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There are three potential N-glycosylation sites in the non-conserved central region of the insulin-like growth factor binding protein-3 (IGFBP-3) sequence (N89AS, N109AS, N172FS). IGFBP-3 exists as two glycoforms which reduce to a single form on enzymatic deglycosylation. To determine the functional significance of the carbohydrate chains, the N-glycosylation sites were mutated singly and in combinations by substituting Asn residues with Ala. Each recombinant glycoform was detected by radioimmunoassay, indicating that glycosylation is not essential for secretion in Chinese hamster ovary cells. Ligand blotting of the conditioned media using [125I]IGF-I indicated that all seven mutants are active. On the basis of the number and molecular masses of the bands detected for each glycoform, there is approximately 4, 4.5 and 5 kDa of carbohydrate on Asn89, Asn109 and Asn172 respectively, with variable occupancy of Asn172. Ternary complex formation by the glycovariants in the presence of ALS and excess IGF-I was not significantly different from that of fully glycosylated recombinant human (rh)IGFBP-3 [Ka (fully glycosylated)=12.5+/-4.1 l/nmol; mean Ka (all mutants)=22.1+/-3.0 l/nmol]. In contrast, Asn to Asp substitutions decreased acid-labile subunit (ALS) binding activity. Cell-surface association experiments indicate that glycosylation may influence the partitioning of IGFBP-3 between the extracellular milieu and the cell surface. Therefore, while the carbohydrate units appear to be non-essential to ALS or IGF binding, they may modulate other biological activities of IGFBP-3.
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Many tumours secrete IGF-II in incompletely processed precursor forms. The ability of these pro-IGF-II forms to complex with the six IGF binding proteins (IGFBPs) is poorly understood. In this study, pro-IGF-II has been extracted from the serum and tumour tissue of two patients with non-islet cell tumour hypoglycaemia. These samples were used to study binary complex formation with IGFBPs-1 to -6 using competitive IGF-II binding assays and ternary complex formation with IGFBP-3 and IGFBP-5. In each case, IGFBPs-1 to -6 showed little difference in their ability to form binary complexes with recombinant IGF-II or tumour-derived pro-IGF-II forms, when the preparations were standardised according to IGF-II immunoreactivity. As previously described, ternary complex formation by acid-labile subunit (ALS) with IGFBP-3 and pro-IGF-II was greatly decreased compared with complex formation with mature IGF-II. In contrast, ALS bound similarly to IGFBP-5 in the presence of pro-IGF-II and mature IGF-II. These studies suggest that pro-IGF-II preferentially forms binary complexes with IGFBPs, and ternary complexes with IGFBP-5, rather than ternary complexes with IGFBP-3 as seen predominantly in normal serum. This may increase the tissue availability of serum pro-IGF-II, allowing its insulin-like potential to be realised.
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The long term therapeutic potential of recombinant human (rh) IGF-I administration in insulin-dependent diabetes mellitus (IDDM) may be determined by changes in the IGF binding proteins (IGFBPs) and thus the bioavailability of IGF-I. We have therefore studied the effects of a single subcutaneous dose of rhIGF-I (40 micrograms/kg at 1800 h), when compared with an untreated control night, in 17 subjects with IDDM, on serum concentrations of IGF-I, IGF-II, IGFBP-3, acid labile subunit (ALS), and IGFBP-3 proteolysis. Mean (+/- S.E.M.) IGF-I levels increased from 242 +/- 30 ng/ml to 399 +/- 26 ng/ml (P = 0.01) after rhIGF-I whereas IGF-II levels declined from 600 +/- 45 ng/ml to 533 +/- 30 ng/ml. There was a small overnight reduction in baseline ALS levels from 48 +/- 2.8 to 44.5 +/- 3.2 micrograms/ml (P = 0.04) after rhIGF-I administration. An early fall in IGFBP-3 concentrations on the control night was not seen after rhIGF-I and overall mean levels were increased (5.2 +/- 0.2 micrograms/ml vs 4.9 +/- 0.2 micrograms/ml, P = 0.04, on the control night). On the baseline night, IGFBP-3 levels correlated with the sum of IGF-I and IGF-II (r = 0.73, P = 0.02) and with levels of the ALS (r = 0.7, P = 0.002). However after rhIGF-I, the sum of IGF-I and IGF-II no longer correlated with IGFBP-3, whereas the relationship with ALS was maintained. Immunoblot studies in six subjects indicated that 60%-70% of the IGFBP-3 was detected as a low molecular weight fragment at 1900 h on both study nights, but the amount of fragment declined to approximately 50% at 0100 h and 45% at 0700 h. In conclusion, despite a slight but significant fall in ALS, IGFBP-3 levels rise after rhIGF-I administration in IDDM. This cannot be explained by alterations in IGFBP-3 proteolysis, and may relate to the relative stability of ALS/IGFBP-3 when complexed principally with IGF-I rather than IGF-II.
