©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Biophysical Characterization of a Recombinant Soluble Interleukin 2 Receptor (Tac)
EVIDENCE FOR A MONOMERIC STRUCTURE (*)

(Received for publication, September 5, 1995; and in revised form, January 26, 1996)

R. P. Junghans (1)(§) A. L. Stone (2) M. S. Lewis (3)

From the  (1)Division of Hematology-Oncology, Harvard Medical School, Biotherapeutics Development Lab, New England Deaconess Hospital, Boston, Massachusetts 02215, the (2)Laboratory of Developmental and Molecular Immunity, NICHD, National Institutes of Health, Bethesda, Maryland 20892, and the (3)Biomedical Engineering and Instrumentation Program, National Center for Research Resources, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha-chain of the receptor (Tac; IL2Ralpha; 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.


INTRODUCTION

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. (^1)A second chain of moderate affinity for IL2 was described. (See Refs. 1 and 2 for review.) A third chain of the receptor, ((c)), 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 beta 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 IL1Ralpha(5) ) or for beta-chain collaboration (as in IL6Ralpha(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 approx 1 h) (8) not expected of a larger molecule and a chromatographic behavior of in vivo- and in vitro-generated Tacbulletanti-Tac complexes (^2)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.


MATERIALS AND METHODS

Soluble Tac

Recombinant human soluble Tac protein (Tac) was purified from the supernatant of mammalian cell transfectants by an anti-Tac affinity column (gift of Dr. John Hakimi, Hoffmann-LaRoche)(9) . Eluted material was >95% monomer on SDS-PAGE (Fig. 1B). Extensive peptide mapping and amino acid analysis of this preparation confirm the intactness of NH(2) and COOH termini(10, 11) . An extinction coefficient of 1.17 A was derived by measurement of optical absorption of a solution quantitated by amino acid analysis; (^3)that calculated from the Wetlaufer procedure as modified by Perkins (12) was 1.13 A. The measured value was used in these studies. The ``anchor-minus'' recombinant protein includes all but four amino acids of the transmembrane domain with a G224P substitution at the carboxyl terminus of the engineered molecule and contains all of the potential extracellular glycosylation sites of the full-length molecule. It is intermediate in size between the naturally occurring soluble Tac (Tac) and the full-length transmembrane form (Tac) and retains their ligand and antibody binding characteristics and low isoelectric point(9, 13) . Where the context is clear, we will refer to the recombinant Tac used in this study simply as Tac.


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 Tacbulletanti-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. (times is the back face of the molecule; other configurations meeting these criteria are possible.) B, HPLC profile of Tacbulletanti-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 3times molar excess of binding sites, iii) 10 µg of anti-Tac + 10 µg of Tac, antigen in 3times 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.



Chromatography

Chromatography was performed on a Waters HPLC unit with TosoHaas TSK-250 and TSK-400 silica sieving columns in buffers described under ``Results'' with a flow rate of 1 or 2 ml/min. The higher flow rate was used when analyzing antigen-antibody complexes to minimize dissociation during the run, which should be minimal given the slow dissociation rate (0.007 min measured at 37 °C(14) ). Column elution times under this condition were less than 4 min. Protein molecular weight markers were thyroglobulin (670,000), IgG (155,000), ovalbumin (44,000), myoglobin (17,500), cyanocobalamin (1,350), as a mixture (Bio-Rad) or as separate components.

Analytical Ultracentrifugation

Equilibrium ultracentrifugation was conducted using a Beckman model E analytical ultracentrifuge equipped with a scanner absorption optical system to determine the molecular mass and partial specific volume of Tac. This procedure is detailed in a separate report. (^4)The protein was in phosphate-buffered saline, pH 7.4. Some samples had the NaCl concentration raised by an additional 0.35 M or 1.00 M. Initial protein concentrations were 0.12, 0.20, and 0.28 absorbance units at 280 nm (0.10, 0.17, and 0.24 mg of protein/ml). The ultracentrifuge was run at 20,000 rpm and 20.0 °C for 71 h, by which time the concentration distributions of all three cells were invariate for 23 h.

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(p) is the concentration in the plateau region, c(b) 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(m) is the radial position of the meniscus, and ^2t 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) .

