Disruption of N-linked glycosylation of bovine luteinizing hormone β-subunit by site-directed mutagenesis dramatically increases its intracellular stability but does not affect biological activity of the secreted heterodimer

Abstract

The single site for N-linked glycosylation of the β-subunit of bovine LH (LHβ) was disrupted by oligonucleotide-directed mutagenesis to assess its potential roles in the biosynthesis, transport, and hormonal activity of the LH α/β heterodimer. Pulse-chase studies performed with stably transfected Chinese hamster ovary cells that expressed both α subunit (fully glycosylated) and nonglycosylated LHβ revealed that turnover, transport, and secretion of newly synthesized, nonglycosylated LHβ were effectively blocked over a 22-h span. Free nonglycosylated LHβ, like free wild-type LHβ was sequestered inside the cell; therefore, the intracellular retention of uncombined LHβ-subunit is not due to a signal located within the N-glycan moiety. Nevertheless, an older pool of unlabeled, nonglycosylated LHβ-subunit was available for combination with newly synthesized α-subunit, as verified by immunoprécipitation of radiolabeled α-subunit from cell lysates and culture medium with anti-LHβ-antiserum. This heterodimer displayed normal kinetics of secretion (t1/2 = 2.4 h) as compared to fully glycosylated LH (t1/2 = 2.1 h). The wild-type and mutant forms of LH were also purified from culture supernatants of the two cell lines, and were compared for their relative abilities to stimulate progesterone secretion in cultured rat Leydig cells. Both proteins displayed similar potency (ED50 = 32 vs. 41 ng/ml, respectively) and maximal stimulation of progesterone release (Pmax = 2.7 vs. 2.5 µg/ml), indicating that N-linked glycosylation of the LHβ-subunit does not play a significant role in LH signal transduction. Collectively, these results indicate that N-linked glycosylation is important for intracellular degradation of free LHβ, but is not essential for either its assembly with α-subunit or the transport and secretion of biologically active heterodimer. © 1989 by The Endocrine Society.