Chem. non-glycosylated peptide (19). With this context, glycosylated mucins could represent important targets for the development of efficient immunotherapies. Using total tumor cell components, we recently explained the enzymatic preparation of MUC6 glycoproteins transporting the Tn antigen (20). The producing MUC6:Tn glycoprotein, prepared with breast tumor cell extract like a source of UDP-the first step of the mucin-type the Tn antigen synthesis (21). In the present study, we designed different mixtures of recombinant MK-4256 ppGalNAc-T1, -T2, and -T7 to carry out GalNAc enzymatic transfer onto the Ser and Thr residues of the MUC6 mucin. Indeed, as opposed to tumor cell components, the use of recombinant ppGalNAc-Ts with overlapping and/or complementary substrate specificities enables the production of and access to various protein glycoforms inside a reproducible and easy manner. A series of MUC6:Tn glycoproteins was MK-4256 produced, characterized, and their immunological properties were analyzed using numerous and assays. This study demonstrates these MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, the Tn glycosylation of the MUC6 protein strongly MK-4256 affected its immunogenicity by partially abrogating Th1 cell reactions and advertising the production of IL-17. Therefore, the design of glycoprotein-based vaccines should take into account the possible immunomodulating properties of glycosylation. EXPERIMENTAL Methods Mice 6- to 8-week-old woman BALB/c or C57BL/6 mice were from CER Janvier or Charles River. Animals were kept in the Pasteur Institute animal house in specific pathogen-free conditions, with water and food supplied and purified as previously explained (20). Briefly, a cDNA clone comprising a partial sequence of the tandem repeat of human being MUC6 was isolated from total cDNA of MCF7 breast tumor cells and indicated in Bli5 by induction with 1 mm isopropyl -d-thiogalactoside. The recombinant protein was purified over Ni2+-nitriloacetic acid columns under denaturing conditions according to the manufacturer’s (Qiagen, Hilden, Germany) instructions. The MUC6 recombinant protein was characterized by amino acid analysis and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), and was quantified by quantitative amino acid analysis (online peptide content). These analyses, together with N-terminal sequencing, showed that it lacks the N-terminal methionine residue. The 15-mer peptides, overlapping by five amino acids and spanning the sequence present in the recombinant MUC6 protein, were synthesized by PolyPeptide (Strasbourg, France). The amino acid sequences of the peptides are demonstrated in Fig. 4KM71H strain, as previously explained (20). Human being ppGalNAc-T2 and -T7 were cloned in pAcGp67 vector and indicated in insect cells, using the baculovirus system, from the Plateforme de Production de Protines Recombinantes (Institut Pasteur, Paris, Rabbit polyclonal to Receptor Estrogen alpha.ER-alpha is a nuclear hormone receptor and transcription factor.Regulates gene expression and affects cellular proliferation and differentiation in target tissues.Two splice-variant isoforms have been described. France). Enzymatic Synthesis of MUC6:Tn Glycoproteins The MUC6:Tn glycoproteins were acquired by enzymatic GalNAc transfer, using ppGalNAc transferases. Optimal conditions for glycosylation of both MUC6 proteins were determined following assays performed at analytical level, using SELDI-TOF MS (Ciphergen Biosystems, Fremont, CA), as explained (22). ppGalNAc-Ts were incubated separately or collectively at 37 C with uridine 5-diphospho-for 5 min. Cells were suspended in total culture medium, consisting of RPMI 1640 with GlutaMAX (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 50 m 2-mercaptoethanol, 100 devices/ml of penicillin, 100 mg/ml of streptomycin. Cells (1 106/well) were cultured for 72 h at 37 C and 5% CO2 in 96-well plates with MUC6 peptides (10 g/ml), MUC6:Tn glycoproteins or non-glycosylated MUC6 (1C10 g/ml). They were then pulsed with [3H]thymidine (ICN Biomedicals Inc.) for the last 18 h of tradition and harvested by an automated cell harvester (Skatron). Proliferation was determined by incorporation of the radioactivity from the cells and the results.