Recombinant human tumor antigen MUC1 expressed in insect cells: structure and immunogenicity.
MUC1, a member of the mucin family of molecules, is a transmembrane glycoprotein abundantly expressed on human ductal epithelial cells and tumors originating from those cells. MUC1 expressed by malignant cells is aberrantly O-glycosylated. Differences in O-glycosylation of the tandem repeat region of MUC1 make tumor and normal forms of this antigen immunologically distinct. The tumor-specific glycoform is, therefore, expected to be a good target for immunotherapy and a good immunogen for generation of antitumor immune responses. We have generated a renewable source of this glycoform by expressing MUC1 cDNA in Sf-9 insect cells using a baculovirus vector. This form of MUC1 (BV-MUC1) is O-glycosylated at a very low level, approximately 0.3% (w/w), and this is not due to the lack of appropriate glycosylotransferases in insect cells. Peptidyl GalNAc-transferases isolated from Sf-9 cells were able to glycosylate in vitro a synthetic MUC1 peptide as efficiently as the transferases isolated from human milk. Neither preparation of peptidyl GalNAc-transferases, however, was able to glycosylate BV-MUC1. This underglycosylated recombinant MUC1 mimics underglycosylated MUC1 on human tumor cells and could serve as an immunogen to stimulate responses that would recognize MUC1 on tumor cells. To test this we immunized mice with Sf-9 cells expressing BV-MUC1. Sera from immunized mice recognized MUC1 on human tumor cells. We also generated MUC1-specific T cells that proliferated in response to synthetic MUC1 peptide.
Soares
M
M
Department of Molecular Genetics & Biochemistry, School of Medicine, University of Pittsburgh, Biomedical Science Tower, Room E 1240, Pittsburgh, PA 15261, USA.
Hanisch
F G
FG
Finn
O J
OJ
Ciborowski
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D005434:Flow Cytometry; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007075:Immunoglobulin M / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018411:Spodoptera / Q000166:cytology / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D020080:Tandem Repeat Sequences
D000939:Epitopes; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D018396:Mucin-1; D011994:Recombinant Proteins