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 α-N-acetylneuraminide α-2,8-sialyltransferase 2 (ST8SiA2) and α-N-acetylneuraminide α-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.
Highlights
From the §Barnett Institute, Northeastern University, Boston, Massachusetts 01225, the ¶Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, the Department of Genetics, Stanford University School of Medicine, Palo Alto, California 94305, and the **Department of Integrated OMICS for Biomedical Science, Yonsei University, Seoul, 120 –749, Korea
The Western blot using anti-epidermal growth factor receptor (EGFR) polyclonal antibody for the A431 total media, EGFR recombinant protein standard, and product of EGFR purification are shown in supplemental Fig. S1A, panel B, along with glycoprotein staining for recombinant EGFR and the product of the EGFR purification
Partial Full Unknown a Data were obtained from the analysis of full-length EGFR in a previous study [24]. b We found unusual glycan structures in secreted EGFR (sEGFR). c We found complex-type glycan structure in circulating EGFR. d We found complex-type glycan structure in sEGFR. e Glycosylation only occurred in sEGFR, in which both Asn-599 and Asn-615 are located in the same Lys-C-digested peptide, and the
Summary
A431 Media Collection—A431 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, 11965) supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere in 5% (v/v) CO2. For EGFR glycopeptides, the likely glycan structure in a glycopeptide was initially assigned by applying the mass obtained from the difference of a glycopeptide and its deglycosylated counterpart to match against the masses of the glycans in the Glycosuite database (Proteome Systems, Sydney, Australia). Among these likely glycostructure candidates, the best assignment was selected from the preferred fragmentation patterns obtained in the related MSn spectra
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