Derivation of Molecular Mass by Ultracentrifugation

Tac was calculated to have a protein mass of 25,253 daltons(9, 20) . The protein is substituted with N- and O-linked sugars and, from pI shift analysis, contains approximately 10 sialic acid residues(21, 22, 23) . In a separate article,^4 we demonstrate a nondenaturing method to derive glycoprotein molecular weights when only the mass of the protein component is known. An abbreviated description is presented here.

The molecular mass of the glycoprotein is M = M(G) + M(P), where M(G) is the molecular mass of the carbohydrate portion of the molecule and M(P) 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(P)) 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.

Stokes' Radius Calculation

The Stokes' radius is calculated from the diffusion coefficient, using the equation,

where the diffusion coefficient is derived from the Svedberg equation,

and where the mass and partial specific volume are from our previously derived values.^4

Circular Dichroism and Estimation of Secondary Structures

Solutions of Tac containing four different concentrations of NaCl in 0.4 mM Tris-HCl buffer, pH 7.2 (B), were prepared by dialysis against solvent for 24 h and measured by UV absorption against the measured value of A = 1.17 (above). Experimental solutions (all in B) were as follows: 1) 0.094 mg/ml, 0.02 M NaCl; 2) 0.233 mg/ml, 0 M NaCl; 3) 0.380 mg/ml, 0.1 M NaCl; 4) 0.540 mg/ml, 2 M NaCl. Higher concentrations were used in higher salt to compensate for decreased signal:noise ratio due to the increase in absorption of light by the solvent. Spectra of Tac were obtained at 24 ± 1 °C using an OLIS-enhanced Cary model 60 automated spectropolarimeter (On-Line Instrument Systems, Inc., Jefferson, GA), equipped with a Hinds photoelastic modulator (Hinds International, Inc., Hillsboro, OR), end-on photomultiplier and a lock-in amplifier having a high energy reserve for small signal detection (Applied Research, Princeton, NJ). Values are reported as an average of three repetitive scans (1.5 nm/min in the range 260-206 nm and 0.3 nm/min below 205 nm). Tac solutions were analyzed at an optical pathlength of 0.5 or 0.1 mm in the ultraviolet range from 260 to 188 nm except for the 2 M NaCl sample, which could only be obtained from 260 to 207 nm. Spectra were corrected for the respective dialysate base-line readings, and molar residue ellipticities ((r)) were based on the mean amino acid residue weight of 113.4 as calculated from the primary sequence(9, 20) ; i.e. [(r)] = 10 times degrees ellipticity/(molar residue concentration times light path in decimeters).

Estimation of the secondary conformation of Tac used a polypeptide model approach(24) . Standard CD spectra for alpha-helix, antiparallel beta-structure, class B type II beta-turns, and extended nonordered structures were used as described(25, 26) . (For a review of secondary structure fitting, see (27) .)


RESULTS

Size Exclusion Chromatography Demonstrates a Large M for Tac

The soluble Tac used in these studies was a recombinant protein (Tac) generated from mammalian cell transfectants (see ``Materials and Methods''). It is intermediate in size between the naturally occurring soluble Tac (Tac) and the full-length transmembrane form (Tac) and retains their ligand and antibody binding characteristics and low isoelectric point(9, 13) . Initial experiments with Tac on an HPLC sieving column (TSK-250) yielded an apparent molecular mass of 180 kDa (Fig. 1A), suggesting a tetrameric structure relative to the nominal 40-45-kDa size on SDS-PAGE reducing (not shown) and nonreducing gels (Fig. 1B) cited for this molecule(9, 13) . Several published studies reported a noncovalent dimeric form for Tac on chromatographic media (see below), but none to our knowledge suggested a native size greater than that of IgG. Either this high molecular weight position for Tac was an artifact of the analysis method or it indicated a novel form for the protein. Yet, HPLC sizing patterns of radiolabeled anti-Tac antibody in humans with high in vivo soluble Tac levels were compatible with a single binding epitope per Tac molecule (not shown),^2 suggesting in contrast that Tac was monomeric in form. We attempted to duplicate these in vivo observations with an in vitro reconstruction to investigate the interaction of Tac with anti-Tac antibody on this separation medium.

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.

