Title
Purification and Characterization of the Mammaglobin/Lipophilin B Complex, a Promising Diagnostic Marker for Breast Cancer
Author
Darrick Carter, John F. Douglass, Charisa D. Cornellison, Marc W. Retter, Jeffrey C. Johnson, Angela A. Bennington, Timothy P. Fleming, Steven G. Reed, Raymond L. Houghton, Deborah L. Diamond, Thomas S. Vedvick
Year
2002
Journal
Biochemistry
Abstract
Mammaglobin, a promising diagnostic marker for breast cancer, forms a covalent complex with lipophilin B. mRNA levels for each component of the complex were determined for a number of breast tumors and normal tissues, and correlation of message expression was highly significant between mammaglobin and lipophilin B (p < 0.0001). The complex was purified by both standard biochemical techniques and immunoaffinity chromatography. N-Terminal sequencing revealed that mammaglobin and lipophilin B are processed as predicted by cleavage of their signal sequence after amino acids 19 and 21, respectively. Three molecular massesrepresenting the fully glycosylated form, the complex without one of the carbohydrate chains, and the deglycosylated proteinsare detected by ProteinChip array SELDI-TOF mass spectrometry after partial enzymatic deglycosylation. This is consistent with the two predicted N-linked glycosylation sites in the primary sequence of mammaglobin and each site having an attached sugar of ∼3500 Da. Reducing agents release lipophilin B from mammaglobin, and the free peptides are seen at their predicted molecular masses in the deglycosylated complex. Molecular modeling, secondary structure prediction, and circular dichroism indicate that the complex is a small α-helical globule that has three disulfide bridges and a carbohydrate chain at each pole. LC-ESI-MS shows that mammaglobin and lipophilin B are bonded in a head to tail orientation. This work describes the biochemistry of the mammaglobin/lipophilin B complex and lays the framework for use of this complex as a novel protein-based serological marker for breast cancer.
Full Article
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Biochemistry