Biochemical characterization of the soluble form of tumor antigen MUC1 isolated from sera and ascites fluid of breast and pancreatic cancer patients.
Transmembrane glycoprotein tumor antigen MUC1 that is overexpressed on pancreatic and breast tumor cells can be found in large amounts in soluble form in serum and ascites fluid. MUC1 has been identified as a target of human antitumor antibody and CTL responses that are generated in the absence of helper T cells. The soluble form of MUC1 should support generation of helper T cells, but we have found recently that this form, although effectively endocytosed by dendritic cells, remains trapped in early endosomes and is not trafficked to antigen-processing compartments. The exact biochemical structure of this form of MUC1 has not been elucidated to date, and it is thus not clear what structural characteristics may be responsible for its retention in early endosomes. We have purified soluble MUC1 from ascites fluid of breast/pancreatic cancer patients (ASC-MUC1) and quantitated O-linked carbohydrates. We have altered ASC-MUC1 by enzymatic treatment: trypsin or clostripain digestion, desialylation, and further in vitro glycosylation. We have found that desialylated ASC-MUC1 was further glycosylated by peptidyl N-acetylgalactosamine transferases and was not when sialic acid was present. These alterations created new forms of ASC-MUC1 that might be handled more efficiently by antigen-presenting cells to generate better tumor-specific immunity and used to identify structures that are directly involved in retention of this antigen in early endosomes.
Beatty
P
P
Department of Molecular Genetics and Biochemistry, School of Medicine, University of Pittsburgh, Pennsylvania 15261, USA.
Hanisch
F G
FG
Stolz
D B
DB
Finn
O J
OJ
Ciborowski
P
P
D001201:Ascites / Q000378:metabolism*; D001943:Breast Neoplasms / Q000097:blood* / Q000378:metabolism*; D002241:Carbohydrates / Q000737:chemistry; D003546:Cysteine Endopeptidases / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D015151:Immunoblotting; D008854:Microscopy, Electron; D008957:Models, Genetic; D009005:Monosaccharides / Q000378:metabolism; D018396:Mucin-1 / Q000096:biosynthesis* / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009439:Neuraminidase / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000097:blood* / Q000378:metabolism*; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D014357:Trypsin / Q000494:pharmacology
D002241:Carbohydrates; C116055:MUC1 tandem repeat peptide; D009005:Monosaccharides; D018396:Mucin-1; D010446:Peptide Fragments; D009439:Neuraminidase; D014357:Trypsin; D003546:Cysteine Endopeptidases; C019913:clostripain; D019158:N-Acetylneuraminic Acid