251 amino acids with a short 13-amino acid COOH-terminal cytoplasmic
domain. The soluble form of the molecule is generated by an active
proteolytic process to generate a molecule of 192 amino acids that
retains the ligand-binding and antibody epitope specificities of the
parent molecule(7) . Using a recombinant soluble form of Tac
(see description under ``Materials and Methods''), the
protein runs as a monomer under nonreducing conditions on denaturing
gels, but its chromatographic behavior in some analyses, including our
own, supported a homodimeric or higher, noncovalent association of the
molecule. Yet, independent studies of Tac led to apparent
contradictions with a multimeric model. These apparent contradictions
included a very rapid renal clearance of Tac in vivo (t
1 h) (8) not expected of a
larger molecule and a chromatographic behavior of in vivo- and in vitro-generated Tac
anti-Tac complexes (
)that suggested a single epitope per Tac molecule or
aggregate.
In the present report, we examine the structure of Tac by HPLC, epitope analysis, analytical ultracentrifugation, and circular dichroism spectroscopy to attempt a definitive assessment of its molecular configuration. The major conclusion of this work is that Tac is a monomer under all conditions.
Figure 1:
Soluble Tac shows
discordant sizes on SDS-PAGE and HPLC. A, HPLC with TSK-250
sieving column chromatography in phosphate-buffered saline. The elution
of Tac is compared with a parallel run of a mixture of protein
standards (thyroglobulin, 670 kDa; IgG, 155 kDa; ovalbumin, 44 kDa;
myoglobin, 22 kDa; vitamin B, 1.35 kDa). Tac runs slightly
ahead of IgG in this mixture (8.19 versus 8.44 min, by
internal Waters peak-detect software). This pattern was confirmed on
separate runs in phosphate-buffered saline on different dates in which
Tac ran ahead of IgG, whether IgG was a single component or part of the
protein standards mixture. See also Fig. 2B. B, SDS-PAGE on 4-20% polyacrylamide gel under
nonreducing conditions. Markers are SeeBlue (Novex) prestained proteins
with approximate molecular masses shown in
kDa.
Figure 2:
SOS
analysis: HPLC of Tacanti-Tac complexes favors monomer. A, definition of the method. Models of monomeric and
oligomeric Tac binding to antibody. With a fixed quantity of anti-Tac
present, three situations are considered: no added Tac, added Tac with
antibody in excess, and added Tac with antigen in excess. The monomeric
model predicts an M
of the complex with
``antibody excess'' that is less than with ``antigen
excess''. The oligomeric model predicts a higher M
with ``antibody excess'' that
decreases with ``antigen excess''. (The same ordered pattern
is predicted with a dimeric model.) The M
of
anti-Tac is given as 150 kDa and that of Tac as 180 kDa, whether
considered as a tetramer or as an aberrantly running monomer. Tac is
shown as a circle with a line and dot to
suggest structural symmetry in the oligomer that is closed and presents
a single antibody binding site per subunit. (
is the back face
of the molecule; other configurations meeting these criteria are
possible.) B, HPLC profile of Tac
anti-Tac complexes.
From left to right are i) 10 µg of
anti-Tac antibody, no added Tac, ii) 10 µg of anti-Tac
+ 1 µg of Tac, antibody in 3
molar excess of binding
sites, iii) 10 µg of anti-Tac + 10 µg of Tac,
antigen in 3
molar excess of binding sites, iv) 1
µg of Tac, no antibody. Elution times are listed. ``Excess
antigen'' generates a larger complex than ``excess
antibody'' and favors the monomeric
model.
