Purification and characterization of 66‐kDa glycoprotein from human breast carcinoma

Abstract

We extracted a 66-kDa glycoprotein (GP-1D8) from breast invasive ductal carcinoma tissues. The monoclonal antibody (mAb) against GP-1D8 was prepared in our laboratory. Western blotting with the purified protein using the mAb demonstrated a single band of 66 kDa. Immunocytochemical and immunohistochemical analysis revealed strong expression of GP-1D8 protein in the cytoplasm of MCF-7 cells and different types of breast carcinoma tissues, but GP-1D8 is absent in normal breast and benign breast tumor tissues. Glycosylation analysis showed GP-1D8 contained methylated salic acid. GP-1D8 was identified using mass-spectrometric techniques and N-terminal sequencing. These data were used to identify the protein through the SWISSPROT protein sequence database and BLAST homology search. These results showed GP-1D8 had some similarity to human albumin precursor. Co-immunoprecipitation assays of lysate from MCF-7 cells and mass spectrometric analysis revealed the interaction of GP-1D8 with beta-tubulin. This is the first time human breast carcinoma tissues and MCF-7 cells have been shown to express a 66-kDa glycoprotein similar to human albumin precursor. These results might be important in the detection of novel potential biomarkers and may provide insight into the molecular mechanisms of tumorigenesis.