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Isolation and characterization of gelatin-binding proteins from goat seminal plasma.

Villemure M, Lazure C, Manjunath P - Reprod. Biol. Endocrinol. (2003)

Bottom Line: Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm.The precipitates were dissolved in ammonium bicarbonate and lyophilised.In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Maisonneuve-Rosemont Hospital, Guy-Bernier Research Centre University of Montreal, Montreal, QC, Canada. mvillemure.hmr@ssss.gouv.qc.ca

ABSTRACT
A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.

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N-terminal sequence comparison between GSP and BSP proteins. Residue numbering is indicated for GSP-20 and GSP-22 kDa, and the other sequences were aligned to show homology. The " - " indicates spaces left for alignment purposes, and "..." indicates continuation of the protein sequence at either end (N- or C-terminal). The identical amino acids are presented in bold.
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Figure 5: N-terminal sequence comparison between GSP and BSP proteins. Residue numbering is indicated for GSP-20 and GSP-22 kDa, and the other sequences were aligned to show homology. The " - " indicates spaces left for alignment purposes, and "..." indicates continuation of the protein sequence at either end (N- or C-terminal). The identical amino acids are presented in bold.

Mentions: N-terminal sequencing of the first few residues of goat seminal plasma proteins showed that GSP-14 kDa and GSP-15 kDa share a lot of similarity with each other, as well as GSP-20 kDa with GSP-22 kDa (Figure 5). In addition, they contain the characteristic sequence found in type II domains [21], particularly the motif CVFPFXY(R/K)X(R/K)(R/K)(H/Y)F that is much conserved among BSPs and other homologs found in boar and stallion seminal plasma proteins [22]. In GSP-20 kDa and GSP-22 kDa, the motif is not strictly respected, markedly the FPF part replaced by FAF, but the general pattern of amino acids is followed.


Isolation and characterization of gelatin-binding proteins from goat seminal plasma.

Villemure M, Lazure C, Manjunath P - Reprod. Biol. Endocrinol. (2003)

N-terminal sequence comparison between GSP and BSP proteins. Residue numbering is indicated for GSP-20 and GSP-22 kDa, and the other sequences were aligned to show homology. The " - " indicates spaces left for alignment purposes, and "..." indicates continuation of the protein sequence at either end (N- or C-terminal). The identical amino acids are presented in bold.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC155548&req=5

Figure 5: N-terminal sequence comparison between GSP and BSP proteins. Residue numbering is indicated for GSP-20 and GSP-22 kDa, and the other sequences were aligned to show homology. The " - " indicates spaces left for alignment purposes, and "..." indicates continuation of the protein sequence at either end (N- or C-terminal). The identical amino acids are presented in bold.
Mentions: N-terminal sequencing of the first few residues of goat seminal plasma proteins showed that GSP-14 kDa and GSP-15 kDa share a lot of similarity with each other, as well as GSP-20 kDa with GSP-22 kDa (Figure 5). In addition, they contain the characteristic sequence found in type II domains [21], particularly the motif CVFPFXY(R/K)X(R/K)(R/K)(H/Y)F that is much conserved among BSPs and other homologs found in boar and stallion seminal plasma proteins [22]. In GSP-20 kDa and GSP-22 kDa, the motif is not strictly respected, markedly the FPF part replaced by FAF, but the general pattern of amino acids is followed.

Bottom Line: Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm.The precipitates were dissolved in ammonium bicarbonate and lyophilised.In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Maisonneuve-Rosemont Hospital, Guy-Bernier Research Centre University of Montreal, Montreal, QC, Canada. mvillemure.hmr@ssss.gouv.qc.ca

ABSTRACT
A family of proteins designated BSP-A1, BSP-A2, BSP-A3 and BSP-30 kDa (collectively called BSP proteins for Bovine Seminal Plasma proteins) constitute the major protein fraction in the bull seminal plasma. These proteins interact with choline phospholipids on the sperm surface and play a role in the membrane stabilization (decapacitation) and destabilization (capacitation) process. Homologous proteins have been isolated from boar and stallion seminal plasma. In the current study we report the isolation and preliminary characterization of homologous proteins from goat seminal plasma. Frozen semen (-80 degrees C) was thawed and centrifuged to remove sperm. The proteins in the supernatant were precipitated by the addition of cold ethanol. The precipitates were dissolved in ammonium bicarbonate and lyophilised. The lyophilised proteins were dissolved in phosphate buffer and loaded onto a gelatin-agarose column, which was previously equilibrated with the same buffer. The column was successively washed with phosphate buffer, with phosphate buffer saline and with 0.5 M urea in phosphate buffer saline to remove unadsorbed proteins, and the adsorbed proteins were eluted with 5 M urea in phosphate buffer saline. Analysis of pooled, dialysed and lyophilised gelatin-agarose adsorbed protein fraction by SDS-PAGE indicated the presence of four protein bands that were designated GSP-14 kDa, GSP-15 kDa, GSP-20 kDa and GSP-22 kDa (GSP, Goat Seminal Plasma proteins). Heparin-affinity chromatography was then used for the separation of GSP-20 and -22 kDa from GSP-14 and -15 kDa. Finally, HPLC separation permitted further isolation of each one from the other. Amino acid sequence analysis of these proteins indicated that they are homologous to BSP proteins. In addition, these BSP homologs bind to hen's egg-yolk low-density lipoproteins. These results together with our previous data indicate that BSP family proteins are ubiquitous in mammalian seminal plasma, exist in several forms in each species and possibly play a common biological role.

Show MeSH
Related in: MedlinePlus