Sedimentation velocity experiments were conducted using a Beckman XL-A analytical ultracentrifuge with a rotor speed of 54,000 rpm at 20.0 °C. Sample concentrations were 0.1 and 0.3 mg of protein/ml. Scans were taken at 10-min increments at a wavelength of 230 nm. Only those scans with a depleted meniscus and a clearly defined plateau region were used for the analysis; nine scans at each concentration were found suitable. Data editing was performed on the XL-A software. The concentration gradients were analyzed by the method of Attri and Lewis (16) to obtain the value of r, the radial position corresponding to the square root of the second moment of the gradient. These radial positions, as functions of rotor angular velocity and time, were used to obtain the uncorrected sedimentation coefficients; these were corrected to standard conditions of water at 20 °C(17) . The buffer densities and viscosities needed for these corrections were calculated from the data of Laue et al.(18) .
Data representation for the sedimentation experiments employs the empirical fitting function,
where r is defined above, c is the concentration in the plateau region, c
is the base-line offset, and n is a parameter whose
value reflects the width of the gradient and is inversely proportional
to the diffusion coefficient. It has been demonstrated that this
fitting function, while empirical and not based on the Lamm equation,
fits the sedimentation velocity gradients of monodisperse solutes quite
well and gives proper values for the sedimentation
coefficients(16) . The sedimentation coefficient is obtained by
fitting the values of r as a function of time
with the equation,
where s is the sedimentation coefficient, r is the radial position of the meniscus, and
t is the integral of the rotor angular
velocity as a function of time from the time the rotor begins turning
until the scan is taken. The computation is obtained by the XL-A
microprocessor and is recorded with each scan.
Data conversion, nonlinear least squares curve fitting, and other computations were performed using MLAB (Civilized Software, Bethesda, MD) for mathematical modeling(19) .
The molecular mass of the glycoprotein is M = M
+ M
,
where M
is the molecular mass of the carbohydrate
portion of the molecule and M
is the molecular
mass of the protein portion calculated from the amino acid sequence.
With appropriate rearrangement of founding equations for sedimentation
analysis, we obtain the equation,
where M` is the measured reduced mass. The
experimentally obtained value of the reduced mass is combined with the
molecular mass (M) and apparent compositional
partial specific volume (v
) of the
protein portion of the glycoprotein (calculated from the amino acid
composition), and with estimates of the partial specific volume (v
) of the carbohydrate portion of
the glycoprotein from analysis of several known structures.
where the diffusion coefficient is derived from the Svedberg equation,
and where the mass and partial specific volume are from our
previously derived values.
Estimation of the secondary conformation of Tac used a
polypeptide model approach(24) . Standard CD spectra for
-helix, antiparallel
-structure, class B type II
-turns,
and extended nonordered structures were used as
described(25, 26) . (For a review of secondary
structure fitting, see (27) .)
Fig. 2A demonstrates the rationale for this test, which we term stoichiometry-ordered size (SOS) analysis. On one hand, the model of antibody excess for a monomer of Tac would yield the smallest complex, and this would increase in size with the addition of further Tac. On the other hand, a tetrameric form with 4-fold symmetry would present four binding faces and would yield the largest complex with small amounts of antigen (antibody excess), and this complex would decrease in size with additional Tac. As seen in Fig. 2B, the experimental complex increases in size with increased Tac, therefore favoring a monomeric form by straightforward symmetry assumptions. It is important to note that SOS analysis does not require precise estimates of size on chromatography, which are not obtained in this range on this column (TSK-250); it only requires that the complexes be ``orderable'' (i.e. have distinguishable retention times) during the titration. All complexes were included on the column and orderable. Identical SOS patterns and conclusions were obtained using a larger pore size column (TSK-400) (not shown).
