Miwa N et al. (2015), Fertilization competence of the egg-coating env...

XB-ART-51083

Sci Rep 2015 Aug 05;5:12672. doi: 10.1038/srep12672.

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Fertilization competence of the egg-coating envelope is regulated by direct interaction of dicalcin and gp41, the Xenopus laevis ZP3.

Miwa N , Ogawa M , Hanaue M , Takamatsu K .

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Fertilization begins with species-restricted interaction of sperm and the egg-coating envelope, which includes a three-dimensional meshwork of filaments composed of glycoproteins (called ZP proteins). Growing evidence has unveiled the molecular nature of ZP proteins; however, the structural property conferring fertilization competence to the egg-coating envelope remains unknown. Here, we show the molecular mechanism that mediates direct interaction between dicalcin, a novel fertilization-suppressive ZP protein-associated protein, and gp41, a Xenopus laevis ortholog of mammalian ZP3, and subsequently demonstrate the structural basis of the envelope for fertilization competence. The interactive regions between dicalcin and gp41 comprised five and nine amino acid residues within dicalcin and twenty-three within gp41 [corrected]. Synthetic peptides corresponding to these regions dramatically affected fertilization: treatment with dicalcin- or gp41-derived peptides decreased or increased fertilization rates, respectively. Prior application of these peptides caused distinct alterations in the in vivo lectin-staining pattern of the envelope as well. Transmission electron microscopy analysis revealed that the dicalcin-derived peptide induced the formation of a well-organized meshwork, whereas the gp41-derived peptide caused the formation of a significantly disorganized meshwork. These findings indicated that the fertilization competence of the egg-coating envelope is crucially regulated by the direct interaction between dicalcin and gp41.

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Species referenced: Xenopus laevis
Genes referenced: dcp1b lamp1 s100a11 zp2 zp3 zp3 zp3.2 zp4

