title: Glycosylated rat prolactin: isolation and structural characterization.
abstract: Isolation of glycosylated 26 kDa rat prolactin and subsequent proper carbohydrate characterization has so far not been reported. In the present work the hormone isoform was isolated to 95% homogeneity by preparative electrophoretic separation on Mini Prep Cell of rat pituitary homogenate. The isoform was then investigated by 2-mercaptoethanol gradient electrophoresis, Cleveland's sequential SDS-PAGE, digestion with endoproteinase Asp-N and N-glycanase. The glycosidic part of the isoform was examined in O-profiling and its monosaccharide composition obtained by FACE and HPAE-PAD analysis. The outcome of the experimental data is: 1) in contrast to unglycosylated 23 kDa rat prolactin, intra-chain S-S bridging is not affected in 26kDa rat prolactin, neither by transiting through a thiol gradient nor in sequential nonreducing/reducing SDS-PAGE; 2) the conformational availability of Asp residues involved in the endoproteinase Asp-N attack is the same in 23- and 26 kDa rat prolactin; the glycan moiety apparently does not cause steric hindrance at this level; 3) no glycosidic N-linkage could be detected, only O-linkage(s); 4) 26 kDa rat prolactin is no glycosyl-phosphaditylinositol-anchored protein; 5) in O-profiling an oligosaccharide chain of Mr +/- 1.4 kDa was recorded; 6) the monosaccharide composition obtained in FACE is peculiar in the sense that next to Fuc, Man, GalNac, GlcNac and NeuAc also Rib was determined; 7) HPAE-PAD analysis identified NeuAc subtypes; 8) in vitro, glycosylation of rat prolactin modulates immune recognition through steric hindrance of the access to the epitope sites.
authors.affiliation: Laboratorium voor Farmacologie, Faculteit Geneeskunde en Farmacie, Vrije Universiteit Brussel, Belgium.
authors.forename: F
authors.initials: F
authors.lastname: Bollengier
authors.forename: A
authors.initials: A
authors.lastname: Mahler
authors.forename: C
authors.initials: C
authors.lastname: Braet
authors.forename: M
authors.initials: M
authors.lastname: Claeyssens
authors.forename: L
authors.initials: L
authors.lastname: Vanhaelst
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D015151:Immunoblotting; D009844:Oligosaccharides / Q000737:chemistry*; D011388:Prolactin / Q000737:chemistry* / Q000302:isolation & purification*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D051381:Rats; D017208:Rats, Wistar
chemical_list: D009844:Oligosaccharides; D020033:Protein Isoforms; D011388:Prolactin