title: D440N mutation in the acid-labile subunit of insulin-like growth factor complexes inhibits secretion and complex formation.
abstract: The acid-labile subunit (ALS) regulates IGF bioavailability by forming heterotrimeric complexes with IGFs and IGF-binding protein-3 (IGFBP-3). A homozygous missense mutation (D440N) resulting in undetectable circulating levels of ALS with a concomitant reduction in IGF-I and IGFBP-3 has been reported to cause mild growth retardation. To understand how this particular mutation affects ALS circulating levels and IGF-transport function, we expressed recombinant ALS and its variants, D440N-ALS, T442A-ALS, and D440N/T442A-ALS, using adenovirus vectors. Compared with wild-type ALS, the secretion of D440N-ALS was 80% lower. The D440N mutation was proposed to generate an N-glycosylation site additional to the seven existing motifs in ALS. D440N-ALS appeared larger than ALS, attributable to N-linked glycans because deglycosylation with N-glycosidase F reduced both proteins to the same molecular mass. When ALS was incubated with IGF-I and IGFBP-3, 70-80% of IGF-I was detected by gel-filtration chromatography in forms corresponding to the 150-kDa ternary complex. In contrast, when D440N-ALS was tested, less than 30% of IGF-I was found in high molecular mass complexes. Two other ALS variants mutated in the same putative glycosylation site, D440N/T442A-ALS and T442A-ALS, showed similar chromatographic profiles to wild-type ALS. The D440N mutation in ALS generates a hyperglycosylated form with impaired secretion and complex formation, potentially leading to dysregulation of endocrine IGF, thus contributing to the growth retardation observed in the affected patient. This is the first study to explain how a natural mutation, D440N, in ALS impairs its function.
authors.affiliation: Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia. sue.firth@sydney.edu.au
authors.forename: Sue M
authors.initials: SM
authors.lastname: Firth
authors.forename: Xiaolang
authors.initials: X
authors.lastname: Yan
authors.forename: Robert C
authors.initials: RC
authors.lastname: Baxter
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D002352:Carrier Proteins / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D006023:Glycoproteins / Q000097:blood / Q000235:genetics* / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism*; D007334:Insulin-Like Growth Factor I / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D006023:Glycoproteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D011994:Recombinant Proteins; C075944:insulin-like growth factor binding protein, acid labile subunit; D007334:Insulin-Like Growth Factor I