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

Takahiko Matsushita; Wataru Takada; Kota Igarashi; Kentaro Naruchi; Risho Miyoshi; Fayna Garcia-Martin; Maho Amano; Hiroshi Hinou; Shin-Ichiro Nishimura

doi:10.1016/j.bbagen.2013.11.009

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

Biochim Biophys Acta. 2014 Mar;1840(3):1105-16. doi: 10.1016/j.bbagen.2013.11.009. Epub 2013 Nov 16.

Authors

Takahiko Matsushita¹, Wataru Takada², Kota Igarashi², Kentaro Naruchi³, Risho Miyoshi³, Fayna Garcia-Martin¹, Maho Amano⁴, Hiroshi Hinou⁴, Shin-Ichiro Nishimura⁵

Affiliations

¹ Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan.
² Sumitomo Bakelite Co., Ltd., Tokyo, Japan.
³ Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan.
⁴ Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan; Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan.
⁵ Field of Drug Discovery Research, Faculty of Advanced Life Science, Hokkaido University, N22, W11 Kita-ku, Sapporo 001-0021, Japan; Medicinal Chemistry Pharmaceuticals, Co. Ltd., N22, W12, Kita-ku, Sapporo 001-0021, Japan. Electronic address: shin@sci.hokudai.ac.jp.

PMID: 24246952
DOI: 10.1016/j.bbagen.2013.11.009

Abstract

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 (Neu5Acα2,3Galβ1,3GalNAcα1→) 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.

Keywords: Autoantibody; Epitope mapping; MUC1 glycopeptide; Microarray; Non-specific protein adsorption.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Antibodies, Monoclonal / immunology
Autoantibodies / blood
Epitope Mapping*
Humans
Molecular Sequence Data
Mucin-1 / immunology*
Peptide Fragments / immunology*
Peptide Library*
Protein Array Analysis / methods*

Substances

Antibodies, Monoclonal
Autoantibodies
Mucin-1
Peptide Fragments
Peptide Library