DISULFIDE BOND ASSIGNMENT IN HUMAN INTERLEUKIN-7 BY MATRIX-ASSISTED-LASER-DESORPTION IONIZATION MASS-SPECTROSCOPY AND SITE-DIRECTED CYSTEINE TO SERINE MUTATIONAL ANALYSIS/

Interleukin-7 (IL-7) is a proteinaceous biological response modifier t hat has a bioactive tertiary structure dependent on disulfide bond for mation, Disulfide bond assignments in human (h)IL-7 are based upon the results of matrix-assisted laser desorption/ionization (MALDI) mass s pectroscopy and Cys to Ser mutational analyses. A gene encoding the hI L-7 was synthesized incorporating Escherichia coli codon usage bias an d was used to express biologically active protein as determined by sti mulation of precursor B-cell proliferation. MALDI mass spectroscopic a nalysis of trypsin-digested hIL-7 was performed and compared with the anticipated results of a simulated tryptic digestion, Many of the anti cipated hIL-7 tryptic fragments were detected including one with a mol ecular mass equivalent to the sum of two polypeptides linked through a disulfide bond formed from Cys residues (Cys(3) and Cys(142)). Subseq uently, Cys to Ser substitution mutational analyses were performed, A hIL-7 variant with all six Cys substituted with Ser was found to be bi ologically inactive (EC50 > 1 x 10(-7) M). In contrast, a family of si ngle disulfide bond-forming variants of hIL-7 were constructed by rein troducing Cys pairs (Cys(3)-Cys(142), Cys(35)-Cys(130), and Cys(48)-Cy s(93)), 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 disulf ide bond-forming mutants of hIL-7, the ability to stimulate cell proli feration was abolished in the presence of 2 mM dithiothreitol, The res ults presented strongly suggest that only a single disulfide bond is r equired for hIL-7 to form a tertiary structure capable of stimulating precursor B-cell proliferation.