We pursued
further efforts to demonstrate whether this high M represented a multimer. If Tac is normally a multimeric,
noncovalent structure, then treatment with SDS or other denaturants and
rechromatography with immediate removal of denaturant should reveal
monomer as a late eluting component. Tac was treated with denaturants
(2% SDS or 8 M guanidine) or heat (95 °C, 15 min),
followed by injection and immediate chromatography in 0.15 M NaCl without denaturant. In each instance, the fast
chromatographic behavior of Tac in saline was recovered, suggesting
that any aggregates are extremely rapidly reassembled (within 0.2 min),
which seemed improbable. Alternatively, the behavior was compatible
with a reversible intramolecular process, a conformational
characteristic of the monomer that is disrupted in the presence of the
negatively charged detergent, SDS, but re-expressed as soon as the
denaturant is removed; or it could have reflected protein-column
interactions. The role of a protein-column interaction is also
suggested by the apparent molecular mass change relative to standards
on the same matrix with a higher exclusion limit. On a TSK-400 column,
Tac ran behind IgG in phosphate-buffered saline with a M
of 125,000 (not shown) versus TSK-250, where Tac ran ahead of IgG with a M
of 180,000 under identical salt conditions ( Fig. 1and Fig. 2).
We then studied the elution behavior of Tac in the
presence of various salt concentrations that might influence such
protein conformation or column interactions. Fig. 3shows a
series of molecular weight standards whose retention times are
monotonically shifted to shorter times at lower salt concentrations.
Relative to these standards, however, Tac is shifted to an even greater
extent in terms of its M and relative to its
predicted monomer molecular mass. As a consequence, the derived M
for Tac decreases with increasing
Na
concentration (Fig. 4). In moderate salt
(0.15 M NaCl), the addition of SDS accelerates the elution
times of all standards except thyroglobulin to faster than even those
in low salt. (Thyroglobulin is at the void and not expected to change;
however, the stability of its elution time is reassuring that the
column matrix is not undergoing gross physical changes during solvent
changes.) Tac, by contrast, is the only protein that is more
accelerated by low salt than by SDS. The M
in
SDS was 33,000 (Fig. 4), closely approximating the true
monomeric molecular mass (see below), in contrast to the high M
obtained on SDS-PAGE (Fig. 1).
Figure 3:
The
HPLC retention time of Tac is more sensitive than standard proteins to
solvent ionic strength. The retention time of Tac on a TSK-250 column
was measured relative to standards (``Materials and
Methods'') in increasing concentrations of NaCl. At the 0.02
Na concentration, ovalbumin did not run separately
from IgG. The influence of ionic strength on Tac retention time is
noted. The sodium concentration is plotted, which equals the ionic
strength for this uni-univalent salt.
Figure 4:
M of Tac on HPLC
decreases with increased ionic strength. The M
of Tac was derived from the data of Fig. 3by analysis of
standard semilog plots.
The longer retention time of monomeric proteins in higher salt may be interpreted as 1) decrease in partial specific volume or change in shape of the proteins, 2) enhancement of hydrophobic interactions, or 3) charge masking on the silica matrix and on the protein that permits better matrix penetration. That Tac is more susceptible to these effects may be a clue to the nature of possible interactions with the chromatographic system. In other words, Tac may 1) have an unusual shape or a large partial specific volume, 2) have less than ``usual'' hydrophobic interaction with the matrix, or 3) have a larger charge-mediated exclusion effect.
Figure 5:
Ultracentrifugation of Tac demonstrates
monomer under all salt conditions. A, equilibrium
sedimentation. Sedimentation was performed in concentrations of 0.15,
0.50, and 1.15 M NaCl, pH 7.4. The concentration profile is
normalized to distance coordinates and plotted in the usual manner.
Data are represented for 0.15 () and 1.15 M NaCl
(
). The 0.15 M curve is displaced +0.1 absorbance
unit for graphing purposes. The upper panel shows the
distribution of residuals for the 0.15 M curve, demonstrating
an excellent fit to the model for a homogeneous, ideal species. All
runs were of high quality and yielded virtually identical mass data (Table 1). B, velocity sedimentation. Data are fit to for the concentration as a function of radial position
obtained after the equivalent of 9194 s of sedimentation at 54,000 rpm
(
t = 2.940
10
s
). The value of r is
6.456 cm. Inset, joint fit of to the values of r as a function of
t for
loading concentrations of 0.1 mg/ml (
) and 0.3 mg/ml
(
).
