Identification of plasma binding proteins for glucose-dependent insulinotropic polypeptide

Abstract

Glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K cells, has potent insulinreleasing and extrapancreatic glucoregulatory activities. However, exogenous GIP has less potent biological effects compared with another incretin hormone, GLP-1, which limits its use for the treatment of type 2 diabetes. The fate and secretion of administered native GIP remain unclear. The aim of this study was to identify plasma binding proteins for human GIP. Fluorescent-labelled GIP was added to fresh human plasma and subjected to clear native polyacrylamide gel electrophoresis (CN-PAGE). Then fluorescent protein bands were in-gel trypsin-digested and subjected to liquid chromatography tandemmass spectrometry (LC-MS/MS) analysis, revealing the presence of albumin, immunoglobulin G (IgG) and transferrin. In contrast to GIP, the binding of fluorescent GLP-1 and glucagon to plasma protein fractions were minimal. CN-PAGE analysis of synthetic GIP incubated with human serum albumin, purified IgG or transferrin, and subsequent western blot analysis revealed that GIP binds to each of these proteins. Taken together, these results indicate that GIP readily binds to albumin, IgG and transferrin, three plasma proteins highly abundant in the human peripheral circulation. Separation of protein complexes using CN-PAGE and the identification of in-gel digested proteins by LC-MS/MS analysis provide a promising strategy to identify plasma binding proteins for bioactive peptides.

ANGT_HUMAN[448–462], an Anorexigenic Peptide Identified Using Plasma Peptidomics

“…To estimate the potential biological functions of ANGT_HUMAN[448–462], which showed significant cell surface binding to several cultured cells, we attempted to identify its cell surface binding proteins using the following strategies. First, the fluorescence-labeled ANGT_HUMAN[448–462] peptide was reacted with cell- and tissue-derived lysates and electrophoresed using CN-PAGE, which allows the migration of high-molecular-weight protein complexes in their native state without dissociating peptides from their carrier proteins/receptors [ 12 , 42 , 43 ]. The fluorescence-positive bands detected were in-gel digested and analyzed using LC-MS/MS.…”

“…Protein complexes separated by CN-PAGE were in-gel trypsin-digested using a previously described protocol [ 12 , 13 ], but with the following modifications. Fluorescent protein spots showing the FAM-ANGT_HUMAN[448–462] complex in CN-PAGE were excised in rectangles of approximately 1 to 2 × 2 mm with a scalpel and washed with deionized water.…”

“…The specific polyclonal antibody against ANGT_HUMAN[448–462] (RRID: AB_2916121) was raised and purified as described previously [ 12 , 17 ], with the following modifications. Two micrograms of [Cys 0 ]-KPEVLEVTLNRPFLF were chemically synthesized, and approximately half was pretreated with a protein cross-linking and fixation reagent, mixed with the remaining 1 μg of untreated peptide, coupled to maleimide-activated mariculture keyhole limpet hemocyanin (Pierce), and immunized on days 1, 15, 29, 43, and 57 into Japanese white rabbits in the laboratory of Scrum Inc (Tokyo, Japan).…”

A Physiologically‐Based Quantitative Systems Pharmacology Model of the Incretin Hormones GLP‐1 and GIP and the DPP4 Inhibitor Sitagliptin

“…Available data on GIP secretion is highly inconsistent. 46 With the limited data available, an accurate characterization of GIP metabolism is not possible. Only one data set 27 reports the concentrations of both, the active and total forms of GIP, but the metabolite has not been directly measured so far.…”