What Is the Significance of IGF-Binding Protein-3 Proteolysis in the Circulation?

Robert C. Baxter

Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney NSW 2065, Australia

Address correspondence to: Robert C. Baxter, Ph.D., D.Sc., Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, Sydney NSW 2065, Australia. E-mail: robaxter{at}med.usyd.edu.au

To the editor:

Lassarre and Binoux (1) recently described an assay for intact IGF-binding protein-3 (IGFBP-3) in which serum is acidified to destroy the acid-labile subunit (ALS) and dissociate IGF-IGFBP complexes, IGFs are removed by ultrafiltration, and active IGFBP-3 is quantitated by radiolabeled IGF-I binding. This assay is described as opening new perspectives in investigating IGFBP-3 proteolysis and IGF bioavailability. The authors state that proteolyzed IGFBP-3 loses affinity for IGFs, which are then "redistributed towards the 40-kDa binary IGFBP-IGF complexes" (1), and point out that in human pregnancy, "virtually all circulating IGFBP-3 is degraded."

There is some disagreement as to the changes in total serum IGFs through pregnancy, with increases in both IGF-I and IGF-II (2), an increase in IGF-I but not IGF-II (3), and a 15% decrease in IGF-I (4) reported. The proportion of total IGF-I detectable in "free IGF-I" assays approximately doubles in pregnancy from the value of about 1% reported for nonpregnant women (4, 5), although the biological significance of these measurements is unclear.

Abundant published evidence refutes the concept that there is a substantial redistribution of IGFs in pregnancy serum away from IGFBP-3-ALS complexes; direct measurement indicates that the proportion of total IGFs in these complexes is unchanged or even slightly increased in pregnancy (6, 7). The size distribution of IGFBP-3 is also unchanged throughout the course of pregnancy, with 85–90% remaining in high molecular weight fractions (8), whereas the total immunoreactive IGFBP-3 concentration is reported to increase (8). This provides important information about the distribution of IGFs, since human IGFBP-3 has an unmeasurably low affinity for ALS unless IGF-I or IGF-II is bound to the IGFBP-3 (9). Thus, the unaltered proportion of pregnancy serum IGFBP-3 in high molecular weight complexes indicates that IGFs are also in these complexes.

It can be demonstrated that, if there is a substantial loss of affinity in the IGF-IGFBP interaction, there would be a decreased binding of IGFs, and consequently a loss of ALS binding (10). In contrast, IGFBP-3 isolated from nonpregnancy or pregnancy serum by immunoaffinity chromatography, and not depleted of its endogenous IGFs, shows normal affinity for ALS (11).

Although IGFBP-3 in pregnancy serum, or isolated from pregnancy serum in the presence of endogenous IGFs, seems to bind and transport IGFs normally, depletion of endogenous IGFs under acidic conditions leads to a loss of affinity as determined by radioligand binding (12)—indeed, this is the basis of the new assay of Lassarre and Binoux (1). While this may in part be explained by the selective effect of altering tyrosine residues 24 and 60 of IGF-I during the iodination process (13), it has been clearly demonstrated that IGF-I affinity is decreased by up to 8-fold, and IGF-II affinity by 2.5-fold, in IGF-depleted pregnancy serum compared with nonpregnancy serum (12, 13). Similarly, IGFBP-3 in pregnancy serum is undetectable by ligand blotting after SDS-PAGE, as first demonstrated over a decade ago (14, 15). Because an 8-fold loss of affinity from the intact IGFBP-3 affinity of 20 liters/nmol would be insufficient to abolish IGF binding on a ligand blot, it must be assumed that further damage to the proteolyzed protein during electrophoresis, or to the IGF-I during iodination (16), contributes to this observation.

What is the biological consequence of this limited proteolysis of IGFBP-3, detectable by radioligand binding in vitro after depletion of bound IGFs, but apparently without effect on IGF transport in the circulation? It is certainly possible that this could alter the bioavailability of IGFs to the tissues, and this may eventually turn out to be true, but there has been no definitive experimental proof of this to date, in which pregnancy-proteolyzed IGFBP-3, not subjected to destructive isolation and processing, has been shown to allow IGFs to pass from circulating complexes to their target tissues more readily than unproteolyzed IGFBP-3. Indeed, the definitive experiment is difficult to envisage.

