Isolation and characterization of two biologically active O-glycosylated forms of human parathyroid hormone produced in Saccharomyces cerevisiae. Identification of a new motif for O-glycosylation.
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 O-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 O-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 O-glycosylation of hPTH with up to seven mannosyl residues/molecule did not affect the biological activity of the hormone, making possible production of hPTH with potential different pharmacokinetic properties.
Olstad
O K
OK
Institute of Medical Biochemistry, University of Oslo, Norway.
Reppe
S
S
Gabrielsen
O S
OS
Hartmanis
M
M
Blingsmo
O R
OR
Gautvik
V T
VT
Haflan
A K
AK
Christensen
T B
TB
Oyen
T B
TB
Gautvik
K M
KM
D000262:Adenylyl Cyclases / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D013058:Mass Spectrometry; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010281:Parathyroid Hormone / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014357:Trypsin / Q000737:chemistry
D000596:Amino Acids; D002241:Carbohydrates; D010281:Parathyroid Hormone; D010455:Peptides; D011994:Recombinant Proteins; D000072235:Mating Factor; D004247:DNA; D014357:Trypsin; D000262:Adenylyl Cyclases