Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

Abstract

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30-35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

Acetylproteomic Analysis Reveals Functional Implications of Lysine Acetylation in Human Spermatozoa (sperm)

“…This suggests that cytoplasmic translation in sperm may be regulated by lysine acetylation. Because of the successful enrichment of lysine-acetylated peptides, we were able to identify novel VDACs, one of which was recently characterized in both bovine and human sperm (50,52). Modification of channel proteins by acetylation is virtually unexplored territory except for a recent report in which incretin regulates ␤-cell function via the acetylation of multiple Kv␣ and Kv␤ subunits (59).…”

VDAC, a multi-functional mitochondrial protein regulating cell life and death

“…Triton X-100 extracts of whole spermatozoa were applied to HTP/Celite, yielding an eluate containing VDAC2 and traces of VDAC3 and VDAC1 (Menzel et al, 2009). The various purification procedures described above have permitted VDAC purification from numerous tissues and organisms (for review, see (Benz, 1994)).…”

Analysis and difference of voltage-dependent anion channel mRNA in ejaculated spermatozoa from normozoospermic fertile donors and infertile patients with idiopathic asthenozoospermia

“…Few recent studies have reported that VDAC is present in mammalian spermatozoa and plays putative roles in spermatogenesis, sperm maturation and fertilization [15][16][17][18][19]. However the respective expression, localization and function of three VDAC subtypes remain uncertain.…”