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.

Publication Type:

Journal Article

Source:

Molecular & cellular proteomics : MCP (2013)

Keywords:

February 2013, Public Health Sciences Division

Abstract:

WWe have used a 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, is also confirmed by the transcriptional analysis (RNA- sequencing). Two unusual types of glycan structures were observed in secreted epidermal growth factor receptor (sEGFR) as compared to 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-acetyl hexosamine attached to a branched fucosylated galactose with N-acetyl glucosamine moieties (HexNAc-(Fuc)Gal-GlcNAc) at Asn 420. These unusual glycans at specific sites were either present at much lower level or not observable in membrane-bound EGFR present in the A431 cell lysate. The observation of these di-sialylated glycan structures is consistent with the observed expression of the corresponding alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 2 (ST8SiA2) and alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 4 (ST8SiA4), by quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR). The connectivity present the branched fucosylated galactose also confirmed by methylation of the glycans followed by analysis with sequential fragmentation in mass spectrometry (MSn). 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 makers of disease progression in blood samples from cancer patients.