Isolation and characterization of two biologically active O‐glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae

Abstract

Expression and secretion of human parathyroid hormone in Saccharomyces cerevisiae were achieved by fusing a cDNA encoding the mature human parathyroid hormone (hPTH) to the preproregion of the yeast mating factor alpha. Purified hPTH from yeast-culture medium was found to contain, in addition to the native unglycosylated form, two mannosylated variants with different molecular masses. The three hPTH forms were processed identically, resulting in the same 84 amino acid polypeptides with amino acid sequences identical to the native hormone. In both the 0-glycosylated forms that were separated by isocratic reverse-phase HPLC, two mannose-linked residues were localized to Thr79. In addition, the most glycosylated form showed a heterogeneous modification of three, four or five mannosyl residues linked at Ser66. Lysine is N-terminally located to Ser66 and probably stimulates this glycosylation, which introduces a possible new motif for 0-glycosylation in yeast. The two glycosylated forms of hPTH had similar biological activity which was identical to the native form of hPTH in a hormone-sensitive adenylate cyclase assay in bone sarcoma cells. Thus, a C-terminal 0-glycosylation of hPTH with upto seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.Parathyroid hormone is the principle regulator of calcium homeostasis in human and has been advanced as an anabolic drug against post-menopausal osteoporosis [l, 21. The hormone, which is produced in the mammalian parathyroid glands, is synthesized as an 11 5-amino-acid precursor that is processed to the mature hormone of 84 amino acids [3].The physiological action of human parathyroid hormone (hPTH) is probably to generate a positive calcium balance and bone formation through its direct effects on the kidney and skeleton and through indirect effects via renal hydroxylation of 25-hydroxy-vitamin D3 to the active 1,25-dihydroxyvitamin D3. Ahhreviations. hPTH, human parathyroid hormone; hPTH-glycl , recombinant human parathyroid hormone containing one 0-glycosylation site; hPTH-glyc2, recombinant human parathyroid hormone containing two 0-glycosylation sites; MFa, yeast mating factor alpha; Pa-n, theoretical fragments after digestion with protease from Stuphylococcus rrureus, where n is the number given to the fragments starting from the N-terminus; T-n, theoretical fragments after digestion with trypsin where n is the number given to the fragments starting from the N-terminus.Due to shortage of supply, only the N-terminal 1-34 amino acid fragment of hPTH has been used in most experiments and clinical trials. One rational was that this peptide fragment, which is not a natural metabolic product, does, however, have biological activity [4].We show that cloned hPTH cDNA [5] expressed in Saccharomyces cerevisiae under the control of the o! mating factor (MFa) promoter [6] results in secretion into the medium of both the intact hormone and two 0-glycosylated forms...

Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae

“…The large majority of the glycosylated material is not truncated at the C terminus, suggesting that 0-glycosylation protects rLDTI from C-terminal degradation. 0-Glycosylation with mannose residues is a common covalent modification of homologous yeast secretory proteins (Kukuruzinska et al, 1987) and has also been reported for heterologous proteins expressed in yeast (Olstad et al, 1992;Yamada et al, 1994). In all cases described, only a portion of the secreted foreign polypeptide is glycosylated and the parameters that determine the amount and extent of glycosylation are poorly understood.…”

Glycosylation of rat NGF receptor ectodomain in the yeast Saccharomyces cerevisiae

“…Little information is so far available on O-glycosylation of mammalian proteins in yeast. Selected sites of the FeE receptor, parathyroid hormone, cell-adhesive lysozyme and insulin-like growth factor, unoccupied in the authentic molecules, were O-glycosylated in yeast [18][19][20][21], whereas the same sites of granulocyte/macrophage colony-stimulating factor were Oglycosylated in yeast and mammalian cells [22].…”

Binding and Cyclic AMP Stimulation by N-Terminally Deleted Human PTHs (3–84 and 4–84) in a Homologous Ligand Receptor System

“…(37,38) In brief, the media were adjusted to pH 3.0 with 1 M HCl before loading. The column was then washed with 0.1 M acetic acid (adjusted to pH 6.0) and eluted with 0.1 M Na 2 HPO 4 , pH 8.5.…”

“…The expression plasmid p␣UXPTH-2 (37,38) constructed for production of authentic hPTH(1-84) in yeast was mod-…”