Abstract
A soluble form of the insulin-like growth factor II/mannose 6-phosphate receptor (sIGF-II/MPR) is present in fetal bovine serum and carries mature 7.5-kDa insulin-like growth factor II (IGF-II) and at least 12 different high molecular weight (Mr) IGF-II isoforms (Valenzano, K. J., Remmler, J., and Lobel, P. (1995) J. Biol. Chem. 270, 16441-16448). In this study, we used gel filtration and anion exchange chromatographies to resolve the isoforms into eight fractions that were characterized with respect to their biochemical, biophysical, and biological properties. Each fraction contained one to three major protein species with apparent sizes ranging from 11 to 17 kDa by SDS-polyacrylamide gel electrophoresis. The 11-kDa species contains no post-translational modifications and consists of an extended IGF-II backbone terminating at Gly-87. The remaining high Mr IGF-II isoforms are also composed of an 87-amino acid IGF-II peptide backbone but contain increasing amounts of sialated, O-linked sugars. Plasmon resonance spectroscopy experiments revealed that all the high Mr isoforms and mature 7.5-kDa IGF-II bound to immobilized recombinant soluble human IGF-I receptor, recombinant human IGF-binding protein 1, and sIGF-II/MPR with similar kinetics. In addition, radiolabeled tracer experiments demonstrated that both mature and high Mr IGF-II isoforms have similar binding profiles in fetal bovine serum and have similar affinities for IGF-II-binding proteins secreted from human fibroblasts. Finally, the biological activity of high Mr IGF-II was shown to be similar to or slightly better than mature IGF-II in stimulating amino acid uptake in fibroblasts and in inducing myoblast differentiation.
Highlights
** To whom correspondence should be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854
Preliminary analysis of the unfractionated high molecular weight (Mr) insulin-like growth factor II (IGF-II) pool isolated from fetal bovine serum revealed that it contained at least 12 different IGF-II isoforms and that all species contained an identical peptide backbone extended beyond the COOH terminus of mature 7.5-kDa IGF-II [14]
The high Mr IGF-II pool was fractionated into nine distinct fractions to facilitate analysis
Summary
** To whom correspondence should be addressed: Center for Advanced Biotechnology and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. A soluble form of the IGF-II/MPR (sIGF-II/MPR) has been detected in serum and urine (9 –13). This protein retains IGF-II and Man-6-P binding activities and is abundant (ϳ5 g/ml) in fetal bovine serum (FBS) [14]. Complex formation with the binding proteins has been shown to both inhibit and potentiate IGF-II’s interactions with its receptors as well as increase the serum half-life of the ligand (for review, see Ref. 8). 7.5-kDa IGF-II; rhIGF-IIE88, rhIGF-II extended to Lys-88; rhIGF-I, recombinant human insulin-like growth factor I; sIGF-IR, soluble IGF-I receptor; IGFBP-1, insulin-like growth factor-binding protein 1; FBS, fetal bovine serum; BSA, bovine serum albumin; HBS, HEPES-buffered saline; PBS, phosphate-buffered saline; AEX, anion exchange; AIB, ␣-aminoisobutyric acid; MES, 2-(N-morpholino)ethanesulfonic acid; bisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
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