Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: Evidence for processing of cell-bound IGFBP-3

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Abstract

Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3(E.coli)) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3(CHO)) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3(E.coli)) or hIGFBP-3(CHO)) produced a dose-dependent inhibition of IGF-I- but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGFBP-3(E.coli) or hIGFBP-3(CHO) potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3(E.coli) for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3']AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3(E.coli) with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 M(r) hIGFBP-3(E.coli) seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3(E.coli) forms of 12,000-27,000 M(r). Cell-associated IGFBP-3(E.coli) (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3(E.coli) in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.

Original languageEnglish (US)
Pages (from-to)3259-3268
Number of pages10
JournalEndocrinology
Volume129
Issue number6
StatePublished - 1991

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Insulin-Like Growth Factor Binding Protein 3
Insulin-Like Growth Factor I
Glycosylation
Escherichia coli
Fibroblasts
Aminoisobutyric Acids
IGF Type 1 Receptor
Insulin-Like Growth Factor Binding Proteins
Cricetulus

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

@article{502803909d474197a7082fe933bde1ce,
title = "Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action: Evidence for processing of cell-bound IGFBP-3",
abstract = "Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3(E.coli)) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3(CHO)) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3(E.coli)) or hIGFBP-3(CHO)) produced a dose-dependent inhibition of IGF-I- but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGFBP-3(E.coli) or hIGFBP-3(CHO) potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3(E.coli) for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25{\%}. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3']AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3(E.coli) with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 M(r) hIGFBP-3(E.coli) seen after 24 h of incubation was reduced approximately 70{\%} after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3(E.coli) forms of 12,000-27,000 M(r). Cell-associated IGFBP-3(E.coli) (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3(E.coli) in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.",
author = "Conover, {Cheryl A}",
year = "1991",
language = "English (US)",
volume = "129",
pages = "3259--3268",
journal = "Endocrinology",
issn = "0013-7227",
publisher = "The Endocrine Society",
number = "6",

}

TY - JOUR

T1 - Glycosylation of insulin-like growth factor binding protein-3 (IGFBP-3) is not required for potentiation of IGF-I action

T2 - Evidence for processing of cell-bound IGFBP-3

AU - Conover, Cheryl A

PY - 1991

Y1 - 1991

N2 - Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3(E.coli)) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3(CHO)) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3(E.coli)) or hIGFBP-3(CHO)) produced a dose-dependent inhibition of IGF-I- but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGFBP-3(E.coli) or hIGFBP-3(CHO) potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3(E.coli) for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3']AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3(E.coli) with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 M(r) hIGFBP-3(E.coli) seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3(E.coli) forms of 12,000-27,000 M(r). Cell-associated IGFBP-3(E.coli) (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3(E.coli) in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.

AB - Insulin-like growth factor binding protein-3 (IGFBP-3) is unique among the IGF binding proteins in its extensive glycosylation in the native state. To determine the functional significance of carbohydrate moieties on IGFBP-3, we examined the effects of nonglycosylated Escherichia coli-derived recombinant human IGFBP-3 (hIGFBP-3(E.coli)) and glycosylated Chinese hamster ovary cell-derived hIGFBP-3 (hIGFBP-3(CHO)) on IGF-I action in cultured bovine fibroblasts. Both hIGFBP-3 preparations bound IGF-I with high affinity and were approximately 5-fold more potent than unlabeled IGF-I in inhibiting [125I]IGF-I binding to bovine fibroblasts. Coincubation of IGF-I and hIGFBP-3(E.coli)) or hIGFBP-3(CHO)) produced a dose-dependent inhibition of IGF-I- but not insulin-stimulated [3H]aminoisobutyric acid (AIB) uptake. In contrast, preincubation of bovine fibroblasts with hIGFBP-3(E.coli) or hIGFBP-3(CHO) potentiated subsequent IGF-I-stimulated [3H]AIB uptake. When cells were preincubated with 50 nM hIGFBP-3(E.coli) for 24 h, [125I]IGF-I binding to bovine fibroblasts increased 2.4-fold, whereas responsiveness to IGF-I was increased only 25%. After a 72-h preincubation, IGF-I cell binding remained increased 2-fold with commensurate enhancement of IGF-I-stimulated [3']AIB uptake. The increase in [125I]IGF-I binding to bovine fibroblast monolayers was primarily due to association of hIGFBP-3(E.coli) with the cell surface; there was no significant change in IGF-I receptor number or affinity under these conditions. Affinity cross-linking experiments indicated that intense binding of [125I]IGF-I to cell-associated 29,000 M(r) hIGFBP-3(E.coli) seen after 24 h of incubation was reduced approximately 70% after 72 h, concomitant with the appearance of smaller bands indicating hIGFBP-3(E.coli) forms of 12,000-27,000 M(r). Cell-associated IGFBP-3(E.coli) (72 h preincubation conditions) had a 10-fold lower affinity for IGF-I compared to hIGFBP-3(E.coli) in solution and a 2-fold lower affinity compared to the IGF-I receptor. These data demonstrate that glycosylation is not obligatory for biologically functional IGFBP-3. Furthermore, they suggest that processing of cell-associated IGFBP-3 to forms with altered affinity for IGF-I peptide may underly the potentiating effect of IGFBP-3 on IGF-I action.

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C2 - 1720092

AN - SCOPUS:0025790353

VL - 129

SP - 3259

EP - 3268

JO - Endocrinology

JF - Endocrinology

SN - 0013-7227

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