N-Linked Glycosylation and Sialylation of the Acid-labile Subunit

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 IG-FBP-3. Desialylation reduced the affinity of ALS for IGFBP-3⅐IGF 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. Insulin-like growth factors (IGF)1 I and II are peptide hormones that regulate the differentiation and proliferation of a large number of cell types and also have a role in glucose homeostasis (1). 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). Recently, IGFBP-5 was also found to form a similar ternary complex with the IGFs and ALS in serum (4). It is thought that the size of the ternary complex restricts the passage of IGFs to target cells, while free IGFs, or IGFs in binary complexes with IGFBPs, can cross the capillary endothelial barrier. Therefore, ALS regulates the hypoglycemic and mitogenic potential of the circulating IGFs via the formation of the ternary complexes. Furthermore, ALS plays a vital role in maintaining a circulating store of the IGFs, IGFBP-3, and possibly IGFBP-5, by significantly increasing their serum half-lives (5, 6).Despite the importance of the ternary complexes in regulating serum IGF bioactivity, nothing is known about the structural aspects of ALS that enable it to interact with IGFBPs. There are two features of ALS structure that may play a part. First, the protein backbone of ALS is made up of repeating blocks each containing 24 amino acids, of which 6 are typically leucine residues. This places ALS in the leucine-rich repeat superfamily of proteins (7), all of which are involved in proteinprotein interactions (8). Second, serum ALS is heavily and heterogeneously glycosylated with N-linked glycan chains (3), and glycosylation is known to influence the interactions of many proteins.Electrophoretic st...

Total Absence of Functional Acid Labile Subunit, Resulting in Severe Insulin-Like Growth Factor Deficiency and Moderate Growth Failure

“…Unlike the first reported case of ALS deficiency in which a frameshift mutation led to early termination of ALS expression (7), in the present case, the missense mutation resulted in a D440N substitution of the ALS prepeptide. Based on molecular modeling of the ALS protein, D440 is within one of the 20 leucine-rich repeat (LRR) motifs (15) that are arranged in a donut-like shape (16,17). The N substitution at residue 440, interestingly, generated a consensus mo- tif for N-glycosylation (Fig.…”

“…The N substitution at residue 440, interestingly, generated a consensus mo- tif for N-glycosylation (Fig. 4) in a protein normally highly glycosylated (16). Overall, the effect(s) of D440N on ALS structure and function remains unclear; the consequence clearly was undetectable levels of circulating ALS.…”

Human Acid-Labile Subunit Deficiency: Clinical, Endocrine and Metabolic Consequences

“…The protein surface contains charged residues, and while charged residues are relatively evenly distributed on the outer regions of the domain, the center hole of the donut is notably lined with large regions of electronegative surfaces. This, together with the negatively charged N-linked carbohydrates on the ALS protein, may provide the necessary electrostatic potential to interact with the IGF-I-IGFBP-3 binary complex [10, 11]. …”

IGF system in children with congenital disorders of glycosylation

“…The conservation of the seven N ‐glycosylation sites in the ALS gene from mouse to human suggests an important role for N ‐glycosylation in ALS function 8 . Enzymatic deglycosylation of ALS reduces the affinity of ALS for IGFBP‐3–IGF complexes by 50–100% depending on the extent of deglycosylation 9 . IGFBP‐3 has three sites of N ‐glycosylation.…”