Purification and characterization of the mammaglobin/lipophilin B complex, a promising diagnostic marker for breast cancer.
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 masses-representing the fully glycosylated form, the complex without one of the carbohydrate chains, and the deglycosylated proteins-are 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 approximately 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 alpha-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.
Carter
Darrick
D
Department of Antigen Discovery, Corixa Corp., 1124 Columbia Street, Suite 200, Seattle, WA 98104, USA. carter@corixa.com
Douglass
John F
JF
Cornellison
Charisa D
CD
Retter
Marc W
MW
Johnson
Jeffrey C
JC
Bennington
Angela A
AA
Fleming
Timothy P
TP
Reed
Steven G
SG
Houghton
Raymond L
RL
Diamond
Deborah L
DL
Vedvick
Thomas S
TS
D000230:Adenocarcinoma / Q000175:diagnosis / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002942:Circular Dichroism; D005260:Female; D005914:Globins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D046911:Macromolecular Substances; D060785:Mammaglobin A; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009185:Myelin Proteins; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D021382:Protein Sorting Signals / Q000502:physiology; D011510:Proteolipids; D060765:Secretoglobins; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014598:Uteroglobin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D015415:Biomarkers; D002352:Carrier Proteins; D046911:Macromolecular Substances; D060785:Mammaglobin A; D009185:Myelin Proteins; D009363:Neoplasm Proteins; D021382:Protein Sorting Signals; D011510:Proteolipids; C556464:SCGB1D2 protein, human; C556448:SCGB2A2 protein, human; D060765:Secretoglobins; D005914:Globins; D014598:Uteroglobin