Screening of peptides with a high affinity to bile acids using peptide arrays and a computational analysis

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Abstract

Bile acid binding peptides have attracted attention for the improvement and prevention of hypercholesterolemia. In this study, screening of bile acid high affinity peptides was investigated using computationally-assisted peptide array analysis. Starting with the screening data obtained from a limited, random 6-mer library (2212 sequences), the peptides with a high affinity to bile acid were characterized by comparison of high- and low-affinity peptides using fuzzy neural network (FNN) analysis. The physical properties of amino acids at specific positions that contribute to bile acid binding activity were extracted as the structural rule; optimization was carried out using three repeated screening cycles of the rule extraction. The extracted structural rule indicates that Trp, Tyr, Phe, Leu, Ile and Val are enriched in bile acid binding peptides. The yields of bile acid binding peptides with an affinity of above the VAWWMY peptide (soystatin, control sequence) were significantly higher in the optimized structural rule (32.5%) compared to that of the random library (3.1%), and 6 peptides were obtained with above 2.0-fold increased binding activity.

Section snippets

Peptide array synthesis

A cellulose membrane (grade 542; Whatman, Maidstone, UK) was activated using β-alanine as the N-terminal basal spacer. Fmoc-11-aminoundecanoic acid (Watanabe Chemical Inc., Hiroshima, Japan) was conjugated as an additional spacer between the candidate peptides and the cellulose. Activated Fmoc amino acids (0.5 M) were spotted to the membrane using a peptide auto-spotter (ASP222; Intavis AG, Köln, Germany) in accordance with the manufacturer's instructions with some modifications. After first

FNN-assisted screening of PEPTIDES WITH HIGH AFFINITY FOR BILE ACIDS

First, we synthesized 2212 random 6-mer peptides on peptide arrays and assayed their affinity for bile acids. Using the bile acid binding capacity of these peptides, the highest (positive) and lowest (negative) 40 peptides were selected and converted to a numerical dataset (78 input parameters) through the data conversion process. Since peptide structural information was converted to input parameters representing physical properties from the exacted sequences, the fuzziness of peptide structure

Discussion

Bile acids play essential roles in the digestion and resorption of fat, fatty acids and lipid soluble vitamins. Bile acids in the enterohepatic circulation contribute to feedback regulation, which are important factor in serum cholesterol homeostasis. Nagaoka et al. (1) reported that a peptic hydrolysate of soy protein has a cholesterol-lowering effect similar to that of intact soy protein. The VAWWMY peptide (soystatin), which is the most hydrophobic region in soy glycinin A1a sequence, has a

Acknowledgments

This study was partially supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Sports, Science and Technology, Japan, No. 20380075 and 21360400.

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    These authors contributed equally to this work.

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    Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.