Glycosylated rat prolactin: isolation and structural characterization.
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.
Bollengier
F
F
Laboratorium voor Farmacologie, Faculteit Geneeskunde en Farmacie, Vrije Universiteit Brussel, Belgium.
Mahler
A
A
Braet
C
C
Claeyssens
M
M
Vanhaelst
L
L
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
D009844:Oligosaccharides; D020033:Protein Isoforms; D011388:Prolactin