These
equilibrium sedimentation data were subjected to an alternative
analysis in which a dimer form was assumed, beginning with a
protein molecular mass of 2 25,253 = 50,506 and solving
for the mass of the total glycoprotein according to . This
yields an estimate of M
of 38,800, which is less
than the mass of the dimeric protein portion alone, thus implying
negative mass for the carbohydrate, which is physically impossible.
This calculation shows that an interpretation that includes dimers or
higher oligomers would not be compatible with the ultracentrifugal data
on this protein. These sedimentation data demonstrate that Tac is a
well behaved, monomeric structure and that chromatographic behavior on
HPLC that suggests higher forms is an anomaly of this analytical
method.
As part of these equilibrium studies, we derived a partial
specific volume (v) for Tac of 0.689
cm/g.
This is a lower value than that of any of
the HPLC standards, which are 0.723 (thyroglobulin), 0.739 (IgG), 0.749
(ovalbumin), and 0.741 cm
/g (myoglobin)(28) . This
contradicts the supposition (supposition 1, above) of a high v for Tac, and this postulated explanation for
the faster base-line chromatographic elution of Tac may thus be omitted
from consideration.
Tac was also studied by velocity sedimentation
analysis, at concentrations of 0.1 and 0.3 mg/ml in 0.15 M salt, which yielded nearly identical s values of 2.64
± 0.02 S and 2.63 ± 0.02 S (Fig. 5B). The
joint fit provides a value of 2.70 ± 0.01 S for s when corrected for viscosity and density to 20 °C in water
and 2.73 ± 0.02 S for s
, extrapolated
to zero concentration. (
)Similarly to the equilibrium
studies, there was no significant effect of salt concentration on the
hydrodynamic behavior of Tac, with a value of 2.62 ± 0.02 S for
s
when the sedimentation was performed in
1.15 M salt. The concentration distribution of the
sedimentation profile is again compatible with a single homogeneous
species without equilibrating dimers or multimers. Using either the
joint fit or the extrapolated sedimentation coefficient with the 34,500
g/mol for the molecular mass and 0.689 cm
/g for the partial
specific volume from the equilibrium data above, a value for f/f
of 1.65 was calculated, compatible
with a relatively elongated structure. Because such structures are more
readily excluded from sieving gels than globular proteins, the
elongated structure of Tac could explain a base-line retention time
that is slightly faster for Tac than other proteins (f/f
values for thyroglobulin, IgG,
ovalbumin, and myoglobin are 1.49 (calculated), 1.26, 1.12
(calculated), and 1.11, respectively(28) ), but the observed
salt dependence of elution behavior would not be expected to be related
to this finding. A Stokes' radius of 35 Å was calculated
according to and under ``Materials and
Methods''; we report the calculation without further comment in
the absence of the requisite hydration data for this molecule.
Figure 6: Circular dichroism shows no salt-dependent conformation changes. A, CD spectrum from 250 to 188 nm of Tac in 0.4 mM Tris, 0.1 M NaCl to assess conformational structure. B, CD spectra from 250 to 205 nm of Tac in 0.4 mM Tris with 0(- - -), 0.1(- -), or 2 M NaCl (-).
It is noted that the
published spectrum in the low UV region for another
-glycoprotein(29) , human complement factor H,
was similar to that of Fig. 6A, including the relative
lack of computed
- or
-structure(30) . Although their
biological functions are unrelated and their molecular sizes are
different (factor H
150-160 kDa), Tac and factor H share
substantial amino acid sequence homology through a large repeating
motif(20) ; our data suggest secondary structure homology as
well.
