Abstract
Glycodelin, also known as placental protein 14 (PP14) or progesterone-associated endometrial protein (PAEP), is a human glycoprotein with potent immunosuppressive and contraceptive activities. In this paper we report the first characterization of glycodelin-derived oligosaccharides. Using strategies based upon fast atom bombardment and electrospray mass spectrometry we have established that glycodelin is glycosylated at Asn-28 and Asn-63. The Asn-28 site carries high mannose, hybrid and complex-type structures, whereas the second site is exclusively occupied by complex-type glycans. The major non-reducing epitopes in the complex-type glycans are: Gal beta 1-4GlcNAc (lacNAc), GalNAc beta 1-4GlcNAc (lacdiNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacNAc), NeuAc alpha 2-6Gal beta 1-4GlcNAc (sialylated lacdiNAc), Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewisx), and GalNAc beta 1-4(Fuc alpha 1-3)GlcNAc (lacdiNAc analogue of Lewisx). It is possible that the oligosaccharides bearing sialylated lacNAc or lacdiNAc antennae may manifest immunosuppressive effects by specifically blocking adhesive and activation-related events mediated by CD22, the human B cell associated receptor. Oligosaccharides with fucosylated lacdiNAc antennae have previously been shown to potently block selectin-mediated adhesions and may perform the same function in glycodelin. The potent inhibitory effect of glycodelin on initial human sperm-zona pellucida binding is consistent with our previous suggestion that this cell adhesion event requires a selectin-like adhesion process. This result also raises the possibility that a convergence between immune and gamete recognition processes may have occurred in the types of carbohydrate ligands recognized in the human.
Highlights
Glycodelin, known as placental protein 14 (PP14) or progesterone-associated endometrial protein (PAEP), is a human glycoprotein with potent immunosuppressive and contraceptive activities
Released glycans were separated from peptides and were analyzed by fast atom bombardment (FAB)-MS after permethylation (Fig. 2, Table I). Notable features of these data are: (i) the major ion at m/z 1557 has the composition of a high mannose structure containing five mannoses; (ii) molecular ions corresponding to complex- and hybridtype structures occur in the mass range from m/z 1800 to m/z 4000 and, taken together, these are significantly more abundant than m/z 1557; (iii) the majority of the molecular ions have compositions consistent with biantennary structures; (iv) the A-type fragment ions in Fig. 2b indicate that both GalGlcNAc and GalNAc-GlcNAc antennae are present; (v) the lacNAc and lacdiNAc antennae may be substituted with either sialic acid or fucose but not both; (vi) minor fragment ions of composition Hex2HexNAc2 and Hex3HexNAc3 are indicative of low levels of poly-N-acetyllactosamine
The majority of the glycodelin N-linked oligosaccharides characterized in this study are not typically found in mammalian glycoproteins
Summary
Isolation of Glycodelin—Isolation and purification procedures were the same as those described elsewhere (Riittinen et al, 1991) using 140 ml of midtrimester amniotic fluid as starting material. After lyophilization tryptic digestion was carried out as described (Dell et al, 1994). Preparation of CNBr Fragments—Glycodelin (160 g) was dialyzed against 2 liters of 50 mM ammonium bicarbonate buffer, pH 8.5 at 4 °C for 12 h, after which time it was dialyzed against 2 liters of water for another 12 h at 4 °C and lyophilized. PNGase F Digestion—PNGase F (EC 3.2.2.18, Boehringer Mannheim) digestion was carried out on tryptic digests of glycodelin (250 g) in ammonium bicarbonate buffer (50 mM, pH 8.4) for 16 h at 37 °C using 0.6 unit of the enzyme. The reaction was terminated by lyophilization and the products were purified on C18-Sep-Pak (Waters Ltd.) as described (Dell et al, 1994). The partially methylated alditol acetates were dissolved in hexanes prior to on-column injection at 65 °C. The sample was dissolved in 0.1% trifluoroacetic acid (buffer A) for injection on the column. After passage through a UV spectrophotometer with a microflow cell, monitoring at 214 nm, the eluant was mixed with a 1:1 mixture of propan-1-ol and 2-methoxyethanol prior to stream splitting 1:9 for ES-MS analysis and collection, respectively
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