A Novel Cysteine Knot Protein for Enhancing Sperm Motility That Might Facilitate the Evolution of Internal Fertilization in Amphibians
(a) Immunoblotting of the SMIS protein in the JE of C. pyrrhogaster. Substances present in the JE were separated through 2D-electrophoresis and transferred to a PVDF membrane. Immunoreaction was performed using an anti-SMIS monoclonal antibody (mAb). The upper and lower photographs show Coomassie brilliant blue staining of the gel and an immunoblot image, respectively. Arrowheads indicate the SMIS proteins. (b) Amplification of SMIS cDNA through reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was purified from the indicated organs using the TRIzol® reagent (Invitrogen), and 1 μg of each sample was reverse-transcribed with an oligo-(dT) primer. Subsequently, PCR was performed using a primer set specific for the SMIS gene. In the control, primers specific for the ß-tubulin gene were used for PCR. (c) Immunoblotting of recombinant SMIS protein with an anti-SMIS mAb. Recombinant SMIS protein was produced in Escherichia coli by transfection of an expression vector (pCold TF, TaKaRa Bio, Tokyo, Japan) containing the open reading frame of SMIS cDNA. The protein was subjected to 1D-electrophoresis and immunoblotted with the anti-SMIS mAb. The left two columns show Coomassie Brilliant Blue staining of the acrylamide gel. The right shows specific binding of the anti-SMIS mAb. Arrowheads and arrows indicate the proteins produced from expression vectors with (SMIS) and without (Mock) SMIS cDNA, respectively. (d) A CK motif in the deduced amino acid sequence of the SMIS. The CK motif includes 6 cysteine residues at the 53rd, 62nd, 66th, 87th, 117th, 119th from N-terminus. Disulfide bonds are formed between cysteines-53 and -87, -62 and -117, and -87 and -119 to produce one ring and 3 loops (loops 1–3) that are supposed to be exposed on the outside. P1-P3 indicate the corresponding sites for preparation of the peptides for sperm motility analysis.