Immunological evidence for in\xa0vivo production of novel advanced glycation end-products from 1,5-anhydro-D-fructose, a glycogen metabolite

Akiko Sakasai-Sakai,Takanobu Takata,Hirokazu Suzuki,Masayoshi Takeuchi,Yoshihiro Motomiya,Ikuro Maruyama

doi:10.1038/s41598-019-46333-2

Akiko Sakasai-Sakai, Takanobu Takata + Show 4 more

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https://doi.org/10.1038/s41598-019-46333-2

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Abstract

The anhydrofructose pathway is an alternate pathway for glycogen degradation by α-1,4-glucan lyase. The sugar 1,5-anhydro-D-fructose (1,5-AF) acts as the central intermediate of this pathway, but its physiological role of in mammals is unclear. Glycation reactions forming advanced glycation end-products (AGEs) are important in the development of complications of diabetes mellitus. We hypothesized that 1,5-AF may contribute to cellular damage by forming 1,5-AF-derived AGEs (AF-AGEs) with intracellular proteins. To clarify the role of 1,5-AF in protein modification, we created a novel antibody targeting AF-AGEs. Serum albumin modified by AF-AGEs was prepared by incubating rabbit serum albumin (RSA) or bovine serum albumin (BSA) with 1,5-AF. After immunizing rabbits with AF-AGEs-RSA, affinity chromatography of anti-AF-AGE antiserum was performed on a Sepharose 4B column coupled with AF-AGEs-BSA or N-(carboxymethyl)/N-(carboxyethyl)lysine-BSA. A novel immunopurified anti-AF-AGE antibody was obtained and was characterized using a competitive enzyme-linked immunosorbent assay. Then an AF-AGEs assay was established using this immunopurified antibody. This assay was able to detect AF-AGEs in human and animal serum samples. Finally, intracellular accumulation of AF-AGEs was shown to be associated with damage to cultured hepatocytes (HepG2 cells). This is the first report about in vivo detection of AF-AGEs with a novel structural epitope.

Highlights

In mammals, including humans, glucose is stored as glycogen to provide energy
The antiserum reacted with AF-advanced glycation end-products (AGEs)-bovine serum albumin (BSA), but not with Glu-AGEs-BSA, Fru-AGEs-BSA or non-glycated BSA incubated without 1,5-AF (Fig. 2a)
The amount of antibody binding to the CML-/CEL-BSA affinity gel was calculated as a percentage of the unbound antibody, revealing that bound anti-CML/CEL antibody accounted for approximately 35% of total antibodies in the antiserum

Summary

Introduction

Glycogen is degraded to glucose-1-phosphate by glycogen phosphorylase, and to glucose by exo-glycosidase (Fig. 1). Advanced glycation end-products (AGEs) are produced as a result of non-enzymatic glycation reactions between ketone or aldehyde groups of reducing sugars, including glucose or fructose, and the ε-amino group of www.nature.com/scientificreports/. Nonenzymatic glycation reactions between 1,5-AF and the ε-amino groups of lysine residues of proteins (or the guanidino groups of arginine residues or the N-terminal α-amino groups of proteins) initially form reversible. The initial phase of the glycation reaction involving 1,5-AF is condensation of its carbonyl group with amino groups of proteins (Fig. 1), and is similar to the reaction for glucose/fructose. 1,5-AF is thought to be more important for AGEs formation than glucose and fructose because their anomerization equilibrium is shifted toward the reactive open chain forms of sugars. In vivo formation of AF-AGEs has been postulated, confirmatory evidence has not been obtained

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