ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 342, No. 2, June 15, pp. 298–305, 1997 Article No. BB979996 Putative Folding Pathway of Insulin-like Growth Factor-I Robert D. Rosenfeld, 1 James A. Miller, Linda O. Narhi, Nessa Hawkins, Viswanatham Katta, Scott Lauren, Michael A. Weiss, and Tsutomu Arakawa Amgen Inc., Amgen Center, Thousand Oaks, California 91320 Received January 16, 1997, and in revised form March 18, 1997 the precursors of the corresponding fully oxidized forms, but their conversions are not energetically a Insulin-like growth factor-1 (IGF-I) has three disul- favorable process.  1997 Academic Press fide bonds and refolding of the fully reduced molecule generates varying ratios of correctly (PII) and incor- rectly (PI) folded forms via several intermediates. All of the intermediates have the disulfide bond between Insulin-like growth factors (IGFs) 2 or somatomed- Cys18 and 61 formed, indicating that formation of this ins are a family of growth hormone-dependent serum disulfide is the first step in refolding. In order to fur- growth factors which circulate bound in high-molecu- ther understand the refolding pathway, two interme- lar-weight complexes (1). There are two types of diate forms, PIII with the additional disulfide Cys(6/47) IGFs, IGF-I and IGF-II, which share a number of formed and PIII a with Cys(6/48) formed, were isolated. The oxidation of the remaining Cys48 and 52 in PIII biological properties as described below (2–7). They and Cys47 and 52 in PIII a would lead to PI and PII, have been shown to regulate skeletal growth in hy- respectively; however, air oxidation of these resulted pophysectomized rats. In addition to these effects on in a rapid reshuffling into other intermediates as well growth in vivo, these factors have a variety of actions as folding into the fully oxidized forms, and this oc- in vitro, including the stimulation of proteoglycan curred whether refolding was started with PIII or sulfation in cartilage tissue, acute insulin-like action PIII a . When oxidation occurred in the presence of an in adipose and muscle tissues, and the stimulation excess of oxidized glutathione, the predominant spe- of mitogenic proliferation activity of a variety of cul- cies generated were various glutathione adducts re- tured cells. gardless of the initial intermediate form, indicating Recombinant IGF-I has been expressed, as either that formation of the last disulfide bond is not a favor- a native or a modified form, in several different host – able process relative to disulfide exchange when ex- vector systems, including yeast and Escherichia coli cess disulfides from oxidized glutathione are present. (4, 8 – 16). Analysis of the final products indicated the Interestingly, if 80 mM copper sulfate, an oxidant, is coexistence of two distinct conformations; the native, added to the refolding buffer, PIII resulted in forma- biologically active form (PII) and the much less active tion of the PI form alone, whereas PIII a resulted in the nonnative state (PI). The nonnative state contains PII form alone. It was concluded from these results mismatched disulfide pairs, occurring with almost that the intermediate forms of IGF-1 can rapidly re- the same frequency as the native state (10, 12, 13, shuffle between different disulfide structures, and that 17). Folding of IGF-I, either by air-oxidation of the formation of the last disulfide bond is not as favorable fully reduced protein or by reductant-catalyzed disul- a process as the conversion to other intermediates. fide exchange of the oxidized protein, generates vari- The oxidation to form the last disulfide bond in PIII ous intermediate species which are transiently pres- or PIII a is accelerated and hence the interconversion to other intermediates is kinetically minimized only ent through the course of the reaction (13, 17). These in the presence of copper sulfate. It appears, therefore, intermediates include a species with one single disul- that the two intermediate forms, PIII and PIII a , are 2 Abbreviations used: IGF, insulin-like growth factor; rp, reverse 1 To whom correspondence should be addressed at Amgen Inc., phase; HPLC, high-performance liquid chromatography; TFA, tri- fluoroacetic acid; MALDI, matrix-assisted laser desorption ioniza- Amgen Center, 14-2-A-223, Thousand Oaks, CA 91320-1789. Fax: 805-499-7464. tion; Ches, 2-(cyclohexylamino) ethanesulfonic acid. 298 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. AID ABB 9996 / 6b37$$$521 05-17-97 01:17:56 arcal