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Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

ABSTRACT: We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sEGFR as compared with membrane-bound EGFR from the A431 cell line. The unusual glycan structures were di-sialylated glycans (sialic acid attached to sialic acid) at Asn-151 and N-acetylhexosamine attached to a branched fucosylated galactose with N-acetylglucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn-420. These unusual glycans at specific sites were either present at a much lower level or were not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures was consistent with the observed expression of the corresponding ?-N-acetylneuraminide ?-2,8-sialyltransferase 2 (ST8SiA2) and ?-N-acetylneuraminide ?-2,8-sialyltransferase 4 (ST8SiA4), by quantitative real time RT-PCR. The connectivity present at the branched fucosylated galactose was also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry. We hypothesize that the presence of such glycan structures could promote secretion via anionic or steric repulsion mechanisms and thus facilitate the observation of these glycan forms in the secreted fractions. We plan to use this model system to facilitate the search for novel glycan structures present at specific sites in sEGFR as well as other secreted oncoproteins such as Erbb2 as markers of disease progression in blood samples from cancer patients.

SUBMITTER: Wu SL

PROVIDER: S-EPMC3650335 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

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Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

Wu Shiaw-Lin SL Taylor Allen D AD Lu Qiaozhen Q Hanash Samir M SM Im Hogune H Snyder Michael M Hancock William S WS

Molecular & cellular proteomics : MCP 20130131 5

We have used powerful HPLC-mass spectrometric approaches to characterize the secreted form of epidermal growth factor receptor (sEGFR). We demonstrated that the amino acid sequence lacked the cytoplasmic domain and was consistent with the primary sequence reported for EGFR purified from a human plasma pool. One of the sEGFR forms, attributed to the alternative RNA splicing, was also confirmed by transcriptional analysis (RNA sequencing). Two unusual types of glycan structures were observed in sE ...[more]

PMID: 23371026

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Project description:A cross-linking method is developed to elucidate glycan-mediated interactions between membrane proteins through sialic acids. The method provides information on previously unknown extensive glycomic interactions on cell membranes. The vast majority of membrane proteins are glycosylated with complicated glycan structures attached to the polypeptide backbone. Glycan-protein interactions are fundamental elements in many cellular events. Although significant advances have been made to identify protein-protein interactions in living cells, only modest advances have been made on glycan-protein interactions. Mechanistic elucidation of glycan-protein interactions has thus far remained elusive. Therefore, we developed a cross-linking mass spectrometry (XL-MS) workflow to directly identify glycan-protein interactions on the cell membrane using liquid chromatography-mass spectrometry (LC-MS). This method involved incorporating azido groups on cell surface glycans through biosynthetic pathways, followed by treatment of cell cultures with a synthesized reagent, N-hydroxysuccinimide (NHS)-cyclooctyne, which allowed the cross-linking of the sialic acid azides on glycans with primary amines on polypeptide backbones. The coupled peptide-glycan-peptide pairs after cross-linking were identified using the latest techniques in glycoproteomic and glycomic analyses and bioinformatics software. With this approach, information on the site of glycosylation, the glycoform, the source protein, and the target protein of the cross-linked pair were obtained. Glycoprotein-protein interactions involving unique glycoforms on the PNT2 cell surface were identified using the optimized and validated method. We built the GPX network of the PNT2 cell line and further investigated the biological roles of different glycan structures within protein complexes. Furthermore, we were able to build glycoprotein-protein complex models for previously unexplored interactions. The method will advance our future understanding of the roles of glycans in protein complexes on the cell surface.

Dataset Information

Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

Publications

Identification of potential glycan cancer markers with sialic acid attached to sialic acid and up-regulated fucosylated galactose structures in epidermal growth factor receptor secreted from A431 cell line.

Similar Datasets

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