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C. Guidry, J. L. King; Characterization of Vitreous Insulin-Like Growth Factor Binding Proteins. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5359.
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Previous studies from this laboratory revealed that insulin-like growth factor (IGF) biological activities increase in Proliferative Diabetic Retinopathy and that this activity is normally attenuated by vitreous insulin-like growth factor binding proteins (IGFBPs). The intent of this study was to identify and characterize the IGFBP species involved.
Human and porcine vitreous, human and porcine plasma, recombinant IGFBP-2 and recombinant IGFBP-3 were separated by one- and two-dimensional gel electrophoresis. The presence of functional IGFBPs was evaluated by western ligand blotting using biotinylated IGF-II. Individual IGFBPs were identified in western blots using IGFBP-specific monoclonal antibodies. Protein glycosylation was evaluated by digestion with the lyase Endoglycosidase F.
Western ligand blots of human and porcine vitreous detected two major proteins at ~35kDa and ~30kDa. Bands matching these sizes were also detected in human and porcine plasma. The larger band migrated to a position similar to that of recombinant IGFBP-2, but neither matched recombinant IGFBP-3 at ~45kDa. Western blots of human and porcine vitreous and plasma confirmed the identity of the ~35kDa band as IGFBP-2 and the ~30kDa band as a fragment of IGFBP-3. Western and western ligand blots of vitreous and plasma proteins separated by two-dimensional gel electrophoresis revealed that the IGFBP-3 fragments in vitreous and plasma have virtually identical profiles. Lyase digestion revealed that the ~30kDa IGFBP-3 fragment is a glycoprotein with a peptide core of ~25kDa.
Normal human and porcine vitreous contain two major IGFBPs that are identified as IGFBP-2 and a previously reported ~30kDa fragment of IGFBP-3. Both vitreous IGFBPs retain biological activity and IGFBP-3 has one or more glycosylation sites with a protein core of ~25kDa. Systematic comparisons between vitreous and plasma indicate that the vitreous IGFBP-3 is similar and perhaps identical to a previously described IGFBP-3 fragment in plasma with reduced growth factor affinities.
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