Abstract
Interleukin-7 (IL-7) is a proteinaceous biological response modifier that has a bioactive tertiary structure dependent on disulfide bond formation. Disulfide bond assignments in human (h)IL-7 are based upon the results of matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and Cys to Ser mutational analyses. A gene encoding the hIL-7 was synthesized incorporating Escherichia coli codon usage bias and was used to express biologically active protein as determined by stimulation of precursor B-cell proliferation. MALDI mass spectroscopic analysis of trypsin-digested hIL-7 was performed and compared with the anticipated results of a simulated tryptic digestion. Many of the anticipated hIL-7 tryptic fragments were detected including one with a molecular mass equivalent to the sum of two polypeptides linked through a disulfide bond formed from Cys residues (Cys3 and Cys142). Subsequently, Cys to Ser substitution mutational analyses were performed. A hIL-7 variant with all six Cys substituted with Ser was found to be biologically inactive (EC50 > 1 x 10(-7) M). In contrast, a family of single disulfide bond-forming variants of hIL-7 were constructed by reintroducing Cys pairs (Cys3-Cys142, Cys35-Cys130, and Cys48-Cys93), and each could stimulate cell proliferation with an EC50 of 4 x 10(-9), 2 x 10(-8), and 2 x 10(-9) M, respectively. In single disulfide bond-forming mutants of hIL-7, the ability to stimulate cell proliferation was abolished in the presence of 2 mM dithiothreitol. The results presented strongly suggest that only a single disulfide bond is required for hIL-7 to form a tertiary structure capable of stimulating precursor B-cell proliferation.
Highlights
Interleukin-7 (IL-7)1 is a pleiotropic cytokine originally detected by its ability to stimulate the growth of precursor B-cells in vivo [1]
The disulfide bond assignments in human IL-7 (hIL-7) were assigned based upon the combination of matrix-assisted laser desorption/ionization (MALDI) mass spectroscopy and site-directed Cys to Ser mutational analyses
Mature hIL-7 contains six cysteine residues and has been reported to lose all biological activity when incubated in the presence of a thiol
Summary
Interleukin-7 (IL-7) is a pleiotropic cytokine originally detected by its ability to stimulate the growth of precursor B-cells in vivo [1]. The observation that IL-7 induced an increase in DNA synthesis in acute myelogenous leukemia cells suggests that the pleiotropic effect of this cytokine is not restricted to cells from the lymphoid lineage [6]. We have biophysically and genetically mapped the disulfide bonds in hIL-7 using a combination of MALDI mass spectroscopy and site-directed cysteine to serine mutational analyses. MALDI mass spectroscopy performed on a tryptic digestion of hIL-7 under nonreducing conditions reveals a disulfide bond between cysteine residues 3 and 142. The results presented demonstrate that cysteine residue pairs (Cys3-Cys142, Cys48-Cys, and Cys35-Cys130) participate in disulfide bond formation. We further demonstrate that only a single disulfide bond between one of these pairs of cysteine residues is sufficient to retain a biologically active conformation
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