Glycosylated equine prolactin and its carbohydrate moiety

Abstract

Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were purified to homogeneity from side fractions obtained during isolation of LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcine PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, NaCl and isoelectric precipitation, and gel filtration. Purification of G-ePRL required additional Con A chromatography. The N-terminal amino acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequences identical to the known primary structure of ePRL. Based on MALDI mass spectrometry analysis and SDS-PAGE mobilities, G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-ePRL displayed only 60% of the immunoreactivity of ePRL in homologous radioimmunoassay. Using the Nb2 lymphoma cell bioassay, ePRL was found to have about 1/30th the mitogenic activity of bovine PRL; G-ePRL was approximately 1/10th as active as ePRL. Glycosylation of G-ePRL at Asn31 was confirmed by isolation and sequence analysis of an enzymatically derived G-ePRL glycopeptide spanning residues 29-37. Monosaccharide compositions of intact G-ePRL and this glycopeptide were very similar (Man3, GlcNAc2, GalNAc1, Fuc0.6, Gal0.2, NeuAc0.15) and resembled that of G-pPRL. The glycopeptide contained one sulfate residue as determined by ion chromatography after acid hydrolysis, indicating the presence of a sulfated monosaccharide. Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the functionally significant Asn31 carbohydrate unit is a fucosylated complex mono- and /or biantennary oligosaccharide terminating with a sulfated GalNAc residue and two or three Man residues.

Synthesis, purification and characterization of recombinant glycosylated human prolactin (G-hPRL) secreted by cycloheximide-treated CHO cells

“…The presence of a unique and partially occupied glycosylation site in Asn-31 in human, monkey, ovine, porcine, dromedary, equine and whale PRL (Sinha, 1995;Butnev et al, 1996) makes it an ideal model of glycosylation for N-glycan studies since it exhibits the simplest type of glycosylation macroheterogeneity, with an occupancy range of 10-30% of G-hPRL relative to the total hPRL of either pituitary or recombinant origin (Shelikoff et al, 1994(Shelikoff et al, , 1996Price et al, 1995;Sinha, 1995; Abbreviations: CHO, Chinese hamster ovary; CRS, chemical reference standard; hPRL, human prolactin; HPSEC, size-exclusion HPLC; MALDI-TOF-MS, matrixassisted laser desorption ionization time-of-flight mass spectrometry; Mr, relative molecular mass; PAGE, polyacrylamide-gel electrophoresis; SDS, sodium dodecyl sulfate; RP-HPLC, reversed-phase HPLC; tR, retention time.…”