Technical Report

Models and Techniques

Assessment of tumor characteristics based on glycoform analysis of membrane-tethered MUC1

  • Laboratory Investigation volume 97, pages 11031113 (2017)
  • doi:10.1038/labinvest.2017.53
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Abstract

Clinical tissue specimens are useful for pathological diagnosis, which is, in some cases, supported by visualization of biomolecule localization. In general, diagnostic specificity in molecular pathology is increased by the acquisition of a probe to distinguish the modification of isomers. Although glycosylation is one of the candidate modifications in a protein, comparative glycan analysis of disease-associated proteins derived from a single tissue section is still challenging because of the lack of analytical sensitivity. Here we demonstrate a possible method for differential glycoform analysis of an endogenous tumor-associated glycoprotein MUC1 by an antibody-overlay lectin microarray. Tissue sections (5 μm thick) of patients with cholangiocarcinoma (CCA; n=21) and pancreatic ductal adenocarcinoma (PDAC; n=50) were stained with an anti-MUC1 antibody MY.1E12 that was established as a monoclonal antibody recognizing an MUC1 glycosylation isoform with a sialyl-core 1 structure (NeuAcα2-3galactosyl β1-3-N-acetylgalactosamine). MY.1E12-positive tissue areas (2.5 mm2) were selectively dissected with a laser capture microdissection procedure. The membrane MUC1 was enriched by immunoprecipitation with MY.1E12 and subjected to lectin microarray analysis. Even though the reactivities of MY.1E12 between CCA and PDAC were similar, the lectin-binding patterns varied. We found Maackia amurensis leukoagglutinin and pokeweed lectin distinguished MY.1E12-reactive MUC1 of CCA from that of PDAC. Moreover, MUC1 with M. amurensis hemagglutinin (MAH) reactivity potentially reflected the degree of malignancy. These results were confirmed with MAH-MY.1E12 double fluorescent immunostaining. These glycan changes on MUC1 were detected with high sensitivity owing to the cluster effect of immobilized lectins on a tandem repeat peptide antigen covered with highly dense glycosylation such as mucin. Our approach provides the information to investigate novel glycodynamics in biology, for example, glycoalteration, as well as diseases related to not only MUC1 but also other membrane proteins.

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Acknowledgements

We thank Ms Azumi Takahashi for assistance in preparing this manuscript. This work was supported by a Grant-in-Aid for Young Scientists (B) (30722590).

Author information

Affiliations

  1. Glycomedicine Technology Research Center (GTRC), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan

    • Atsushi Matsuda
    • , Tomomi Nakagawa
    • , Atsushi Kuno
    •  & Hisashi Narimatsu
  2. Department of Pathology, Field of Oncology, Graduate School of Medical and Dental Sciences, Kagoshima University, Sakuragaoka, Kagoshima, Japan

    • Michiyo Higashi
    • , Seiya Yokoyama
    •  & Suguru Yonezawa

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Competing interests

The authors declare no conflict of interest.

Corresponding authors

Correspondence to Atsushi Kuno or Hisashi Narimatsu.

Supplementary information

Supplementary Information accompanies the paper on the Laboratory Investigation website (http://www.laboratoryinvestigation.org)