To address the specific issue of change in conformation, the CD spectra between 250 and 206 nm were examined for Tac protein in 0, 0.02, 0.1, and 2 M NaCl (Fig. 6B and Table 2; data for 0.02 M are omitted but are comparable with represented data). The CD spectra of Tac in the various aqueous solutions are similar, with negligible quantitative variation at the peak. Overall, circular dichroism spectroscopy shows that the apparent conformations did not differ under conditions varying widely in salt concentration. Thus, conformational alterations in the protein are unlikely to account for the pattern of retention times on HPLC with increasing NaCl content of the solvent.
The molecular form of Tac has been examined in several studies by different modalities with a lack of consistent conclusions. Some estimates suggest a monomeric form, while others suggest a dimer or higher than dimer mass, and some studies have suggested the presence of mixed forms, depending upon the source of protein, as we shall discuss. The presence of a free cysteine at position 192 makes possible the formation of covalent dimers, but higher forms than dimer must invoke noncovalent association of monomers and/or dimers.
Early
studies using SDS-PAGE showed that the native transmembrane Tac protein
was monomeric under reducing and nonreducing conditions (21) , ()indicating that there was little covalent dimerization prior to shedding. In studies of soluble Tac
(Tac
) released from Tac-expressing malignant T cell
lines, Robb and Kutny (7) showed that >98% of the material
was monomer on nonreducing SDS-PAGE even after the prolonged in
vitro incubation preceding media harvest. Similarly, the soluble
Tac (Tac
) used in the present study was >95% monomer
by nonreducing SDS-PAGE ( (9) and Fig. 1). Covalent
dimerization (presumably through Cys-SH
) is catalyzed in
crystals of soluble Tac but is absent in the saturated mother liquor of
the crystallization droplets(13) . Taken together, these data
indicate that formation of significant covalent dimer is not part of
the normal biology of Tac protein.
Other reports suggested higher
apparent molecular weights of the native protein by nondenaturing
analyses. One report (32) showed dimer (65 kDa) and larger
(>90 kDa) forms by gel filtration of soluble Tac in serum of
rheumatoid arthritis patients. Two other reports showed gel filtration
sizes of 40-50 kDa (taken as monomer) for Tac in the serum of
such patients(33, 34) , with 80-100 kDa or
50-200 kDa forms in sinovial fluid, either alone (33) or
in addition to the monomer weight form(34) . Symons et al.(33) developed further data suggesting the higher
molecular weight form in sinovium was due to binding to sinovial
components rather than dimerization of the protein to itself. Jacques et al.(35) , employing the same protein product
(Tac) as in our studies, examined Tac by size exclusion
chromatography, sucrose gradient velocity sedimentation, and other
modalities, concluding that Tac was a noncovalent dimer, as the soluble
form in solution and, by inference, as the complete transmembrane
molecule.
In this report we also observed a high M for Tac by sieving chromatography,
suggesting an oligomer. Yet two independent observations of our own
conflicted with this interpretation and prompted additional studies.
The first conflicting observation was that complexes of endogenous
Tac
with radiolabeled anti-Tac in vivo in human
therapies showed a pattern indicative of a single epitope per molecule.
The second was that Tac
was efficiently filtered by the
kidney with a resultant rapid catabolic t
of 1 h
in murine models(8) ; proteins the size of the putative
oligomers are too large to pass the glomerular filter.
Our further investigation confirmed the in vivo epitope pattern using in vitro reconstructions with the recombinant soluble Tac protein of this study. This information was developed applying a new type of evaluation, which we term stoichiometry-ordered size (SOS) analysis, which predicts binding patterns based on symmetry assumptions that are independent of precise size data. We subsequently presented evidence that the chromatographic behavior of Tac was anomalous, with an accelerated elution relative to standards that was a function of the pore size of the chromatographic support and of the salt conditions of the chromatography. In our case, the size discrepancy may have been greater due to the use of a genuine HPLC matrix, which was not employed by prior studies. The advantages of HPLC in resolution conferred by dense matrix packing that yields vastly increased numbers of analytical plates also increase the opportunity for interaction between solute and matrix. Other differences in the media (e.g. silica versus agarose) may have had additional influence.
