Spermadhesins of the AQN and AWN families, DQH sperm surface protein and HNK protein in the heparin-binding fraction of boar seminal plasma

Abstract

Heparin-binding proteins (designated BHB-2-BHB-9) were isolated from boar seminal plasma by affinity chromatography on heparin immobilized on polyacrylamide gel, followed by reverse phase HPLC. According to their N-terminal amino acid sequences, BHB-3-BHB-5 belong to the AQN family of spermadhesins and BHB-7-BHB-9 to the AWN family. BHB-6 is composed of two different proteins. The dominant protein (14 kDa) has the N-terminal amino acid sequence HNKQEGRDHD that is identical to the sequence of human semenogelin at positions 85-94. The minor proteins (16 and 17 kDa) belong to the AWN family of spermadhesins. The 14 kDa HNK protein does not crossreact with antibodies against AQN or AWN spermadhesins. BHB-2 also binds to the acrosome of boar epididymal spermatozoa but has the N-terminal sequence DQH. Therefore, basic protein BHB-2 belongs to a new family of DQH sperm surface proteins that are homologous to the acidic proteins from bull and stallion seminal plasma, to the collagen binding domain II in fibronectin and to the leucocyte cell-cell adhesion regulator, but are not homologous to AQN or AWN spermadhesins. Nevertheless, anti-AQN-1 spermadhesin antibodies crossreact strongly with DQH protein. All boar heparin-binding proteins bind concanavalin A indicating their glycoprotein nature, which was proved by the detection of glucosamine and galactosamine residues in their molecules. Furthermore, spermadhesins interact with zona pellucida, protease inhibitors and a polyacrylamide derivative of heparin. Affinity chromatography experiments showed that the DQH protein bound to gelatin-agarose together with the AWN proteins and that the DQH protein and AQN-1 spermadhesin belong to the phosphoryl choline binding proteins.

Determination of the complete covalent structure of the major glycoform of DQH sperm surface protein, a novel trypsin‐resistant boar seminal plasma O‐glycoprotein related to pB1 protein

“…N-terminal sequence of DQH protein matches exactly the determined N-terminal sequence~32 amino acid residues! of pAIF-1~Hadjisavas et al, 1994;Jonáková et al, 1998!. On the other hand, it is obvious that the DQH protein described here and the pB1 protein described previously belong to the same family of seminal plasma proteins as bovine BSP A1, BSP A3 Seidah et al, 1987!, andstallion HSP-1~Calvete et al, 1995!. These proteins are all members of the large family of cell and matrix adhesion proteins, which includes seminal plasma proteins, colagenases, fibronectins, blood coagulation factors, and large cell surface receptors such as the macrophage mannose receptor, and the cation independent mannose-6-phosphate receptors~Hadjisa-vas et al, 1994!. Unlike in seminal plasma proteins, where the tandemly arranged fibronectin type II repeats encompass most of the protein sequence, in the other proteins of this family, this repeat represents only a small portion of the entire protein sequence.…”

“…It has been shown that boar seminal plasma contains proteins that are not members of AQN and AWN families~Fernlund et al, 1994;Hadjisavas et al, 1994;Jonáková et al, 1998!. One of them, the phosphorylcholine-binding protein pB1, has been recently characterized~Calvete et al, 1997!.…”

Glycobiology of fertilization in the pig

“…Mannose-binding sites and AQN-1 molecules (Jonakova et al, 1998) are localized over the acosomal region of the sperm. GNA-binding proteins of the oviductal membrane bind to the same region of the sperm surface when tested by fluorescence microscopy after treatment of a sperm suspension with the biotinylated protein fraction (Fig.…”

Affinity liquid chromatography and capillary electrophoresis of seminal plasma proteins

“…These fractions were subjected to CE. Individual protein components of these fractions were obtained using RP-HPLC separation as described previously [14]. These individual proteins were used as standards for CE.…”

“…Among the ligands used, immobilized heparin is often applied to separate H+ and heparin-nonbinding fraction of seminal plasma (H -) proteins [7,13,14]. Affinity techniques not only are useful for the protein isolation but also represent a valuable and essential tool for the study and analysis of binding properties of protein components that participate in the fertilization process.…”