Abstract
A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.
Highlights
A procedure that combined ion exchange, gel per- Based on the deduced sequence [1, 2], human pro-insulinmeation, and insulin-like growth factor-binding pro- like growth factor I1 (IGF-11)’is predicted to be a M, = 17,627 tein 3 (IGF-BP-3) affinity chromatography with chro- polypeptide that contains an amino-terminal sequence of67 matofocusing and reversed-phasehigh pressure liquid amino acids that is homologous to thereceptor binding B and chromatography was used to isolate high molecular A chains of insulin and theB, C, A, and D domains of IGF-I
Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent M, = 15,000IGF-I1 (PI 5.1),following its digestion with the endoprotease Lys-C, inand immunological analysis of the different forms of IGF-I1 in rat [4] and human serum [3, 5, 6] and in the conditioned media of cultured rat liver cells [7] indicated that pro-IGF-I1 is endoproteolytically processed within the E domain to yield mostly M, = 7,500mature IGF-I1and some partially processed higher M, forms
E domain extension that was described initially by Zumstein et al [6] for the CGD/Ser33pro-IGF-I1 variant. These results suggest that apparent M, = 15,000 IGF-I1 should have a M, value based on its amino acid composition of 9,804 (Table I)
Summary
Baker Inc. and acetonitrile from EM Science; Sephadex G-50 superfine, SP-SephadexC-25, the Mono-P HR 5/20 column, and pH 4.06.5 ampholytes were purchased from Pharmacia LKB Biotechnology Inc.; Spectra-Por-3 dialysis tubing was from Spectrum Medical Industries; neuraminidase (Arthrobacter) and Lys-C were from Boehringer Mannheim; endo-a-N-acetylgalactosaminidase(0-glycosidase) was obtained from Genzyme; trifluoroacetic acid, constant boiling 6 N HCI and phenylisothiocyanate were from Pierce Chemical Co.; electrophoresis reagents werefrom Bio-Rad; sequencing reagents were from AppliedBiosystems; and goat biotinylated anti-rabbit IgG and alkaline phosphatase coupled to avidin D wasfromVector Laboratories, Inc. Recombinant human IGF-binding protein 3 was a gift from BioGrowth, Inc. It was coupled to CNBr-activated Sepharose 4B according to the manufacturer's directions to make an IGFbinding protein affinity resin. All other chem- different PI values, a chromatofocusing procedure wasemployed icals were reagent-grade
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