Size Analysis by Analytical Ultracentrifugation Shows Monomer under All Conditions

We then employed analytical ultracentrifugation as an independent approach to assess the M(r) of soluble Tac under physiologic salt and nondenaturing conditions, which required development of new procedures.^4 This approach permits direct measurement of molecular mass and oligomerization constants by analysis of equilibrium sedimentation profiles. These studies all showed a homogeneous, noninteracting, well behaved species (Fig. 5). A mass of 34,500 ± 430 was estimated under a formula that incorporates assumptions of ranges of carbohydrate composition and the known amino acid sequence of the protein. Paralleling the chromatography studies, the mass was estimated under varying salt conditions but, in contrast, yielded virtually identical values in 0.15, 0.50, and 1.15 M NaCl (Table 1). There is accordingly no molecular mass change on ultracentrifugation that parallels the salt-dependent changes in M on HPLC seen in Fig. 4.


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 (box) and 1.15 M NaCl (circle). 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 (^2t = 2.940 times 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 (circle) and 0.3 mg/ml (box).





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 times 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^0, extrapolated to zero concentration. (^5)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^0 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^3/g for the partial specific volume from the equilibrium data above, a value for f/f(0) 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(0) 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.

Circular Dichroic Analysis Demonstrates No Salt-dependent Conformation Changes

If Tac exhibited unusual base-line structural characteristics in conjunction with major salt-dependent secondary structure changes, it might suggest a mechanism for the unusual chromatographic behavior other than a change in mass of the molecule. To this end, CD spectroscopy of Tac was undertaken in the low UV spectral region. Fig. 6A shows the CD spectrum from 250 to 188 nm of Tac in 0.4 mM Tris buffer, pH 7.2, containing 0.1 M NaCl. Because of a sharp decrease in signal:noise ratio below 190 nm, the data at 188 nm are a qualitative estimate of that point. The spectra of Tac in 0.00 and 0.02 M NaCl were similar (not shown). Conformational content was computed on the basis of polypeptide models from 250 to 190 or 188 nm. This analysis overall indicated the following: that the ordered conformation was 0% alpha-helix, 8-19% beta-structure, and 7% beta-turns and that the remainder (80%) was extended nonordered structure. The polypeptide model has been applied appropriately for proteins having a small degree of secondary structure(26) . CD models based on related proteins of known three-dimensional structure might be more appropriate for a given protein, but there are no proteins in the crystallographic data base that are comparable with Tac. The percentage deviations from the best-fit for the Tac conformations were <10%, except for the beta-structure, which was 15%. The double negative peak at 190/200 nm may be due to contributions of disulfide bonds and/or glycosidic linkages that absorb in this region.


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 beta(2)-glycoprotein(29) , human complement factor H, was similar to that of Fig. 6A, including the relative lack of computed alpha- or beta-structure(30) . Although their biological functions are unrelated and their molecular sizes are different (factor H approx 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.




DISCUSSION

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) , (^6)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 <10^3M to avoid detection in our analysis. This effectively precludes noncovalent dimerization under solution conditions in vivo, for which the upper limit of normal is <10M(36) , and the highest documented serum level in any individual was still only 10M(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 approx 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(0)) 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 alpha- and beta-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) (^7)(13) and 2) the presence of negative charges on the chromatographic matrix, which behaves as a weak cation exchanger in low salt. (^8)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 alpha-helices and small amounts of beta-sheet and beta-turn. Additionally, the f/f(0) (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 beta2-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 alpha- and beta-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) .^4

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.


FOOTNOTES

*
This work was supported in part by grants from the Milheim Foundation for Cancer Research (to R. P. J.) and a Clinical Oncology Career Development Award from the American Cancer Society (to R. P. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: New England Deaconess Hospital, 99 Brookline Ave., Room 301, Boston, MA 02215. Tel.: 617-632-0943; Fax: 617-632-0998.

(^1)
The abbreviations used are: IL, interleukin; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; SOS, stoichiometry-ordered size analysis.

(^2)
R. P. Junghans, J. A. Carrisquillo, T. A. Waldmann, manuscript in preparation.

(^3)
Y. Pan and P. Bailan, personal communication.

(^4)
M. S. Lewis and R. P. Junghans, submitted for publication.

(^5)
Given that the individual s values lie within 1 S.E. of each other, an assumption of a concentration dependence is tenuous.

