A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides

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• 作者：Takahiko Matsushita ; Wataru Takada ; Kota Igarashi ; Kentaro Naruchi ; Risho Miyoshi ; Fayna Garcia-Martin ; Maho Amano ; Hiroshi Hinou ; Shin-Ichiro Nishimura
• 关键词：MUC1 glycopeptide ; Microarray ; Epitope mapping ; Non-specific protein adsorption ; Autoantibody
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：March, 2014
• 年：2014
• 卷：1840
• 期：3
• 页码：1105-1116
• 全文大小：1446 K

文摘

Background

Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear.

Methods

A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies.

Results

Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Ac伪2,3Gal尾1,3GalNAc伪1鈫? at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats.

Conclusion

We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption.

General significance

The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.