title: Characterization of truncated insulin-like growth factor-binding protein-2 in human milk.
abstract: 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 Mr 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 Mr, 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 Mr. 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.
authors.affiliation: Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St. Leonards, New South Wales, Australia.
authors.forename: P J
authors.initials: PJ
authors.lastname: Ho
authors.forename: R C
authors.initials: RC
authors.lastname: Baxter
mesh_terms: D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000737:chemistry / Q000302:isolation & purification*; D007335:Insulin-Like Growth Factor II / Q000378:metabolism; D008895:Milk, Human / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D018971:Insulin-Like Growth Factor Binding Protein 2; D010446:Peptide Fragments; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II