Interaction of Insulin-like Growth Factor II (IGF-II) with Multiple Plasma Proteins: HIGH AFFINITY BINDING OF PLASMINOGEN TO IGF-II AND IGF-BINDING PROTEIN-3

Sandra Oesterreicher,Werner F Blum,Bernhard Schmidt,Thomas Braulke,Bernd Kübler

doi:10.1074/jbc.m411754200

Abstract

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of > or =150 kDa. To investigate molecular interactions between proteins involved in IGF.IGFBP complexes, Cohn fraction IV of human plasma was subjected to IGF-II affinity chromatography followed by reversed-phase high pressure liquid chromatography and analysis of bound proteins. Mass spectrometry and Western blotting revealed the presence of IGFBP-3, IGFBP-5, transferrin, plasminogen, prekallikrein, antithrombin III, and the soluble IGF-II/mannose 6-phosphate receptor in the eluate. Furthermore, an IGFBP-3 protease cleaving also IGFBP-2 but not IGFBP-4 was co-purified from the IGF-II column. Inhibitor studies and IGFBP-3 zymography have demonstrated that the 92-kDa IGFBP-3 protease belongs to the class of serine-dependent proteases. IGF-II ligand blotting and surface plasmon resonance spectrometry have been used to identify plasminogen as a novel high affinity IGF-II-binding protein capable of binding to IGFBP-3 with 50-fold higher affinity than transferrin. In combination with transferrin, the overall binding constant of plasminogen/transferrin for IGF-II was reduced 7-fold. Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of > or =440 kDa. The present data indicate that IGFs, low and high affinity IGFBPs, several IGFBP-associated proteins, and IGFBP proteases can interact, which may result in the formation of binary, ternary, and higher molecular weight complexes capable of modulating IGF binding properties and the stability of IGFBPs.

Highlights

In the circulation, most of the insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases are bound in high molecular mass complexes of >150 kDa
Size exclusion chromatography of the IGF-II matrix eluate revealed that transferrin, plasminogen, and the IGFBP-3 protease are present in different high molecular mass complexes of >440 kDa
To examine whether IGFBP proteases were co-purified with IGFBPs by IGF-II affinity chromatography, the eluted protein fraction was tested for [125I]IGFBP-3 protease activity using nonglycosylated recombinant [125I]IGFBP-3 as substrate

Summary

EXPERIMENTAL PROCEDURES

Materials—IGFBP-4 and IGFBP-5 were kindly provided by Dr J. Purification of Serum Proteins Eluted from IGF-II Affinity Columns—Cohn fraction IV of human plasma was fractionated as described previously [28]. The elution of IGFBP-3 was monitored by a competitive binding protein assay as described previously [28]. Radiolabeled IGFBP-3 zymography and ligand blotting of RP-HPLC fractions (12 ␮l) using monobiotinylated IGF-II were carried out as described previously [12, 29]. Surface Plasmon Resonance Interaction Analysis—Interaction measurements were carried out as described previously [32] using IGF-IIand IGFBP-3-coupled CM5 sensor chips in a BIAcore 3000 (Amersham Biosciences). To analyze the formation of protein-transferrin complexes in vitro, transferrin (200 ng) was incubated with or without single or multiple recombinant proteins (each at 200 ng), identified in the eluate of the IGF-II affinity matrix, in phosphate-buffered saline in a total volume of 20 ␮l. The reaction was terminated by the addition of 50 mM Tris/HCl, pH 7.4, for 15 min followed by transferrin immunoblotting

RESULTS

Amino acids

KD nM

DISCUSSION