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	Figure 1. Identification of the amino acid regions mediating the suppressive action of dicalcin on fertilization.(a) A schematic representation of deleted mutants of dicalcin. The primary structure of dicalcin contains four calcium-binding domains (EF-hand) and linker region (hatched) that links the N-terminal and C-terminal halves. The numbers in the boxes refer to the amino acid positions. One mutant (Δ160C) was histidine-tagged at the N-terminus. On the basis of deleted regions, the sequence of dicalcin was divided into four regions (regions 1, 2, 3 and 4). (b) Purification of wild type and mutants of dicalcin. Purified recombinants were electrophoresed and silver-stained. (c) Binding of wild type and mutants to gp41 and gp37. Blots of VE proteins were probed by biotinylated proteins. Intensities for gp41 were converted to the molar amounts of bound proteins. The molar amount of the wild-type was set to 100% and the data were normalized (n = 10, mean ± s.e.m). (d) Binding activities of four regions of dicalcin. Binding activities of each region were estimated by referring to normalized binding activities in (c). (e) A schematic diagram of the role of each region for gp41-binding. N-terminal region of dicalcin is required for gp41-binding, whereas C-terminal region suppresses the binding (see Supplementary Fig. S3). (f) Binding of the synthetic peptides on gp41 and gp37. The sequence of N-terminal region (regions 1 and 2) was divided into seven regions. Biotin-coupled peptides corresponding to each region were probed on VE proteins. (g) Binding of shorter peptides on gp41 and gp37. (Left) The amino acid region of dcp4 was divided into two regions (dcp11 and dcp12). (Right) The amino acid region containing dcp7 was divided into three regions (dcp13, dcp14, and dcp15). (h) Effect of pretreatment with dicalcin-derived peptides on the fertilization rate. Ovulated eggs were pretreated with BSA (control), peptides (dcp1, dcp11, dcp15 and 1:1 mixture of dcp11 and dcp15) followed by insemination. The fertilization rate was scored as percentage of fertilized eggs/all eggs and normalized (n = 7; *p < 0.02, Student’s t-test). (i) Concentration dependence of inhibitory effect of dcp11 and dcp15. The graph shows mean data (n = 10, mean ± s.e.m).
	Figure 2. Identification of amino acid region of gp41 essential in mediating the action of dicalcin.(a) A schematic representation of the wild-type, ZP-N and ZP–C domains of gp41. The numbers in the boxes refer to the amino acid positions. All recombinants were histidine-tagged at the N-terminus. (b) Binding activities of each mutant to dicalcin-derived peptides. Each of the wild type and mutants was expressed in E. coli, and the blots of their lysates were probed either by anti-histidine antibody (Anti-His Ab), biotinylated dicalcin (Dicalcin), dcp11, or dcp15. Arrowheads indicated positions of recombinants for the wild type, ZP-N, and ZP-C. (c) A schematic representation of ZP-C and deleted mutants. (d) Binding of dicalcin-derived peptides to each mutant. Each of the wild type and mutants was expressed in E. coli, and the blots of their lysates were probed by anti-histidine antibody (Anti-His Ab), dcp11, or dcp15. (e) The binding activities of dicalcin-derived peptides to ZP-C and mutants. Intensities of dcp11- (Left) and dcp15- (Right) binding to mutants were normalized by those to ZP-C (n = 10, mean ± s.e.m). (f) Binding of gp41-derived peptide on dicalcin. The amino acid sequence (residues 486–518) was divided into two regions (gpp1 and gpp2), and their binding to dicalcin was examined. CBB, CBB-stained dicalcin; gpp1, treated with biotinylated gpp1; gpp2, treated with biotinylated gpp2. (g) Inhibition of the binding of dicalcin-derived peptides to gp41 by gpp2. Blots of VE proteins were probed with biotinylated dicalcin-derived peptides (dcp11 and dcp15, 1 μM) either in the presence (1 μM) or absence of gpp2. Co-incubation with gpp2 reduced the binding (n = 10; p < 0.03, Student’s t-test). (h) Effect of pretreatment with gpp2 on fertilization. Normalized fertilization rate increased up to 132% of control (n = 30–40, mean ± s.e.m., p = 0.031. **p = 0.015, Student’s t-test). (i) Mapping of the amino acid region of gp41 in mediating the action of dicalcin. Each monomer of gp41 was shown in green and white. The region for gpp2 was shown in blue (residues 242–264 in each monomer of chicken ZP3). ZP-C subdomain was shown in violet (residues 250–256, and 312–343).
	Figure 3. Dicalcin- and gp41-derived peptides induce alterations in in vivo lectin reactivities of the VE.(a) Blot of VE proteins treated with RCAI. CBB, CBB-stained VE; RCAI, Rhodamine-labeled RCAI blot. (b) Representative confocal images of a Xenopus egg treated with RCAI and averaged intensities across the VE. (Left) RCAI stained the outermost region of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of RCAI staining across the VE (dashed line in Control in the left, n = 15). The position where RCAI-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (c) RCAI staining of the VE pretreated with dcp11. (Left) RCAI reactivity of the VE pretreated with dcp11. (Right) Intensities across the VE (black) (n = 15). RCAI reactivities pretreated with dicalcin (blue) and BSA (red) were also shown. (d) RCAI staining of the VE pretreated with gpp2. (Left) RCAI reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15). (e) Blot of VE proteins treated with WGA. WGA recognized gp41 as well as gp69/64 and gp120. CBB, CBB-stained VE; WGA, FITC-labeled WGA blot. (f) Representative confocal images of a Xenopus egg treated with WGA and averaged intensities across the VE. (Left) WGA stained the outermost and midsection regions of the VE following treatment with BSA (Control) or dicalcin (DC). Scale bar: 50 μm. (Right) Intensities of WGA staining across the VE (n = 15). The position where WGA-signal starts to rise is designated as 0 μm in the x axis (arrow in the left). (g) WGA staining of the VE pretreated with dcp11. (Left) WGA reactivity of the VE pretreated with dcp11. (Right) Intensities of the VE across the VE (black) (n = 15). WGA reactivities of the VE pretreated with dicalcin (blue) and BSA (red) were also shown. (h) WGA staining of the VE pretreated with gpp2. (Left) WGA reactivity of the VE pretreated with gpp2. (Right) Intensities of the VE across the VE (black) (n = 15).
	Figure 4. Dicalcin-and gp41-derived peptides induce distinct nanoscale ZP meshworks. TEM analysis of the VE treated with dcp11 and gpp2.(Upper) Low magnified images of the VE treated with peptides (dcp1 as control, dcp11 and gpp2; 4 μM; n = 3). Note that intensities were enhanced to obtain fine details of ZP filaments. Scale bar: 500 nm. (Lower) Higher magnified images. Scale bar: 30 nm.
	Figure 5. Reversible transition of the ZP meshwork status by substitution treatment with dcp11 and gpp2.(a) RCAI staining of the VE substitutionally treated with dcp11 and gpp2. gpp2 → dcp11; eggs were pretreated first with gpp2 (4 μM), followed by rinse and treatment with dcp11 (8 μM). dcp11 → gpp2; eggs were pretreated first with dcp11 (4 μM), followed by rinse and treatment with gpp2 (8 μM). (Left) Representative confocal image of unfertilized egg. (Right) Intensities across the VE (black) (n = 15). RCAI reactivity of the VE pretreated with dcp11 (blue) and gpp2 (red) were also shown. (b) A schematic model of the transition of ZP meshwork between fertilization competent and incompetent statuses. Our TEM studies revealed that ZP filaments pretreated with dcp11 were arranged parallel to the egg plasma membrane, exhibiting a “pin-stripe” pattern, while ZP filaments pretreated with gpp2 were arranged oblique to the egg plasma membrane, occasionally forming a “herring-bone” pattern. These results implies that the ZP filaments pretreated with dcp11 was a well-organized sheet-like structure, while the ZP filaments pretreated with gpp2 was randomly disoriented organization. On the basis of these two-dimensional images, we hypothesized three-dimensional meshwork model of ZP filaments. Treatment with gpp2 induces a randomized disoriented ZP meshwork that allows sperm to fit into the three-dimensional structure (i.e. capture of sperm), and enables acrosome reaction, while treatment with dcp11 alters a better-organized meshwork, forming parallel sheet of ZP filaments where sperm may not fit to the structure and therefore sperm move away from the VE, resulting in fertilization failure.

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Bork, A large domain common to sperm receptors (Zp2 and Zp3) and TGF-beta type III receptor. 1992, Pubmed