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The IGFs are believed to be important in pregnancy and are implicated in the pathophysiology of pre-eclampsia. In adults the IGFs circulate primarily with IGF-binding protein-3 (IGFBP-3) and an acid-labile glycoprotein (ALS) in a 140 kDa complex which limits IGF bioavailability. Less than 10% of IGFBP-3 is in lower molecular weight forms. We have investigated the developmental regulation of the IGF/IGFBP system in normal and pre-eclamptic pregnancies with particular emphasis on the IGFBP-3 ternary complex. Circulating levels of IGF-I, IGFBP-3 and ALS, and their degree of association in the ternary complex in the fetus increased with gestational age. In neonatal serum from deliveries <35 weeks' gestation IGFBP-3 was predominantly in 30-50 kDa form(s) and ALS was a limiting factor for ternary complex formation. In serum from deliveries >35 weeks both ALS and IGFs were limiting but approximately 25% of IGFBP-3 was unable to form the ternary complex even in the presence of excess ALS and IGF-I. Serum IGFBP-1, -2 and -6 concentrations tended to decrease with increasing gestational age. In pre-eclamptic pregnancies, amniotic fluid IGFBP-2, -3 and -6 levels decreased with gestational age while IGFBP-1 levels did not show the normal decline. We speculate that the endocrine IGF system develops in the fetus during the third trimester of pregnancy when ALS levels increase.
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Insulin-like growth factor-binding protein-3 (IGFBP-3) is a member of a family of structurally conserved proteins (IGFBP-1 to -6) which act as carriers and regulators of the mitogenic peptide hormones IGF-I and IGF-II. Members of the IGFBP family share conserved cysteine-rich amino- and carboxyl-terminal regions. The amino-terminal domain of these proteins is recognised to contain an IGF-binding determinant, but evidence to support a binding site in the carboxyl-terminal region of the protein is less rigorous. To further investigate this, we have synthesised both the amino-terminal (residues 1-88; N-88) and carboxyl-terminal (residues 165-264; C-165) domains of human IGFBP-3 in bacteria, as fusion proteins with a carboxyl-terminal FLAG peptide. Although only C-165 showed binding to IGF-I and -II by solution-binding assays, both N-88 and C-165 demonstrated binding to IGF-I and -II by biosensor analysis albeit with reduced affinities compared with full-length IGFBP-3. Only the carboxyl-terminal fragment (C-165) was able to form hetero-trimeric complexes with IGF-I and the acid-labile subunit (ALS). We conclude that the carboxyl-terminal domain of IGFBP-3 contains an IGF-binding determinant and can form ternary complexes with ALS.
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The acid-labile subunit (ALS) of the high molecular weight insulin-like growth factor binding protein complex is a liver-derived glycoprotein which is regulated by growth hormone and serves as a serum marker of growth hormone action. We have compared the measurement of ALS by four immunoassay methods (two RIAs, two ELISAs) utilizing various polyclonal and monoclonal antibodies raised against natural or recombinant human ALS, or synthetic ALS peptides. Despite the variety of methodologies and reagents, results obtained by the four methods were highly correlated for 125 sera from various patient groups, and when compared for individual groups of sera from healthy children and adults, growth hormone-deficient children and adults, and subjects with acromegaly. Some weaker correlations among methods were seen when measuring ALS levels in groups of sera from pregnant subjects and subjects with chronic renal failure. An assay using antibodies raised against recombinant ALS yielded lower apparent values than the other methods in patient sera, the discrepancy probably being attributable to a difference in standardization. We conclude that a variety of assay formats and reagents can yield serum ALS values of potential clinical utility.
