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Characterization of Truncated Insulin-Like Growth Factor-Binding Protein-2 in Human Milk¹

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia

Address all correspondence and requests for reprints to: Dr. P. Jean Ho, Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, New South Wales 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au

Truncated forms of insulin-like growth factor (IGF)-binding protein-2 (IGFBP-2) have been purified from human milk and shown to retain partial IGF-binding activity. By affinity chromatography on agarose-IGF-I and HPLC, truncated IGFBP-2 of apparent M_r 14,000–16,000 resolved into two peaks. Both peaks bound radioiodinated IGF-II on ligand blotting. Within both peaks, two sequences were identified, starting at Gly¹⁶⁹ and Lys¹⁸¹ of hIGFBP-2 (predicted M_r, 13,786 and 12,502, respectively, if both extend to Gln²⁸⁹). Mass spectrometry of a fraction predominantly containing Gly¹⁶⁹ peptides yielded two major species, 13,840 and 13,425 M_r. Prolonged incubation of radioiodinated recombinant human (rh) IGFBP-2 with human milk failed to reveal any degradation, suggesting the formation of the fragments within the mammary gland. By solution binding assay, truncated IGFBP-2 showed less than 10% binding of [¹²⁵I]IGF-I and 25% binding of [¹²⁵I]IGF-II at pH 7.0 compared with rhIGFBP-2. No binding activity was seen at pH 4.0, in contrast to intact IGFBP-2, which showed peak binding from pH 4.0 to at least pH 9.0. The IGF-II association constant for truncated IGFBP-2 (6.5 nM^-1) was 10-fold lower than that for intact IGFBP-2 (58 nM^-1). Des(1–6)-IGF-II was totally inactive in displacing IGF-II tracer from the IGFBP-2 fragment, but displaced tracer from rhIGFBP-2 with 10% the activity of IGF-II. Thus, the amino-terminal hexapeptide of IGF-II is required for interaction with the carboxy-terminal domain of IGFBP-2. The presence of active IGFBP-2 fragments in milk suggests a role for milk IGFBP-2 in modifying IGF activity in the neonatal gut.

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