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Biophysical characterization of a recombinant soluble interleukin 2 receptor (Tac). Evidence for a monomeric
R P Junghans1, A L Stone, M S Lewis
1Division of Hematology-Oncology, Harvard Medical School, New England Deaconess Hospital, Boston, Massachusetts 02215, USA.
Insights
The soluble interleukin 2 receptor alpha-chain (Tac) is monomeric under physiological conditions. Previous studies suggesting larger forms were artifacts due to the molecule's acidic nature and high carbohydrate content.
Area of Science:
- Immunology
- Molecular Biology
- Biochemistry
Background:
- The interleukin 2 receptor (IL2R) is crucial for T cell, B cell, and NK cell activation.
- The IL2R alpha-chain (Tac, CD25) is shed from activated cells, forming a soluble form.
- Previous studies reported conflicting native structures for soluble Tac, including monomeric, dimeric, and tetrameric forms.
Purpose of the Study:
- To definitively determine the native molecular structure of soluble Tac.
- To resolve discrepancies in previous structural assessments of soluble Tac.
- To understand the implications of Tac's structure on its biological function and clearance.
Main Methods:
- High-performance liquid chromatography (HPLC) sieving chromatography.
- Stoichiometry-ordered size (SOS) analysis of antibody-antigen complexes.
- Analytical ultracentrifugation with glycoprotein-specific analysis.
- Circular dichroism (CD) spectroscopy.
Main Results:
- HPLC suggested higher molecular weight forms, potentially tetrameric.
- SOS analysis indicated a monomeric form with a single epitope per Tac molecule.
- Analytical ultracentrifugation consistently demonstrated monomeric masses under various salt conditions.
- CD spectroscopy revealed no salt-dependent conformational changes.
- Artifacts from ionic exclusion and glycoprotein properties explained elevated HPLC apparent masses.
Conclusions:
- Soluble Tac exists as a monomer under physiological conditions.
- Previous findings of higher molecular weight forms were attributed to experimental artifacts.
- The acidic nature and high carbohydrate content of Tac influence its behavior in chromatographic and sedimentation analyses.
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; IL2R alpha; 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(r)(app) 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(r)(app) 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(r)(app) 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.
