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

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 mm) 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.

Discovery of Pancreatic Ductal Adenocarcinoma-Related Aberrant Glycosylations: A Multilateral Approach of Lectin Microarray-Based Tissue Glycomic Profiling With Public Transcriptomic Datasets

“…Previously this analysis could only be done using the method described for the glycan analyses of the O-glycoproteins in FFPE tissue sections (22,25). A high level of sialylated glycans, which is one of distinctive features of cancer cells (36), was successfully detected in the present tissue glycomic profiling, indicating the validity of these results.…”

“…After the incubation, 20 µg of human serum IgG (Sigma-Aldrich) was added to each well to block the interaction of free lectins with detection antibody for 30 min at 20 • C. After washing with PBSTx, the LecChip was incubated with the biotinylated anti-basigin antibody (100 ng) for 1 h at 20 • C. After washing with PBSTx, the LecChip was further incubated with Cy3conjugated streptavidin (200 ng; GE Healthcare) for 20 min at 20 • C. After washing with PBSTx, the LecChip was scanned using GlycoStation Reader 1200. The data processing was performed as described above to obtain the mean-normalized signal intensities as the glycan profiles of basigin (25). It should be noted that the normalized values of the glycan profiling data for basigin could not be compared between those of the tissue glycomic profiling data, owing to the batch-to-batch variations of the lectin array chips used for these analyses.…”

“…For the discovery of diseasespecific glycosylation alterations, the feasibility and utility of this tissue glycomic profiling has been demonstrated using diseased tissues (15)(16)(17)(18)(19)(20)(21). Since lectin microarray can be used for glycan analysis not only of crude samples but also of target glycoproteins enriched by immunoprecipitation (22), this technology has been utilized for the verification of diseaserelevant alterations of glycans attached to glyco-biomarker candidates (23)(24)(25)(26).…”

Aberrant glycosylation and cancer biomarker discovery: a promising and thorny journey

“…Fortunately, lectins have been considered as an extremely useful tool for glycomic and glycoproteomic study that can be used alone or in conjunction with other methods such as MS and CE because of the wide availability, affinity and relative specificity [93]. Several approaches based on lectins including lectin blot [94,95], lectin histochemistry/cytochemisty [29,96], lectin-antibody sandwich enzyme-linked immunosorbent assay (ELISA) [33,44,97], lectin affinity chromatography [98,99], lectin microarray [100][101][102] and lectin microfluidics [103] have been applied to capture the specific glycan of the glycoproteins [102,[104][105][106], enhancing the understanding of aberrant glycosylation and carcinogenesis, accelerating biomarkers recognition and quantitative detection. The Gao group has investigated the diagnostic and prognostic value of fucosylated fetuin A in HCC patients by AAL-based ELISA [107].…”

Glycoprotein biomarkers for the detection of pancreatic ductal adenocarcinoma

“…These authors also identified changes in MUC1 glycosylation, as the glycoform MUC1-T observed in normal pancreas became MUC1-STn in neoplastic stages. Recently, the study of immunoprecipitated MUC1 glycoforms using lectins has exposed its potential to discriminate between PaC and cholangiocarcinoma patients[ 124 ]. As mucins can be cleaved from cells and released to the bloodstream, all the specific glycoforms detected in tissues could be potentially detected in serum to be exploited as biomarkers.…”