title: Properties of native and in vitro glycosylated forms of the glucagon-like peptide-1 receptor antagonist exendin(9-39).
abstract: The intestinal hormone glucagon-like peptide-1-(7-36)-amide (GLP-1) has recently been implicated as a possible therapeutic agent for the management of type 2 non-insulin-dependent diabetes mellitus (NIDDM). However, a major difficulty with the delivery of peptide-based agents is their short plasma half-life, mainly due to rapid serum clearance and proteolytic degradation. Using a peptide analog of GLP-1, the GLP-1 receptor antagonist exendin(9-39), we investigated whether the conjugation of a carbohydrate structure to exendin(9-39) would generate a peptide with intact biological activity and improved survival in circulation. The C-terminal portion of exendin(9-39) was reengineered to generate an efficient site for enzymatic O-glycosylation. The modified exendin(9-39) peptide (exe-M) was glycosylated by recombinant UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase 1 (GalNAc-T1) alone or in conjunction with a recombinant GalNAc alpha2,6-sialyltransferase (Sialyl-T), resulting in exe-M peptides containing either the monosaccharide GalNAc or the disaccharide NeuAc alpha2,6GalNAc. The nonglycosylated and glycosylated forms of exe-M competed with nearly equal potency (> 90% of control) with the binding of [125I]GLP-1 to human GLP-1 receptors expressed on stably transfected COS-7 cells. In addition, each peptide was equally effective for inhibiting GLP-1-induced cyclic adenosine monophosphate (cAMP) production in vitro. Studies with rats demonstrated that the modified and glycosylated forms of exendin(9-39) could antagonize exogenously administered GLP-1 in vivo. Interestingly, glycosylated exendin(9-39) homologs were more than twice as effective as the nonglycosylated peptide for inhibiting GLP-1-stimulated insulin production in vivo, suggesting a longer functional half-life in the circulation for glycosylated peptides. Results from in vivo studies with 3H-labeled peptides suggest that the glycosylated peptides may be less susceptible to modification in the circulation.
authors.affiliation: Unit of Protein Research, Pharmacia & Upjohn, Kalamazoo, MI, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Meurer
authors.forename: J R
authors.initials: JR
authors.lastname: Colca
authors.forename: P S
authors.initials: PS
authors.lastname: Burton
authors.forename: A P
authors.initials: AP
authors.lastname: Elhammer
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D000242:Cyclic AMP / Q000037:antagonists & inhibitors; D003924:Diabetes Mellitus, Type 2 / Q000188:drug therapy; D005934:Glucagon / Q000037:antagonists & inhibitors*; D052216:Glucagon-Like Peptide 1; D006031:Glycosylation; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D010446:Peptide Fragments / Q000037:antagonists & inhibitors* / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology; D011498:Protein Precursors / Q000037:antagonists & inhibitors*; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D010446:Peptide Fragments; D011498:Protein Precursors; C083773:exendin (9-39); D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D000242:Cyclic AMP