Abstract
We used a targeted proteomics approach to investigate whether introduction of new N-linked glycosylation sites in a chimeric protein influence the glycosylation of the existing glycosylation sites. To accomplish our goals, we over-expressed and purified a chimeric construct that contained the Fc region of the IgG fused to the exons 7 & 8 of mouse ZP3 (IgG-Fc-ZP3E7 protein). Immunoglobulin heavy chain (IgG-HC protein) was used as control. We then analyzed the IgG-HC and IgG-Fc-ZP3E7 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and by Western blotting (WB). We concluded that in control experiments, the glycosylation site was occupied as expected. However, in the IgG-Fc-ZP3E7 protein, we concluded that only one out of three NXS/T glycosylation sites is occupied by N-linked oligosaccharides. We also concluded that in the IgG-Fc-ZP3E7 protein, upon introduction of additional potential NXS/T glycosylation sites within its sequence, the original NST/S glycosylation site from the Fc region of the IgG-Fc-ZP3E7 protein is no longer glycosylated. The biomedical significance of our findings is discussed.
Copyright © 2013 Elsevier B.V. All rights reserved.
Publication types
-
Research Support, Non-U.S. Gov't
-
Research Support, U.S. Gov't, Non-P.H.S.
MeSH terms
-
Amino Acid Motifs
-
Amino Acid Sequence
-
Animals
-
Chromatography, High Pressure Liquid
-
Egg Proteins / chemistry*
-
Egg Proteins / genetics
-
Egg Proteins / metabolism
-
Glycosylation
-
Immunoglobulin Fc Fragments / chemistry*
-
Immunoglobulin Fc Fragments / genetics
-
Immunoglobulin Fc Fragments / metabolism
-
Immunoglobulin G / chemistry*
-
Immunoglobulin G / genetics
-
Immunoglobulin G / metabolism
-
Membrane Glycoproteins / chemistry*
-
Membrane Glycoproteins / genetics
-
Membrane Glycoproteins / metabolism
-
Mice
-
Molecular Sequence Data
-
Oligosaccharides / chemistry*
-
Oligosaccharides / metabolism
-
Receptors, Cell Surface / chemistry*
-
Receptors, Cell Surface / genetics
-
Receptors, Cell Surface / metabolism
-
Recombinant Proteins / chemistry*
-
Recombinant Proteins / genetics
-
Recombinant Proteins / metabolism
-
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization*
-
Zona Pellucida Glycoproteins
Substances
-
Egg Proteins
-
Immunoglobulin Fc Fragments
-
Immunoglobulin G
-
Membrane Glycoproteins
-
Oligosaccharides
-
Receptors, Cell Surface
-
Recombinant Proteins
-
Zona Pellucida Glycoproteins
-
Zp3 protein, mouse