IDENTIFICATION OF A GLYCOSYLATED RELAXIN-LIKE MOLECULE FROM THE MALE ATLANTIC STINGRAY, DASYATIS-SABINA

The alkaline gland fluid of the Atlantic stingray (Dasyatis sabina) co ntains a molecule that cross-reacts weakly to anti-porcine relaxin ant ibodies. This material was isolated and purified to homogeneity by rev ersed-phase high-performance liquid chromatography. In SDS gel electro phoresis, the molecule showed an apparent molecular mass of 13 kDa whi ch upon reduction formed two polypeptide chains of 4 and 9 kDa, respec tively. Sequence analyses revealed a 27-amino acid residue A chain and a 54-amino acid residue B chain which contained an N-glycosylation si te in position B37. The distribution of the six cysteines and possibly the disulfide bonding is identical to that found in insulins and most relaxins. Although the stingray relaxin-like molecule contains the st ructurally relevant glycine residues within the A chain, in the midreg ion of the B chain it has only one of the two requisite binding site a rginines, which explains the lack of relaxin bioactivity in standard m ammalian assays. Stingray relaxin is the first member of the relaxin f amily identified in a nonhomeotherm male. Carbohydrate analysis of rel axin revealed an N-linked asialo, agalacto, bisected biantennary, and a core-fucosylated oligosaccharide in the position of Asn B37 which ma kes it the first reported glycosylated relaxin-like molecule.