Purification and characterization of 66-kDa glycoprotein from human breast carcinoma.
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.
Feng
Renqing
R
Department of Biochemistry, College of Life Sciences, Peking University, Beijing 100871, China. rqfeng@pku.edu.cn
Liu
Xiang
X
Guo
Zhenquan
Z
Li
Jinfeng
J
D000595:Amino Acid Sequence; D000818:Animals; D014408:Biomarkers, Tumor / Q000737:chemistry / Q000302:isolation & purification*; D001943:Breast Neoplasms / Q000737:chemistry* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000737:chemistry* / Q000473:pathology; D045744:Cell Line, Tumor; D005260:Female; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry / Q000302:isolation & purification*; D010446:Peptide Fragments / Q000737:chemistry; D011498:Protein Precursors / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry
D014408:Biomarkers, Tumor; D006023:Glycoproteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; D011498:Protein Precursors; D012709:Serum Albumin