Characterisation of recombinant glycosylation variants of insulin-like growth factor binding protein-3

Abstract

There are three potential N-glycosylation sites in the non-conserved central region of the insulin-like growth factor binding protein-3 (IGFBP-3) sequence (N 89 AS, N 109 AS, N 172 FS). IGFBP-3 exists as two glycoforms which reduce to a single form on enzymatic deglycosylation. To determine the functional significance of the carbohydrate chains, the N-glycosylation sites were mutated singly and in combinations by substituting Asn residues with Ala. Each recombinant glycoform was detected by radioimmunoassay, indicating that glycosylation is not essential for secretion in Chinese hamster ovary cells. Ligand blotting of the conditioned media using [

Effect of recombinant porcine IGF-binding protein-3 on proliferation of embryonic porcine myogenic cell cultures in the presence and absence of IGF-I

“…There are three potential glycosylation sites in the IGFBP-3 molecule and the 39 kDa glycosylation variant is glycosylated at two of these sites while the 43 kDa variant is glycosylated at all three sites (Firth & Baxter 1999). Non-glycosylated IGFBP-3 migrates as a single band with a molecular weight of approximately 29 kDa (Firth & Baxter 1999). Since the rpIGFBP-3 in this study was produced and secreted from a eukaryotic cell system, the protein should be glycosylated.…”

“…It is well established that IGFBP-3 exists as a glycoprotein; however, glycosylation does not appear to be required for biological activity (Conover 1992, Firth & Baxter 1999. Native porcine IGFBP-3 exists as two glycosylation variants migrating at apparent molecular weights of 39 and 43 kDa on non-reducing SDS polyacrylamide gels (Yang et al 1999).…”

“…Native porcine IGFBP-3 exists as two glycosylation variants migrating at apparent molecular weights of 39 and 43 kDa on non-reducing SDS polyacrylamide gels (Yang et al 1999). There are three potential glycosylation sites in the IGFBP-3 molecule and the 39 kDa glycosylation variant is glycosylated at two of these sites while the 43 kDa variant is glycosylated at all three sites (Firth & Baxter 1999). Non-glycosylated IGFBP-3 migrates as a single band with a molecular weight of approximately 29 kDa (Firth & Baxter 1999).…”

Multiple post-translational modifications of mouse insulin-like growth factor binding protein-6 expressed in epithelial Madin–Darby canine kidney cells

“…The central regions show low similarity among the different IGFBPs. Post-translational modifications such as proteolytic cleavage, N-and O-glycosylation (Chelius et al, 2002;Firth and Baxter, 1999;Neumann et al, 1998), and phosphorylation (Coverley and Baxter, 1997;Graham et al, 2007;Hoeck and Mukku, 1994;Jones et al, 1993;Jones et al, 1991) have been reported to occur in the central domain (Firth and Baxter, 2002). Both phosphorylation and proteolysis have been shown to affect the IGF binding affinity of IGFBPs (Bunn and Fowlkes, 2003;Jones et al, 1991).…”

“…Both phosphorylation and proteolysis have been shown to affect the IGF binding affinity of IGFBPs (Bunn and Fowlkes, 2003;Jones et al, 1991). In contrast, N-and O-glycosylation appear to be non-essential for binding of IGFBP-3 or IGFBP-6 to IGFs, but have the potential to modulate cell surface binding and binding to extracellular glycosaminoglycans, respectively (Firth and Baxter, 1999;Marinaro et al, 2000b). At present, it is unclear whether post-translational modifications play a role in transport of IGFBPs in the secretory pathway or in differential sorting and M a n u s c r i p t Shalamanova et al 4 secretion of IGFBPs in polarized cells (Remacle-Bonnet et al, 1995;Shalamanova et al, 2001).…”