No abstract

The acid-labile subunit (ALS) is a glycosylated 85-kDa member of the leucine-rich repeat (LRR) protein superfamily and circulates in ternary complexes with the insulin-like growth factors (IGFs) and their binding proteins (IGFBPs). These complexes are thought to regulate the serum IGFs by restricting IGF movement out of the circulation. However, little is known about how ALS binds to IGFBP-3 or -5, which link the IGFs to ALS. To investigate potential sites of interaction, the ALS structure has been modeled with the crystal structure of the LRR protein porcine ribonuclease inhibitor as a template. ALS is predicted to be a donut-shaped molecule with an internal diameter of 1.7 nm, an external diameter of 7.2 nm, and a thickness of 3.6 nm. These dimensions are supported by rotary shadowing electron microscopy of ALS. The internal face is lined with a substantial region of electronegative surface potential that could interact with the positively charged region on IGFBP-3 known to be involved in ALS binding. The model also predicts that three potential N-linked oligosaccharide sites within the LRR domain are clustered together, which may be important in light of recent studies showing ALS glycan involvement in complex formation with IGFBP-3.The majority of serum insulin-like growth factors (IGFs) 1 circulate within 130 -150-kDa ternary complexes containing either IGF-I or -II, IGF-binding protein (IGFBP)-3, and the acid-labile subunit (ALS), an 85-kDa glycoprotein. It is thought that the size of these complexes prevents IGF access to target cells, while free IGFs and IGFs in binary complexes with the IGFBPs can easily cross the capillary endothelial barrier. Furthermore, the ALS-containing complex significantly increases the serum half-lives of both the IGFs and IGFBP-3 and in this way maintains a circulating store of these molecules (1, 2). Therefore, the association of ALS to the IGF complex is an important event in serum IGF regulation. ALS binding is the limiting step in complex formation, since the affinity of ALS for the IGF⅐IGFBP-3 complex is up to 2000-fold less than the affinity of IGFBP-3 for the IGFs in physiological salt concentrations, pH and temperature (3). Recently, Twigg and Baxter (4) showed that ALS and the IGFs can also form a ternary complex in vitro with IGFBP-5 (which has high homology to IGFBP-3 in the ALS binding domain) and that this IGFBP-5 complex is found in low concentrations in serum.Although the structures of the IGFs have been solved (5, 6), the structures of the proteins which interact with and regulate the serum IGFs have not been elucidated. Therefore, it is not yet clear how ALS interacts physically with IGFBP-3 to form the ternary complexes. However, one major structural feature of ALS is that around 75% of its residues are ordered into 18 tandem repeats of 24 amino acids plus two partial repeats, all of which contain the consensus motif for the leucine-rich repeat (LRR) superfamily of proteins. (7). All the members of this superfamily are involved in protein-protein interactions...

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...

While immunisation rates were 94.63% for the whole Nepean Blue Mountains region of New South Wales in 2017, coverage for 1 year olds in the upper Blue Mountains was under 80%. There is a known relationship between vaccine-hesitant parents and complementary and alternative medicine (CAM) use; however, little is known about how CAM practitioners fit within the information-seeking pathway of parents. This exploratory study sought to address this knowledge gap. Qualitative semi-structured interviews with vaccine-hesitant and vaccine rejecting parents and CAM practitioners purposively sampled from the Blue Mountains area revealed three sets of themes: 1) Parents' search for further information about immunisation; 2) Parents' use of CAM practitioners as an immunisation information source; and 3) CAM practitioners' engagement with parents about immunisation. CAM practitioners form a definite, if complex, part of vaccinehesitant and vaccine-rejecting parents decision-making pathway in the Blue Mountains area. The notion of patient choice is crucial to vaccine discussions. Development of support materials, such as decision resources which give impartial and detailed information while acknowledging and supporting patient choice, are needed to support both CAM practitioners and parents in making informed vaccination decisions.