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IN VITRO MODULATION OF OLIGOSACCHARIL-TRANSFERASE EXPRESSION BY PROGESTERONE MB Pradosa, J Caramelob and S Mirandaa(smiranda@ffyb.uba.ar). aIDEHU (CONICETUBA), bFundacio´n Inst. Leloir-Buenos Aires, Argentina. Asymmetric antibodies (AAb) are antibodies that have an extra N-linked oligosaccharide in one of the two Fab regions. It has been demonstrated that progesterone (P4) can modulate its synthesis by a murine hybridome. However, the mechanisms involved in this regulation are not understood. Oligosaccharyltransferase (OST) is an integral membrane protein that catalizes N-linked glycosylation of nascent polipeptides in the lumen of the endoplasmic reticulum. Mammalian organisms express several OST isoforms. OST isoforms that incorporate STT3-B as a subunit are more active and have a reduced ability to discriminate between donor substrates than those OST that incorporate STT3-A. It has been suggested that differences in expression on the enzymatically distinct OST isoforms might provide an explanation for tissue specific glycan heterogeneity.To investigate the involvement of OST in AAb synthesis, here we studied the influence of progesterone on STT3-B and STT3-A expression in a murine hybridoma able to secrete both symmetric and AAb. The cells were incubated with P4 (0, 10-5, 10-6, 10-8, 10-9 and 10-10M) for 48 h. STT3-B and STT3-A expression was measured by western blot using two polyclonal antibodies that specifically recognize STT3-B or STT3-A in cell microsomal fractions. Our results indicate that P4 10-8 M increased STT3-B expression (around 70%) while STT3-A expression was not modified. P4 10-9 M also augmented STT3-B expression but diminished that of STT3-A. P4 10-7 M only diminished STT3-B (aprox. 70%). P4 10-5 M and 10-6 M did not modify the expression of any isoform. These preliminary results suggested that P4 might modulate OST activity, and in this way, this hormone could be involved in immunoglobulin glycan heterogeneity. Asymmetric antibodies (AAb) are antibodies that have an extra N-linked oligosaccharide in one of the two Fab regions. It has been demonstrated that progesterone (P4) can modulate its synthesis by a murine hybridome. However, the mechanisms involved in this regulation are not understood. Oligosaccharyltransferase (OST) is an integral membrane protein that catalizes N-linked glycosylation of nascent polipeptides in the lumen of the endoplasmic reticulum. Mammalian organisms express several OST isoforms. OST isoforms that incorporate STT3-B as a subunit are more active and have a reduced ability to discriminate between donor substrates than those OST that incorporate STT3-A. It has been suggested that differences in expression on the enzymatically distinct OST isoforms might provide an explanation for tissue specific glycan heterogeneity.To investigate the involvement of OST in AAb synthesis, here we studied the influence of progesterone on STT3-B and STT3-A expression in a murine hybridoma able to secrete both symmetric and AAb. The cells were incubated with P4 (0, 10-5, 10-6, 10-8, 10-9 and 10-10M) for 48 h. STT3-B and STT3-A expression was measured by western blot using two polyclonal antibodies that specifically recognize STT3-B or STT3-A in cell microsomal fractions. Our results indicate that P4 10-8 M increased STT3-B expression (around 70%) while STT3-A expression was not modified. P4 10-9 M also augmented STT3-B expression but diminished that of STT3-A. P4 10-7 M only diminished STT3-B (aprox. 70%). P4 10-5 M and 10-6 M did not modify the expression of any isoform. These preliminary results suggested that P4 might modulate OST activity, and in this way, this hormone could be involved in immunoglobulin glycan heterogeneity. a, J Caramelob and S Mirandaa(smiranda@ffyb.uba.ar). aIDEHU (CONICETUBA), bFundacio´n Inst. Leloir-Buenos Aires, Argentina. Asymmetric antibodies (AAb) are antibodies that have an extra N-linked oligosaccharide in one of the two Fab regions. It has been demonstrated that progesterone (P4) can modulate its synthesis by a murine hybridome. However, the mechanisms involved in this regulation are not understood. Oligosaccharyltransferase (OST) is an integral membrane protein that catalizes N-linked glycosylation of nascent polipeptides in the lumen of the endoplasmic reticulum. Mammalian organisms express several OST isoforms. OST isoforms that incorporate STT3-B as a subunit are more active and have a reduced ability to discriminate between donor substrates than those OST that incorporate STT3-A. It has been suggested that differences in expression on the enzymatically distinct OST isoforms might provide an explanation for tissue specific glycan heterogeneity.To investigate the involvement of OST in AAb synthesis, here we studied the influence of progesterone on STT3-B and STT3-A expression in a murine hybridoma able to secrete both symmetric and AAb. The cells were incubated with P4 (0, 10-5, 10-6, 10-8, 10-9 and 10-10M) for 48 h. STT3-B and STT3-A expression was measured by western blot using two polyclonal antibodies that specifically recognize STT3-B or STT3-A in cell microsomal fractions. Our results indicate that P4 10-8 M increased STT3-B expression (around 70%) while STT3-A expression was not modified. P4 10-9 M also augmented STT3-B expression but diminished that of STT3-A. P4 10-7 M only diminished STT3-B (aprox. 70%). P4 10-5 M and 10-6 M did not modify the expression of any isoform. These preliminary results suggested that P4 might modulate OST activity, and in this way, this hormone could be involved in immunoglobulin glycan heterogeneity.-5, 10-6, 10-8, 10-9 and 10-10M) for 48 h. STT3-B and STT3-A expression was measured by western blot using two polyclonal antibodies that specifically recognize STT3-B or STT3-A in cell microsomal fractions. Our results indicate that P4 10-8 M increased STT3-B expression (around 70%) while STT3-A expression was not modified. P4 10-9 M also augmented STT3-B expression but diminished that of STT3-A. P4 10-7 M only diminished STT3-B (aprox. 70%). P4 10-5 M and 10-6 M did not modify the expression of any isoform. These preliminary results suggested that P4 might modulate OST activity, and in this way, this hormone could be involved in immunoglobulin glycan heterogeneity.

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