Rates of proximal and distal glycosylation of luteinizing hormone by cultured rat pituitary cells.
The purpose of the present work was to study the rate of basal luteinizing hormone (LH) glycosylation, discriminating the co-translational (proximal) and the post-translational (distal) glycosylation. The experiments were performed to determine the temporal relationship between the biosynthesis of the peptide chains (by [14C]leucine incorporation: [14C]Leu-LH) and the proximal (by [3H]mannose incorporation: [3H]Man-LH) and distal (by [3H]galactose incorporation: [3H]Gal-LH) glycosylation of LH, by rat pituitary cells in primary culture. In addition, the effects of cycloheximide (translation inhibitor) and tunicamycin (glycosylation inhibitor) on the rates of synthesis and release of [3H]Man-LH and [3H]Gal-LH were studied. The rates of synthesis and release (between 0 and 2 h and between 2 and 5 h) were calculated by regression analysis and the statistical significance of differences was determined by the slope test. The rate of synthesis of [3H]Man-LH (slope = 45.59) parallelled that of [14C]Leu-LH (slope = 41.39), which was in agreement with the assumption that the addition of the high mannose core is a co-translational event. Release of [3H]Man-LH (slope = 4.32) as well as that of [14C]Leu-LH (slope = 2.53) showed a lag period of approximately 2 h. The dynamics of [3H]Gal-LH secretion over the course of incubation, with a slower rate of synthesis (slope = 26.40) and a faster rate of release (slope = 6.34), differed from that of [3H]Man-LH. LH labeled with [3H]Gal was released from the early times of the incubation, indicating that galactose is added in the final stages of the secretory process into LH molecules, which are immediately released. LH translation blockage induced by cycloheximide was associated with a corresponding decrease of [3H]Man incorporation. On the other hand, addition of tunicamycin to the pituitary cells inhibited the rates of synthesis and release of [3H]Man-LH, without affecting those of [14C]Leu-LH. These findings show that proximal glycosylation depends on synthesis of the peptide chains, whereas the addition of the polymannose core is not a condition for translation. The rates of synthesis and release of [3H]Gal-LH were less affected by the antibiotics, and the inhibition was only significant at higher doses and long-time treatments. The present results demonstrate the independence of both steps of LH glycosylation, the rate of [3H]Gal-LH synthesis being 1.7-fold slower and that of release 1.5-fold faster than those of [3H]Man-LH, respectively. The data also suggest that glycosylation is not an essential step in the LH secretory process since the hormone, which is normally secreted in a glycosylated form, was synthesized, transported, and released without the carbohydrate side chains.
Pérez
G T
GT
Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Argentina.
Apfelbaum
M E
ME
D000818:Animals; D002250:Carbon Radioisotopes; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D005260:Female; D005690:Galactose / Q000378:metabolism / Q000493:pharmacokinetics; D006031:Glycosylation / Q000187:drug effects; D007930:Leucine / Q000378:metabolism / Q000493:pharmacokinetics; D007986:Luteinizing Hormone / Q000096:biosynthesis* / Q000378:metabolism; D008358:Mannose / Q000378:metabolism / Q000493:pharmacokinetics; D010902:Pituitary Gland / Q000378:metabolism*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D014316:Tritium; D014415:Tunicamycin / Q000494:pharmacology
D002250:Carbon Radioisotopes; D011500:Protein Synthesis Inhibitors; D014316:Tritium; D014415:Tunicamycin; D007986:Luteinizing Hormone; D003513:Cycloheximide; D007930:Leucine; D008358:Mannose; D005690:Galactose