(^6)
The single report of higher fractions of dimer, cell-associated Tac may be due to oxidative conditions of the iodination procedure rather than representing a native cellular component(31) . It is likely that the intramembrane cysteine is not accessed and blocked by N-ethylmaleimide, whereas the oxidative radicals will be expected to penetrate the intramembrane phase where the cysteine formation likely takes place (e.g. as with T cell receptor chain dimerization).

(^7)
This is lower even than that of the complete transmembrane Tac (pI 4.2-4.5) (9, 21) due to removal of the several cytoplasmic basic residues in the soluble form.

(^8)
TosoHaas, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. John Hakimi (Hoffmann-La Roche, Nutley, NJ) for supplying affinity-purified soluble human Tac protein. R. P. J. is grateful to Dr. Thomas A. Waldmann (NCI, National Institutes of Health) for support during a portion of this work.


REFERENCES

  1. Smith, K. A. (1989) Annu. Rev. Cell Biol. 5, 397-425 [CrossRef]
  2. Waldmann, T. A., Pastan, I. H., Gansow, O. A., and Junghans, R. P. (1992) Ann. Intern. Med. 116, 148-160 [Medline] [Order article via Infotrieve]
  3. Takeshita, T., Asao, H., Ohtani, K., Ishii, N., Kumaki, S., Tanaka, N., Munakata, H., Nakamura, M., and Sugamura, K. (1992) Science 257, 379-382 [Medline] [Order article via Infotrieve]
  4. Dower, S. K., Smith, C. A., and Park, L. S. (1990) J. Clin. Immunol. 10, 289-300 [Medline] [Order article via Infotrieve]
  5. Fanslow, W. C., Sims, J. E., Sassenfeld, H., Morrissey, M. H., Gillis, S., Dower, S., and Widmer, M. B. (1990) Science 248, 739-742 [Medline] [Order article via Infotrieve]
  6. Taga, T., Hibi, M., Hirata, Y., Yamasaki, K., Yasukawa, K., Matsuda, T., Hirano, T., and Kishimoto, T. (1989) Cell 58, 573-581 [Medline] [Order article via Infotrieve]
  7. Robb, R. J., and Kutny, R. M. (1987) J. Immunol. 139, 855-862 [Abstract/Free Full Text]
  8. Junghans, R. P., and Waldmann, T. A. (1996) J. Exp. Med. 183, in press
  9. Hakimi, J., Seals, C., Anderson, L. E., Podlaski, F. J., Lin, P., Danho, W,, Jenson, J. C., Perkins, A., Donadio, P. E., Familletti, P. C., Pan, Y. C. E., Tsien, W. H., Chizzonite, R. A., Casabo, L., Nelson, D. L., and Cullen, B. R. (1987) J. Biol. Chem. 262, 17336-17341 [Abstract/Free Full Text]
  10. Miedel, M. E., Hulmes, J. D., Weber, D. V., Bailon, P., and Pan, Y. C. (1988) Biochem. Biophys. Res. Commun. 154, 372-379 [Medline] [Order article via Infotrieve]
  11. Miedel, M. C., Hulmes, J. D., and Pan, Y-C. E. (1989) J. Biol. Chem. 264, 21097-21105 [Abstract/Free Full Text]
  12. Perkins, S. J. (1986) Eur. J. Biochem. 157, 169-180 [Abstract]
  13. Lambert, G., Stura, E. A., and Wilson, I. A. (1989) J. Biol. Chem. 264, 12730-12736 [Abstract/Free Full Text]
  14. Depper, J. M., Leonard, W. J., Kronke, M., Noguchi, P. D., Cunningham, R. E., Waldmann, T. A., and Greene, W. C. (1984) J. Immunol. 133, 3054-3061 [Abstract/Free Full Text]
  15. Saragovi, H., and Malek, T. R. (1987) J. Immunol. 139, 1918-1926 [Abstract/Free Full Text]
  16. Attri, A., and Lewis, M. S. (1992) Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E., Rowe, A. J., and Horton, J. C., eds) pp. 138-146, The Royal Society of Chemistry, Cambridge, UK
