Chemoenzymatic synthesis of glycosylated glucagon-like peptide 1: effect of glycosylation on proteolytic resistance and in vivo blood glucose-lowering activity.
Glucagon-like peptide 1 (7-36) amide (GLP-1) has been attracting considerable attention as a therapeutic agent for the treatment of type 2 diabetes. In this study, we applied a glycoengineering strategy to GLP-1 to improve its proteolytic stability and in vivo blood glucose-lowering activity. Glycosylated analogues with N-acetylglucosamine (GlcNAc), N-acetyllactosamine (LacNAc), and alpha2,6-sialyl N-acetyllactosamine (sialyl LacNAc) were prepared by chemoenzymatic approaches. We assessed the receptor binding affinity and cAMP production activity in vitro, the proteolytic resistance against dipeptidyl peptidase-IV (DPP-IV) and neutral endopeptidase (NEP) 24.11, and the blood glucose-lowering activity in diabetic db/db mice. Addition of sialyl LacNAc to GLP-1 greatly improved stability against DPP-IV and NEP 24.11 as compared to the native type. Also, the sialyl LacNAc moiety extended the blood glucose-lowering activity in vivo. Kinetic analysis of the degradation reactions suggested that the sialic acid component played an important role in decreasing the affinity of peptide to DPP-IV. In addition, the stability of GLP-1 against both DPP-IV and NEP24.11 incrementally improved with an increase in the content of sialyl LacNAc in the peptide. The di- and triglycosylated analogues with sialyl LacNAc showed greatly prolonged blood glucose-lowering activity of up to 5 h after administration (100 nmol/kg), although native GLP-1 showed only a brief duration. This study is the first attempt to thoroughly examine the effect of glycosylation on proteolytic resistance by using synthetic glycopeptides having homogeneous glycoforms. This information should be useful for the design of glycosylated analogues of other bioactive peptides as desirable pharmaceuticals.
Discovery Research Laboratories, Settsu Plant, and Pharmaceutical Research Division, Shionogi & Co., Ltd., Toyonaka, Osaka 561-0825, Japan.
Taichi
T
Ueda
Kazuyoshi
K
Tomita
Yoshihide
Y
Notsu
Takaomi
T
Ito
Masataka
M
Fumoto
Tomoaki
T
Takakura
Hirofumi
H
Nagatome
Akio
A
Takimoto
Shin-Ichi
S
Mihara
Hiroko
H
Togame
Keiko
K
Kawamoto
Takanori
T
Iwasaki
Kenji
K
Asakura
Takeo
T
Oshima
Kohji
K
Hanasaki
Shin-Ichiro
S
Nishimura
Hirosato
H
Kondo
D000818:Animals; D001786:Blood Glucose / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D003921:Diabetes Mellitus, Experimental; D018819:Dipeptidyl Peptidase 4 / Q000737:chemistry / Q000378:metabolism; D004195:Disease Models, Animal; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry* / Q000378:metabolism*; D006031:Glycosylation; D051379:Mice; D008820:Mice, Obese; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D055550:Protein Stability; D013997:Time Factors
D001786:Blood Glucose; D052216:Glucagon-Like Peptide 1; D018819:Dipeptidyl Peptidase 4; D015260:Neprilysin