Miyagawa Yu
Determination of trypsin-digested α-casein peptides that effectively inhibit Concanavalin A aggregation
Waseda University

A number of neurodegenerative diseases including Alzheimer's disease are known to involve protein aggregation.Thus, the inhibition of aggregation in protein solutions is currently a subject of great interest in many research fields that range from the study of protein-misfolding related diseases to pharmaceutics, biotechnology, and food science.It has been shown that α-casein, one of the four types of caseins that are the largest protein component of bovine milk, hinders the aggregation process of several proteins, including amyloid β-peptide, which is involved in Alzheimer's disease.In this context, we performed an experimental study on Concanavalin A (Con A) aggregation and used it as a model to study the aggregation processes.To shed light into the mechanisms by which α-casein exerts this chaperone-like protective action, we examined which sequences of α-casein inhibited Con A aggregation, by means of a turbidity assay , a MTT assay and MALDI-TOF MS.We first confirmed that the peptides released by the trypsin digestion of α-casein were potent in blocking Con A aggregation and preventing cytotoxicity in neuronal NG108-15 cells.These peptides were pooled and applied to Con A-Sepharose, and Con A-sepharose binding peptides were eluted with 0.1 M methyl a-D-mannopyranoside.The turbidity assay and MTT assays showed that the peptides inhibited Con A aggregation and were neuroprotective.When these peptides were analyzed by mass spectrometry, only a few peaks were detected.The sequences of the peaks corresponded to those generated by the trypsin digestion of α-casein.Therefore, these peptides offer an encouraging opportunity to prevent protein aggregation.