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Abstract:

Avian prolactin glycosylation on N-X-C sequon; interferes with consensus disulfide bond Prolactin (PRL) is a simple polypeptide hormone of 199 amino acids with three intramolecular disulfide bonds. It is involved in a variety of functions, such as lactogenesis in mammals or osmoregulation in fish. In turkey hens, a rise in circulation PRL levels induces incubation activity. Turkey PRL has been shown to exist in three isoforms: a non-glycosylated isoform of about 22,500 Da, and two glycosylated isoforms of about 24,500 Da, one containing only O-linked glycosylation, and the other containing both N- and O-linked glycosylation. Although there is no N-X-S/T sequon present in the sequence, there are two N-X-C sequons. Glycosylation at one of these sites could interfere with the disulfide bonds in which the C residues in these sequons are normally involved. This might result in significant conformational changes. Purified non-glycosylated (NG-PRL) and glycosylated (G-PRL) prolactin were analysed using in-solution reduction, alkylation and digestion. Neural network prediction, peptide mapping of tryptic and endoproteinase Glu-C digests, and glycopeptide analysis, were used to reveal the glycosylation site. Glycan composition and structure was derived using manual interpretation combined with the GlycoMod and Sweet Substitute tools. Disulfide bond state was investigated using alkylation without prior reduction. All analyses were done using nano-ESI on a Micromass Q-Tof instrument. Glycosylation site prediction and peptide mapping pointed to N-linked glycosylation at N56, an N-X-C sequon. Precursor ion discovery for daughter ions at m/z 204 and 366 revealed the glycopeptides, of which glycan composition and structure were derived using CID MS/MS data. Both G-PRL and NG-PRL also showed Maillard-type non-enzymatic glycations in their molecular spectra. Full molecular masses were inconclusive regarding disulfide bond state; therefore, samples were alkylated without prior reduction, and compared to reduced and alkylated samples. These experiments revealed that only two out of three consensus disulfide bonds are present in G-PRL: the central disulfide bond is present in NG-PRL, but absent in G-PRL. During biosynthesis of the polypeptide, the N-linked glycan probably hinders the formation of this central disulfide bond.