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The CUB domain is a widespread 110-amino-acid module found in functionally diverse, often developmentally regulated proteins, for which an antiparallel β-barrel topology similar to that in immunoglobulin V domains has been predicted. Spermadhesins have been proposed as a subgrou0 of this protein family built up by a single CUB domain architecture. To test the proposed structural model, we have analyzed the structural organization of two members of the spermadhesin protein family, porcine seminal plasma proteins I/II (PSP-I/PSP-II) heterodimer and bovine acidic seminal fluid protein (aSFP) homodimer, using differential scanning calorimetry, far-ultraviolet circular dichroism and Fourier-transform infrared spectroscopy. Thermal unfolding of PSP-I/PSP-II and aSFP were irreversible and followed a one-step process with transition temperatures (Tm) of 60.5°C and 78.6°C, respectively. The calorimetric enthalpy changes (ΔHcat) of thermal denaturation were 439 kJ/mol for PSP-I/PSP-II and 660 kJ/mol for aSFP dimer. Analysis of the calorimetric curves of PSP-I/PSP-II showed that the entire dimer constituted the cooperative unfolding unit. Fourier-transform infrared spectroscopy and deconvolution of circular dichroic spectra using a convex constraint analysis indicated that β-structure and turns are the major structural element of both PSP-I/PSP-II (53% of β-sheet, 21% of turns) and aSFP (44% of β-sheet, 36% of turns), and that the porcine and the bovine proteins contain little, if any, α-helical structure. Taken together, our results indicate that the porcine and the bovine spermadhesin molecules are probably all-β-structure folds, such as the Greek-key pattern characteristic of many cabohydrate-binding protein domains cannot be eliminated. Finally, the same combination of biophysical techniques was used to characterize the residual secondary structure of thermally denatured forms of PSP-I/PSP-II and aSFP, and to emphasize the aggregation tendency of these forms. [ABSTRACT FROM AUTHOR]

We have analyzed the oligosaccharide recognition ability of boar spermadhesin AWN-1 using biotinylated glycoproteins with defined carbohydrate chains as probes. Our results show that AWN-1 bound to proteins containing O-linked NeuAcα(2-3/6)-Gal β(1-3)-GalNAc (PDC-109 and fetuin) with a Kd = 0.7 microM. AWN-1 also bound to NeuAc α(2-3/6)-Gal β(1-4)-GlcNAc sequences in N-linked triantennary structures of fetuin, but not to the same oligosaccharide in the diantennary structures of IgG or fibrinogen. The absence of terminal sialic acid decreased fivefold the binding affinity. By competitive ELISA, peptides containing the N-linked oligosaccharide sequence inhibited the binding of the parent glycoprotein to immobilized AWN-1 5-45 times less effectively than those carrying O-linked NeuAc α(2-3/6)-Gal β(1-3)-GalNAc structures. In addition, AWN-1 bound with a Kd = 0.3 μM to solubilized, biotinylated porcine zona pellucida glycoproteins. PDC-109 competed effectively with zona pellucida glycoproteins for AWN binding, whereas fetuin was a poor competitor. On the other hand, AWN epitopes were demonstrated on in vitro capacitated boar spermatozoa which were able to bind to, and penetrate, zona-encased oocytes. These data indicate that spermadhesin AWN-1 may play a role in pig fertilization as a sperm-associated lectin of broad specificity though preferential affinity for certain O-linked oligosaccharide structures of the oocyte's zona pellucida. [ABSTRACT FROM AUTHOR]

Boar spermadhesins AQN-1, AQN-3 and AWN form a recently described protein family, synthesized by the sexual accessory glands, and become associated with the sperm head upon ejaculation. They contain 109–133 amino acid residues, two conserved disulphide bridges, are not glycosylated, and have 40–60% primary structure identity. These boar polypeptides are multifunctional proteins, which possess heparin-, serine-protease-inhibitor- and/or zona-pellucida-glycoprotein-binding capability and have, therefore, been implicated inunique among boar spermadhesins in that it is the only member of the family which is known to be glycosylated and which possesses weak zona-pellucida-binding but not seminal-plasma-inhibitor-binding ability. In this study we report the structural and functional characterization of the two glycoproteins contained in the AQN-2 fraction. One component is identical with PSP-I, a major porcine seminal plasma protein whose function has not yet been identified, while the second protein is a glycosylated isoform of AQN-3. Here we show that the inability of the glycosylated boar spermadhesins to bind seminal-plasma protease inhibitors as well as the weak binding of glycosylated AQN-3 to zona pellucida glycoproteins is due to the presence of the oligosacharide chain on a conserved asparagines residue. This indicates that modification of a spermadhesin polypeptide framework may serve to modulate its ligand-binding capabilities. [ABSTRACT FROM AUTHOR]

Gamete recognition and adhesion are essential steps in the complex process of fertilization. In mammals and in other species, increasing evidence indicates that carbohydrate-binding proteins on the sperm surface play a pivotal role as counter-receptors for certain oligosaccharide moieties attached to the oocyte zona pellucida glycoproteins. Although different sperm-associated zona-pellucidabinding proteins have been identified in a number of species, few of them have been isolated and structurally characterized. In this paper we report the primary structural characterization of AQN-1, a 12-kDa boar-sperm-associated carbohydrate-binding and zona-pellucida-binding protein. The molecular mass of AQN-1 was determined by time-of-flight plasma-desorption mass spectrometry. Determination of its amino acid sequence and location of disulphide bridges were accomplished by a combination of proteochemical and mass spectrometric methods. The primary structure of AQN-1 failed to show any significant similarity to the protein structures deposited with the Martinsried Institute for Protein Sequences data bank, indicating that it may belong to a novel protein family involved in fertilization. AQN-1 shares extensive structural, as welt as functional, similarity with two other boar sperm zona-pellucida-binding proteins, AQN-3 and AWN, which we have recently characterized. To name this protein family, we have coined the term spermadhesin. Our data may be relevant for identification of spermadhesins in other species, and thus may contribute to a better understanding of the species-specific sperm-egg recognition mechanism. [ABSTRACT FROM AUTHOR]