Analysis of a heterogeneous group of human breast carcinoma associated glycoproteins bearing the Tn determinant.
The Tn determinant (GalNAc alpha-O-Ser/Thr) is expressed by about 90% of human carcinomas, but is cryptic in most normal human tissues. A murine monoclonal antibody (MAb) 83D4, developed following immunization with human breast carcinoma cells, reacts with a Tn-related epitope. In the present study we characterized the glycoprotein antigen identified by 83D4 in the human breast carcinoma cell line MCF-7. We further showed that the 83D4 antigenic determinant is masked in human milk fat globule membranes (HMFGM), and can be exposed upon mild m-periodate treatment after desialylation. Western-blot analysis resolved the 83D4 antigen from MCF-7 into two main components of 120-190 kD and > 500 kD respectively. Non equilibrium pH gradient electrophoresis/SDS PAGE revealed the acidic nature of the reactive glycoproteins (pI 4.43-4.70). 83D4 antigenic activity resolved by CsCl gradient ultracentrifugation layered on a wide range of densities (1.30-1.46 g/ml) including typical densities of mucin-like glycoproteins but also lower densities. The amino acid composition of the antigen, relatively rich in serine but poor in threonine and proline, confirmed the divergence from other mucin-like carcinoma-associated glycoproteins. Dicarboxylic amino acids were abundant, accounting in part for the acidic nature of the molecules. ELISA and Western-blot analysis of the subcellular fractions from MCF-7 cells revealed that the 83D4 antigen is mainly contained in plasma membranes (85%) from which it may be resolved into two broad bands (slow and fast migrating components). These results provide information on a group of breast carcinoma associated glycoproteins related to but different from typical mucins, and provide data on alteration of O-glycosylation in tumor cells.
Osinaga
E
E
Division D'Immuno-Cytologie Appliquée, CNRS URA 1442, Université de Technologie de Compiègne, France.
Pancino
G
G
Porchet
N
N
Berois
N
N
De Cremoux
P
P
Mistro
D
D
Aubert
J P
JP
Calvo
F
F
Roseto
A
A
D000230:Adenocarcinoma / Q000737:chemistry* / Q000276:immunology; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000951:Antigens, Neoplasm / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000032:analysis* / Q000276:immunology; D001943:Breast Neoplasms / Q000737:chemistry* / Q000276:immunology; D002240:Carbohydrate Sequence; D000939:Epitopes / Q000276:immunology; D005260:Female; D006023:Glycoproteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008895:Milk, Human / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018396:Mucin-1; D009077:Mucins / Q000032:analysis / Q000276:immunology; D009363:Neoplasm Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D009439:Neuraminidase / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D013347:Subcellular Fractions / Q000737:chemistry; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015295:Antigens, Tumor-Associated, Carbohydrate; D000939:Epitopes; D006023:Glycoproteins; D037102:Lectins; D008562:Membrane Glycoproteins; D018396:Mucin-1; D009077:Mucins; D009363:Neoplasm Proteins; C040286:Tn antigen; D009439:Neuraminidase