Insulin and IGF binding by IGFBP-3 fragments derived from proteolysis, baculovirus expression and normal human urine

J Clin Endocrinol Metab. 1998 Apr;83(4):1392-5. doi: 10.1210/jcem.83.4.4858.

Abstract

Recombinant human IGFBP-3 was proteolysed with different concentrations of plasmin for various periods of time. The major IGFBP-3 fragment resulting from this digestion migrated at ca. 15 kDa in nonreducing SDS-PAGE. Following the identification of this fragment as an N-terminal IGFBP-3 fragment, by use of N-terminus-specific monoclonal antibody and amino acid sequence analysis, we constructed and expressed a similar fragment in a baculovirus expression system. The fragments resulting from plasmin digestion, as well as the baculovirus-expressed recombinant human IGFBP-3(1-97), retain weak IGF binding and show specific insulin binding on cross-linking and western ligand blot. RhIGFBP-3(1-97) can inhibit insulin receptor autophosphorylation in insulin receptor-overexpressing NIH 3T3 cells. Insulin and IGF binding to IGFBP-3 fragments could be further demonstrated in normal urine. These data indicate the physiological significance of IGFBP-3 fragments derived from proteolysis in vivo.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Baculoviridae / genetics*
  • Child, Preschool
  • Female
  • Fibrinolysin / metabolism*
  • Humans
  • Hydrolysis
  • Insulin / metabolism*
  • Insulin-Like Growth Factor Binding Protein 3 / chemistry*
  • Insulin-Like Growth Factor Binding Protein 3 / urine
  • Peptide Fragments / metabolism*
  • Phosphorylation
  • Protein Binding
  • Recombinant Proteins / biosynthesis
  • Reference Values
  • Somatomedins / metabolism*

Substances

  • Insulin
  • Insulin-Like Growth Factor Binding Protein 3
  • Peptide Fragments
  • Recombinant Proteins
  • Somatomedins
  • Fibrinolysin