Abstract
Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with IGF-binding protein-3 (IGFBP-3) and the 84-86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate. The ternary complexes regulate IGF availability to the tissues. Since interactions of glycoproteins can be influenced by their glycan moieties, this study aimed to determine the role of ALS glycosylation in ternary complex formation. Complete deglycosylation abolished the ability of ALS to associate with IGFBP-3. To examine this further, seven recombinant ALS mutants each lacking one of the seven glycan attachment sites were expressed in CHO cells. All the mutants bound IGFBP-3, demonstrating that this interaction is not dependent on any single glycan chain. Enzymatic desialylation of ALS caused a shift in isoelectric point from 4.5 toward 7, demonstrating a substantial contribution of anionic charge by sialic acid. Ionic interactions are known to be involved in the association between ALS and IGFBP-3. Desialylation reduced the affinity of ALS for IGFBP-3. IGF complexes by 50-80%. Since serum protein glycosylation is often modified in disease states, the dependence of IGF ternary complex formation on the glycosylation state of ALS suggests a novel mechanism for regulation of IGF bioavailability.
Highlights
Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with Insulin-like growth factors (IGF)-binding protein-3 (IGFBP-3) and the 84 – 86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate
Since we demonstrated that the N-linked sugars on ALS have a role in ternary complex formation, we used sitedirected mutagenesis to investigate whether any single glycan chain was solely responsible for the binding of ALS to the IGF
NANase III-treated ALS Has a Reduced Affinity for IGFBP3—Having established that ALS glycosylation has a role in ternary complex formation, we investigated whether negatively charged sialic acid moieties may be involved in ALS binding to the IGF1⁄7IGFBP-3 complex
Summary
Over 75% of the circulating insulin-like growth factors (IGF-I and -II) are bound in 140-kDa ternary complexes with IGF-binding protein-3 (IGFBP-3) and the 84 – 86-kDa acid-labile subunit (ALS), a glycoprotein containing 20 kDa of carbohydrate. At least 75% of the total circulating IGFs are carried in 130 –150-kDa ternary complexes containing IGFbinding protein-3 (IGFBP-3) [2] and an 85-kDa glycoprotein, the acid-labile subunit (ALS) [3]. We have shown that the removal of basic residues in the carboxylterminal region of IGFBP-3 decreased its affinity for ALS by 90%, indicating the importance of positive charge in this region [15] From these observations, it is likely that negative charges on ALS may be involved in the interaction. At physiological pH, ALS binds tightly to weak anion exchange columns, indicating that it has a net negative charge [3]
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