248
J. J. CALVETE and L. SANZ
6
The ~-Galactoside Recognition Site of Spermadhesin PSP-JI?
Asparagine SO is a key residue for expression of the carbohydrate- and zona pellucida glycoprotein-binding capability of boar spermadhesins (Calvete et a1.l995;
T6pfer-Petersen et al. 1996). These activities are blocked in PSP-I due to glycosylation of Asn so • Asparagine 50 of PSP-II is not glycosylated and the glycoprotein
indeed possesses zona pellucida glycoprotein-binding capability, suggesting that
the carbohydrate-binding pocket might be located near this residue. Mechanisms
for carbohydrate binding have evolved independently in diverse protein structural frameworks, but nevertheless share some key features. Although the actual
arrangement of the carbohydrate-binding residues of PSP-II can not be established from the present PSP-I!PSP-II structure, polar residues hydrogen-bonded
to carbohydrate hydroxyl groups and an aromatic residue involved in stacking
interaction with the sugar ring are usually found in the sugar combining site of
leguminous and animallectins of the galectin family. Such arrangement is potentially found in the peripheral domain of the dimer interface composed of PSP-I
Glnll2, and Tyr 48 and AsnSOy of PSP-II. Similar to legume and animallectins, the
putative carbohydrate recognition domain ofPSP-II is located at a shallow groove
on the protein surface. Residues PSP-II Tyr 48 and Asn so co-ordinate a structural
water molecule, which may playa dual role contributing to the stabilisation of the
heterodimer interface and maintaining the conformation of the side chains of the
carbohydrate recognition domain in the absence of sugar ligands. Structural
studies on a variety of lectins have revealed that the binding sites of lectins
appear to be preformed with ordered water molecules forming hydrogen bonds
with the unligated proteins in a pattern that closely mimics the hydrogen bonding by sugar hydroxyl groups. Binding of sugars often displaces those ordered
water molecules. Mutagenesis experiments can now be carried out to investigate
more directly the carbohydrate binding role of PSP-I Glnll2, and Tyr 48 and Asn so
of PSP-II, and additional residues from the surroundings. In addition, we are
engaged in generating PSP-I!PSP-II-oligosaccharide complexes for studying
protein-carbohydrate interactions, which may provide clues on the molecular
mechanism underlying sperm-egg binding mediated by spermadhesin molecules.
7
Structural Characterisation of the Glycan Chains of PSP-I and PSP-II
The structures of the N-linked oligo saccharides of PSP-I and PSP-II were elucidated by combination of methylation analysis and collision-induced dissociation
by tandem electrospray ionisation mass spectrometry of reduced and permethylated oligo saccharides, released by hydrazine treatment of the isolated proteins,
and purified by anion-exchange and amino-bonded phase chromatography
(Nimtz M, Calvete JJ, in preparation). Selected proposed structures were further
confirmed by IH-NMR spectroscopy. Both glycoproteins bear the same neutral,
mono- and disialylated glycans although in different molar ratios. 22 neutral oligosaccharides, 11 monosialylated glycans, and 3 disialylated carbohydrate chains
J. J. CALVETE and L. SANZ
6
The ~-Galactoside Recognition Site of Spermadhesin PSP-JI?
Asparagine SO is a key residue for expression of the carbohydrate- and zona pellucida glycoprotein-binding capability of boar spermadhesins (Calvete et a1.l995;
T6pfer-Petersen et al. 1996). These activities are blocked in PSP-I due to glycosylation of Asn so • Asparagine 50 of PSP-II is not glycosylated and the glycoprotein
indeed possesses zona pellucida glycoprotein-binding capability, suggesting that
the carbohydrate-binding pocket might be located near this residue. Mechanisms
for carbohydrate binding have evolved independently in diverse protein structural frameworks, but nevertheless share some key features. Although the actual
arrangement of the carbohydrate-binding residues of PSP-II can not be established from the present PSP-I!PSP-II structure, polar residues hydrogen-bonded
to carbohydrate hydroxyl groups and an aromatic residue involved in stacking
interaction with the sugar ring are usually found in the sugar combining site of
leguminous and animallectins of the galectin family. Such arrangement is potentially found in the peripheral domain of the dimer interface composed of PSP-I
Glnll2, and Tyr 48 and AsnSOy of PSP-II. Similar to legume and animallectins, the
putative carbohydrate recognition domain ofPSP-II is located at a shallow groove
on the protein surface. Residues PSP-II Tyr 48 and Asn so co-ordinate a structural
water molecule, which may playa dual role contributing to the stabilisation of the
heterodimer interface and maintaining the conformation of the side chains of the
carbohydrate recognition domain in the absence of sugar ligands. Structural
studies on a variety of lectins have revealed that the binding sites of lectins
appear to be preformed with ordered water molecules forming hydrogen bonds
with the unligated proteins in a pattern that closely mimics the hydrogen bonding by sugar hydroxyl groups. Binding of sugars often displaces those ordered
water molecules. Mutagenesis experiments can now be carried out to investigate
more directly the carbohydrate binding role of PSP-I Glnll2, and Tyr 48 and Asn so
of PSP-II, and additional residues from the surroundings. In addition, we are
engaged in generating PSP-I!PSP-II-oligosaccharide complexes for studying
protein-carbohydrate interactions, which may provide clues on the molecular
mechanism underlying sperm-egg binding mediated by spermadhesin molecules.
7
Structural Characterisation of the Glycan Chains of PSP-I and PSP-II
The structures of the N-linked oligo saccharides of PSP-I and PSP-II were elucidated by combination of methylation analysis and collision-induced dissociation
by tandem electrospray ionisation mass spectrometry of reduced and permethylated oligo saccharides, released by hydrazine treatment of the isolated proteins,
and purified by anion-exchange and amino-bonded phase chromatography
(Nimtz M, Calvete JJ, in preparation). Selected proposed structures were further
confirmed by IH-NMR spectroscopy. Both glycoproteins bear the same neutral,
mono- and disialylated glycans although in different molar ratios. 22 neutral oligosaccharides, 11 monosialylated glycans, and 3 disialylated carbohydrate chains