The role of glycosylation in the action of IGFBP-3.
There are three potential N-glycosylation sites (Asn-X-Ser/Thr) located in the nonconserved central region of the human IGFBP-3 sequence (Asn89, Asn109, Asn172-sites 1, 2 and 3, respectively). Upon ligand blotting with IGFs, IGFBP-3 appears as two bands (40-45 kDa) representing different glycosylated forms. We have mutated the N-glycosylation sites in permutations of three single, three double and one triple mutations and expressed these variant cDNAs. Each mutant protein was detected by radioimmunoassay, indicating that glycosylation is not required for the secretion of the protein from CHO cells. Ligand blotting using [125I]IGF-I indicated that all seven mutants retained IGF-I binding. Based on the molecular weights of the variant proteins, there are approximately 4, 5 and 6 kDa of carbohydrate on sites 1, 2 and 3, respectively. Furthermore, the two forms of IGFBP-3 represent the protein glycosylated either at all three sites or at Asn89 and Asn109 only. There appears to be no difference between the mutants and the fully-glycosylated rhIGFBP-3 in their acid-labile subunit (ALS) binding. Analysis of variance confirmed that the association constant for ALS was not significantly changed by any mutation [Ka (fully-glycosylated) = 12.5 +/- 4.1 nM-1; mean Ka (all mutants) = 22.1 +/- 3.0 nM-1]. While glycosylation does not appear to play a role in IGFBP-3 ligand binding, it may affect the turnover rate of the protein or be involved in rendering the protein resistant to proteolysis.
Firth
S M
SM
Kolling Institute of Medical Research, Royal North Shore Hospital, St. Leonards, NSW, Australia.
Baxter
R C
RC
D000818:Animals; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D006224:Cricetinae; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D018972:Insulin-Like Growth Factor Binding Protein 3 / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D013002:Somatomedins / Q000378:metabolism
D002352:Carrier Proteins; D006023:Glycoproteins; D018972:Insulin-Like Growth Factor Binding Protein 3; D013002:Somatomedins; C075944:insulin-like growth factor binding protein, acid labile subunit