title: Isolation and characterization of vitreous insulin-like growth factor binding proteins.
abstract: OBJECTIVE
  Previous studies from this laboratory revealed that vitreous insulin-like growth factor (IGF) biological activity increases in proliferative diabetic retinopathy and that this activity is normally attenuated by IGFBPs. The goal of this study was to identify and characterize the species involved.
  
  
  METHODS
  Human and porcine vitreous, plasma, recombinant IGFBP-2, and IGFBP-3 were separated by gel electrophoresis. Functional IGFBPs were detected in Western ligand blots with biotinylated IGF-II. IGFBPs were identified using IGFBP-specific antibodies.
  
  
  RESULTS
  Western ligand blots of normal vitreous and plasma detected two major proteins at ∼35 kDa and ∼29 kDa. Western blot analysis of human and porcine vitreous and plasma confirmed the identity of the ∼35-kDa band as IGFBP-2 and the ∼29-kDa band as a fragment of IGFBP-3. Western blot and Western ligand blot analyses 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 ∼29-kDa IGFBP-3 fragment is a glycoprotein with a peptide core of ∼25 kDa. N-terminal sequence data obtained from vitreous IGFBP-3 revealed that the protein is proteolytically truncated at the C terminus. CONCLUSIONS. Normal human and porcine vitreous contain two major IGFBPs, IGFBP-2 and an ∼29-kDa fragment of IGFBP-3. Both IGFBPs retain biological activity, and IGFBP-3 has one or more glycosylation sites with a protein core of ∼25 kDa. Systematic comparisons indicate that the vitreous IGFBP-3 is similar to and perhaps identical with a previously described IGFBP-3 fragment in plasma with reduced growth factor affinities.
authors.affiliation: Department of Ophthalmology, University of Alabama School of Medicine, Birmingham, AL, USA. cguidry@uab.edu
authors.forename: Clyde
authors.initials: C
authors.lastname: Guidry
authors.forename: Jeffery L
authors.initials: JL
authors.lastname: King
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D018971:Insulin-Like Growth Factor Binding Protein 2 / Q000032:analysis / Q000302:isolation & purification*; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins / Q000032:analysis / Q000302:isolation & purification*; D008970:Molecular Weight; D013552:Swine; D014822:Vitreous Body / Q000737:chemistry*
chemical_list: C515497:IGFBP3 protein, human; D018971:Insulin-Like Growth Factor Binding Protein 2; D018972:Insulin-Like Growth Factor Binding Protein 3; D018969:Insulin-Like Growth Factor Binding Proteins
references.citation: Invest Ophthalmol Vis Sci. 2003 Mar;44(3):1355-63
references.pmid: 12601069
references.citation: Am J Physiol Endocrinol Metab. 2000 Jun;278(6):E967-76
references.pmid: 10826997
references.citation: Invest Ophthalmol Vis Sci. 2004 Dec;45(12):4535-42
references.pmid: 15557465
references.citation: Endocr Rev. 2002 Dec;23(6):824-54
references.pmid: 12466191
references.citation: Invest Ophthalmol Vis Sci. 1996 Apr;37(5):740-52
references.pmid: 8603859
references.citation: Br J Ophthalmol. 1993 Nov;77(11):731-3
references.pmid: 8280690
references.citation: Prog Retin Eye Res. 2005 Jan;24(1):75-86
references.pmid: 15555527
references.citation: Cell Mol Life Sci. 2007 Jul;64(14):1870-80
references.pmid: 17593323
references.citation: Curr Eye Res. 1995 Feb;14(2):127-35
references.pmid: 7539353
references.citation: Ophthalmologe. 1992 Oct;89(5):387-90
references.pmid: 1304218
references.citation: Invest Ophthalmol Vis Sci. 2009 Apr;50(4):1929-39
references.pmid: 19117921
references.citation: Mol Cell Endocrinol. 1998 May 25;140(1-2):19-24
references.pmid: 9722163
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Horm Metab Res. 2001 May;33(5):312-6
references.pmid: 11440279
references.citation: J Endocrinol. 1999 Mar;160(3):379-87
references.pmid: 10076184
references.citation: Horm Metab Res. 2000 May;32(5):196-200
references.pmid: 10871161
references.citation: Invest Ophthalmol Vis Sci. 1997 Feb;38(2):456-68
references.pmid: 9040479
references.citation: Endocr J. 1996 Oct;43 Suppl:S43-8
references.pmid: 9076341
references.citation: Diabetes. 1998 Mar;47(3):445-9
references.pmid: 9519752
references.citation: Br J Ophthalmol. 1994 Mar;78(3):219-22
references.pmid: 7511932
references.citation: Ophthalmic Res. 1993;25(5):289-94
references.pmid: 8259261
references.citation: Diabetes. 1986 Apr;35(4):416-20
references.pmid: 2420665
references.citation: Curr Eye Res. 2001 Nov;23(5):368-71
references.pmid: 11910526
references.citation: Ophthalmology. 1989 Jun;96(6):835-41
references.pmid: 2662103
references.citation: Diabetes Care. 2000 Jan;23(1):80-3
references.pmid: 10857973
references.citation: Horm Metab Res. 1999 Feb-Mar;31(2-3):192-202
references.pmid: 10226802
references.citation: Diabetes. 2004 Sep;53(9):2428-35
references.pmid: 15331555
references.citation: Br J Ophthalmol. 1997 Mar;81(3):228-33
references.pmid: 9135388
references.citation: Gen Comp Endocrinol. 2005 May 15;142(1-2):44-52
references.pmid: 15862547
references.citation: Invest Ophthalmol Vis Sci. 1997 Sep;38(10):2053-63
references.pmid: 9331269
references.citation: Endocrinology. 2009 Nov;150(11):4968-76
references.pmid: 19734278
references.citation: J Clin Invest. 1993 Dec;92(6):2620-5
references.pmid: 7504689
references.citation: N Engl J Med. 2004 Jan 1;350(1):48-58
references.pmid: 14702427