A new strategy for
glycoprotein mass determination by equilibrium analytical
ultracentrifugation showed that Tac was 34,500 ±
430 daltons, with 25,253 daltons of protein and 9,250 ± 430
daltons of carbohydrate under nondenaturing conditions, with an
uncertainty equivalent to approximately ±3 sugar residues. This
implies a carbohydrate content for this preparation that is 37%
relative to protein and 27% relative to total mass. This provides a
more accurate measure of molecular mass than SDS-PAGE, which typically
overestimates the size of glycoproteins because carbohydrate
contributes mass and resistance without a proportionate contribution of
charge, lacking the SDS binding characteristic of protein. Velocity
sedimentation similarly confirmed a nonaggregating monomer form. These
sedimentation analyses showed that Tac exists solely as a monomer with
no detectable tendency to reversible association under any conditions
of salt and protein concentration tested. We estimate that any affinity
between monomers must be <10
M
to avoid detection in our analysis. This effectively precludes
noncovalent dimerization under solution conditions in vivo,
for which the upper limit of normal is <10
M(36) , and the highest documented serum level
in any individual was still only 10
M(37) .
This ultracentrifugal mass determination by nondenaturing methods (34,500) was closely approximated by the value obtained on sieving chromatography in SDS (33,000), where column-protein interactions are rendered uniform. Within the limits of this single comparison, this concordance suggests that glycoprotein total mass may be reasonably estimated by sizing chromatography in SDS, in contrast to the overestimates routinely obtained in SDS-PAGE.
Our velocity sedimentation data may be
reinterpreted in terms of the partial specific volume of Tac (v = 0.689) versus that for
pure protein standards (v 0.74), which
illustrates an important point in deriving glycoprotein masses by
nonanalytical centrifugation methods. By the Svedberg equation (), s
(1 - v
), and the velocity acceleration of the
Tac glycoprotein relative to pure proteins of equal total mass can be
shown to be approximately +20% in water (
= 1.000) and
+30% in typical salt plus sucrose density media (e.g.
= 1.080). Accordingly, this effect directly predicts
important overestimates of total mass for the glycoprotein when
compared with pure protein standards, unless corrected for protein
partial specific volumes and solute densities. Applying these factors
to the only other published data on Tac sedimentation(35) , an
s
value of 3.01 ± 0.22 is derived
relative to the protein standards in that study. Within the
uncertainties of converting data from nonanalytical methods, this
normalized value reasonably approximates our determination of 2.73
± 0.02 for the s
for Tac monomer.
Having demonstrated that Tac is a monomer, three possibilities were
considered to explain its anomalous behavior on HPLC (accelerated
elution time) and its salt dependence: 1) unusual conformation or large
partial specific volume that is salt-sensitive, 2) less than usual
hydrophobic interaction with the matrix, or 3) a protein surface charge
distribution that inhibits matrix penetration. The first of these was
addressed by ultracentrifugation and circular dichroism. Analytical
centrifugation showed a small, rather than large, partial specific
volume. The derived frictional ratio (f/f) of 1.65 suggests an elongated shape
for the molecule, which would make it more readily excluded than
globular molecules at base line, but the effect should be modest and
would not explain a salt dependence. The secondary structure inferred
by circular dichroism was unusual in having little
- and
-structure, but there were no conformational changes by this
technique under extreme salt conditions to correlate with the salt
sensitivity of Tac's chromatographic behavior. Thus, partial
specific volume and shape changes seemed not to be primary factors in
the unusual chromatographic behavior. The next of the possibilities, a decreased hydrophobic interaction of Tac with the matrix,
could explain a base-line elution that is faster for mass than the
standards. However, the observed greater mobility suppression by salt (i.e. increase to ``normal'' column interaction) for
Tac relative to other proteins of putative higher hydrophobicity would
suggest higher hydrophobicity of Tac instead, thus yielding an
internal contradiction to this model. Therefore, we favor the final
explanation, an unusual surface charge on Tac that inhibits matrix
penetration, accelerates elution, and leads to overestimates of protein
size. This has some plausibility given 1) the extremely acidic nature
of the Tac
glycoprotein (pI 3.5) (
)(13) and 2) the presence of negative charges on
the chromatographic matrix, which behaves as a weak cation exchanger in
low salt. (
)The role of the higher salt is to provide charge
masking between the matrix and protein. Also compatible with this model
is our observation that the mobility acceleration was less marked with
a larger pore size using the same matrix (see ``Results'').
