GLYCOSYLATED EQUINE PROLACTIN AND ITS CARBOHYDRATE MOIETY

Citation
Vy. Butnev et al., GLYCOSYLATED EQUINE PROLACTIN AND ITS CARBOHYDRATE MOIETY, Journal of protein chemistry, 15(5), 1996, pp. 413-426
Citations number
56
Categorie Soggetti
Biology
ISSN journal
02778033
Volume
15
Issue
5
Year of publication
1996
Pages
413 - 426
Database
ISI
SICI code
0277-8033(1996)15:5<413:GEPAIC>2.0.ZU;2-9
Abstract
Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were p urified to homogeneity from side fractions obtained during isolation o f LH/FSH from horse pituitaries. Both PRL forms were isolated together in high yield by the isolation procedure used for glycosylated porcin e PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, N aCl 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 sequence s identical to the known primary structure of ePRL. Based on MALDI mas s spectrometry analysis and SDS-PAGE mobilities, -G-ePRL and ePRL had estimated molecular weights of 25,000 and 23,000 Da, respectively. G-e PRL displayed only 60% of the immunoreactivity of ePRL in homologous r adioimmunoassay, 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 Asn (31) was confirmed by isolation and sequence analysis of an enzymatica lly derived G-ePRL glycopeptide spanning residues 29-37. Monosaccharid e compositions of intact G-ePRL and this glycopeptide were very simila r (Man(3), GlcNAc(2), GalNAc(1), Fuc(0.6), Gal(0.2), NeuAc(0.15)) and resembled that of G-pPRL. The glycopeptide contained one sulfate resid ue as determined by ion chromatography after acid hydrolysis, indicati ng the presence of a sulfated monosaccharide, Comparative carbohydrate analysis of G-ePRL and other G-PRL preparations suggests that the fun ctionally significant Asn(31) carbohydrate unit is a fucosylated compl ex mono- and/or biantennary oligosaccharide terminating with a sulfate d GalNAc residue and two or three Man residues.