  17. Svedberg, T., and Pedersen, K. O. (1940) The Ultracentrifuge , pp. 35-36, Clarendon Press, Oxford, UK
  18. Laue, T. M., Shah, B. D., Ridgeway, T. M., and Pelletier, S. L. (1992) in Analytical Ultracentrifugation in Biochemistry and Polymer Science (Harding, S. E., Rowe, A. J., and Horton, J. C., eds) pp. 90-125, The Royal Society of Chemistry, Cambridge, UK
  19. Knott, G. D. (1979) Comput. Programs Biomed. 10, 271-280 [Medline] [Order article via Infotrieve]
  20. Leonard, W. J., Depper, J. M., Kanehisa, M., Kronke, M., Peffer, N. J., Svetlik, P. B., Sullivan, M., and Greene, W. C. (1985) Science 230, 633-639 [Medline] [Order article via Infotrieve]
  21. Leonard, W. J., Depper, J. M., Robb, R. J., Waldmann, T. A., and Greene, W. C. (1983) Proc. Natl. Acad. Sci. 80, 6957-6961 [Abstract]
  22. Wano, Y., Uchiyama, T., Fukui, K., Maeda, M., Uchino, H., and Yodoi, J. (1984) J. Immunol. 132, 3005-3010 [Abstract/Free Full Text]
  23. Wano, Y., Uchiyama, T., Yodoi, J., and Uchino, H. (1985) Microbiol. Immunol. 29, 45-128
  24. Chen, Y.-H., Yang, J. T., and Chau, K. H. (1974) Biochemistry 13, 3350-3359 [Medline] [Order article via Infotrieve]
  25. Woody, R. W. (1985) in The Peptides (Udenfriend, S., ed) Vol. 7, pp. 15-114, Academic Press, Inc., Orlando, FL
  26. Stone, A. L., Park, J. Y., and Martenson, R. E. (1985) Biochemistry 24, 6666-6673 [Medline] [Order article via Infotrieve]
  27. Yang, J. T., Wu, S.-S. C., and Martinez, H. M. (1986) Methods Enzymol. 130, 208-269 [Medline] [Order article via Infotrieve]
  28. Smith, M. H. (1968) in Handbook of Biochemistry (Sober, H. A., ed) pp. C-3 to C-35, Chemical Rubber Co., Cleveland
  29. Paulson, J. C. (1990) in Proteins: Form and Function (Bradshaw, R. A., Purton, M., ed) pp. 209-218, Elsevier Trends Journals, Cambridge, UK
  30. Discipio, R. G., and Hugli, T. E. (1982) Biochim. Biophys. Acta 709, 58-64 [Medline] [Order article via Infotrieve]
  31. Kato, K., and Smith, K. A. (1987) Biochemistry 26, 5359-5364 [Medline] [Order article via Infotrieve]
  32. Manoussakis, M. N., Papadopoulos, G. K., Drosos, A. A., and Moutsopoulos, H. M. (1989) Clin. Immunol. 50, 321-322
  33. Symons, J. A., Wood, N. C., Di Giovine, F. S., and Duff, G. W. (1988) J. Immunol. 141, 2612-2618 [Abstract/Free Full Text]
  34. Miossec, P., Elhamiani, M., Edmonds-Alt, X., Sany, J., and Hirn, M. (1990) Arthritis Rheum. 33, 1688-1694 [Medline] [Order article via Infotrieve]
  35. Jacques, Y., Le Mauff, B., Godard, A., Naulet, J., Concino, M., March, H., Ip, S., and Soulillou, J. P. (1990) J. Biol. Chem. 265, 20252-20258 [Abstract/Free Full Text]
  36. Nelson, D. L. (1986) Annu. Intern. Med. 105, 560-572 [Medline] [Order article via Infotrieve]
  37. Waldmann, T. A., White, J., Goldman, C. K., Top, L., Grant, A., Bamford, R., Roessler, E., Horak, I., Zaknoen, S., Kasten-Sportes, C., England, R., Horak, E., Mishra, B., Dipre, M., Hale, P., Fleisher, T., Junghans, R. P., Jaffe, E. S., and Nelson, D. L. (1993) Blood 82, 1701-1712 [Abstract]
  38. Barth, H. G. (1980) J. Chromatogr. Sci. 18, 409-429
  39. Rusk, C. M., Neeper, M. P., Kuo, L.-M., Kutny, R. M., and Robb, R. J. (1988) J. Immunol. 140, 2249-2259 [Abstract/Free Full Text]

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