We note that ovalbumin also had a disproportionate mobility
acceleration in low salt (Fig. 3), and it is the most acidic (pI
4.8) of the marker proteins studied. Similar phenomena with acidic
proteins have been documented previously, explained as ``ion
exclusion'' due to repulsive interactions with fixed negative
charges on silica and organic gels used for chromatographic
media(38) . The present instance is unusual in that the effect
was detectable in moderate to high salt concentrations, which we would
explain by the exceptionally acidic character of the Tac protein.
One comment on the Tac structure may be warranted that derives from
these studies. The protein is unusual in its relative lack of ordered
secondary structure (20%), with CD suggesting no
-helices and
small amounts of
-sheet and
-turn. Additionally, the f/f
(1.65) from the velocity
sedimentation studies suggests a relatively extended molecule, although
it is not possible to assign an axial ratio in the absence of protein
hydration data. The presence of five disulfide bridges in this small
molecule is compatible with a highly constrained folding that could
maintain a nonglobular form and suppress other secondary structure
features. This is given plausibility by data with the homologous
2-glycoprotein, complement factor H, which shares some CD and
structural characteristics of Tac. Factor H undergoes major
conformation changes after disulfide reduction, assuming a more typical
structure that now includes significant
- and
-structure(30) . We did not repeat this evaluation with
Tac, but the analogy with factor H suggests the importance of the
disulfides in its overall structure; other mutational analysis data
show abrogation of IL2-binding ability with disturbance of any of the
Tac disulfides(39) . Although important efforts have been made
to study Tac by x-ray diffraction, the crystals to date have not
allowed sufficient resolution(13) . The ultimate x-ray solution
of this unusual and biologically important molecule will likely yield
important protein structural information for general application.
In
summary, prior conclusions, including our own, of a dimeric or higher
form for Tac on sieving chromatography (32, 33, 34, 35) or on sucrose
gradient velocity sedimentation (35) may be explained by the
highly acidic character of the glycoprotein monomer, which fosters
ionic exclusion effects on matrices with trace fixed anions, and a high
molecular density (low partial specific volume) that accelerates
sedimentation relative to pure protein standards in density media. The
conclusion of a monomer form for Tac accordingly would limit
alternative models for IL2 interaction that depend upon preformed
dimeric or higher states of this
molecule(15, 35) .
The Tac glycoprotein
(Tac) used in these experiments and by others (9, 10, 11, 13, 35) is a
recombinant, truncated form released from mammalian cell transfectants.
By criteria of IL2 binding and epitope preservation, this molecule is
indistinguishable from the native
product(7, 9, 35) . By size, it is between
native transmembrane Tac
and the shed form of soluble
Tac
released from cells in vitro and in
vivo. Although post-translational differences in carbohydrate
addition are discernible between different human malignant T cell
lines, there is otherwise a dominant common isoelectric and
electrophoretic pattern with which this product appears to
conform(7, 9, 13, 21, 22, 35) .
In addition, the relative size of complexes in our antigen-antibody
titration corresponds to patterns in vivo in humans involving
native soluble Tac. Therefore, we believe that our results are
representative of the biologic behavior of Tac and that Tac is
essentially only monomer under all conditions studied in vitro and under physiologic conditions in vivo.