Even if this accelerated release of IGFs does occur following proteolysis, it is unclear whether the released IGFs would have increased availability to tissue receptors (local IGFBPs might rapidly re-bind them), or would simply be lost through degradative clearance, for example through the kidneys. Therefore, it appears that there is insufficient evidence at present to state, as Lassarre and Binoux (1) do, that "limited proteolysis of IGFBP-3 is a fundamental mechanism in the regulation of IGF-I bioavailability in the bloodstream."

Received May 24, 2001.

References

  1. Lassarre C, Binoux M 2001 Measurement of intact insulin-like growth factor-binding protein-3 in human plasma using a ligand immunofunctional assay. J Clin Endocrinol Metab 86:1260–1266[Abstract/Free Full Text]
  2. Wilson DM, Bennett A, Adamson GD, et al. 1982 Somatomedins in pregnancy: a cross-sectional study of insulin-like growth factors I and II and somatomedin peptide content in normal human pregnancies. J Clin Endocrinol Metab 55:858–861[Abstract]
  3. Langford K, Nicolaides K, Miell JP 1998 Maternal and fetal insulin-like growth factors and their binding proteins in the second and third trimesters of human pregnancy. Hum Reprod 13:1389–1393[Abstract]
  4. Hasegawa T, Hasegawa Y, Takada M, Tsuchiya Y 1995 The free form of insulin-like growth factor I increases in circulation during normal human pregnancy. J Clin Endocrinol Metab 80:3284–3286[Abstract]
  5. Hizuka N, Takano K, Asakawa K, et al. 1991 Measurement of free form of insulin-like growth factor I in human plasma. Growth Regul 1:51–55[Medline]
  6. Davies SC, Holly JM, Coulson VJ, et al. 1991 The presence of cation-dependent proteases for insulin-like growth factor binding proteins does not alter the size distribution of insulin-like growth factors in pregnancy. Clin Endocrinol (Oxf) 34:501–506[Medline]
  7. Gargosky SE, Owens PC, Walton PE, et al. 1991 Most of the circulating insulin-like growth factors-I and -II are present in the 150 kDa complex during human pregnancy. J Endocrinol 131:491–497[Abstract]
  8. Suikkari A-M, Baxter RC 1992 Insulin-like growth factor binding protein-3 is functionally normal in pregnancy serum. J Clin Endocrinol Metab 74:177–183[Abstract]
  9. Baxter RC, Martin JL, Beniac VA 1989 High molecular weight insulin-like growth factor binding protein complex. Purification and properties of the acid-labile subunit from human serum. J Biol Chem 264:11843–11848[Abstract/Free Full Text]
  10. Baxter RC, Bayne ML, Cascieri MA 1992 Structural determinants for binary and ternary complex formation between insulin-like growth factor-I (IGF-I) and IGF binding protein-3. J Biol Chem 267:60–65[Abstract/Free Full Text]
  11. Baxter RC, Suikkari AM, Martin JL 1993 Characterization of the binding defect in insulin-like growth factor binding protein-3 from pregnancy serum. Biochem J 294:847–852[Medline]
  12. Lassarre C, Binoux M 1994 Insulin-like growth factor binding protein-3 is functionally altered in pregnancy plasma. Endocrinology 134:1254–1262[Abstract]
  13. Baxter RC, Skriver L 1993 Altered ligand specificity of proteolysed insulin-like growth factor binding protein-3. Biochem Biophys Res Commun 196:1267–1273[CrossRef][Medline]
  14. Giudice LC, Farrell EM, Pham H, Lamson G, Rosenfeld RG 1990 Insulin-like growth factor binding proteins in maternal serum throughout gestation and in the puerperium: effects of a pregnancy-associated serum protease activity. J Clin Endocrinol Metab 71:806–816[Abstract]
  15. Hossenlopp P, Segovia B, Lassarre C, Roghani M, Bredon M, Binoux M 1990 Evidence of enzymatic degradation of insulin-like growth factor-binding proteins in the 150K complex during pregnancy. J Clin Endocrinol Metab 71:797–805[Abstract]
  16. Suikkari AM, Baxter RC 1991 Insulin-like growth factor (IGF) binding protein-3 in pregnancy serum binds native IGF-I but not iodo-IGF-I. J Clin Endocrinol Metab 73:1377–1379[Abstract]




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