Fig. 2
From: Molecular basis of egg coat cross-linking sheds light on ZP1-associated female infertility
The cross-linking function of ZP1 maps to its N-terminal ZP-N1 domain. a Domain architecture of cZP1, with feature names abbreviated as in Fig. 1a. b Coomassie-stained SDS-PAGE analysis of insoluble (pellet; P) and soluble (S) fractions of native chicken egg coat material that was either untreated or incubated with a sperm protease extract prepared from acrosome-reacted sperm44. c Immunoblot analysis using anti-cZP1 N-terminal fragment polyclonal57 confirms the presence of cZP1-N1 in the soluble fraction of egg coat material treated with sperm extract. d Silver-stained SDS-PAGE analysis of a size-exclusion chromatography (SEC) separation of the sperm protease-solubilised egg coat material. e SEC fractions 44-54 contain cZP1-N1 as evidenced by ELISA analysis using anti-cZP1 N-terminal fragment polyclonal57. Bands corresponding to the dimer and monomer of cZP1-N1 released from the egg coat were in-gel digested with chymotrypsin and trypsin, followed by MALDI-MS/MS to confirm their protein sequences. f Immunoblot analysis of SEC fractions 45–53 indicates that the ~35 kDa protein in these fractions is a disulphide-linked dimer of the ~21 kDa protein containing cZP1-N1. g, h Anti-5His immunoblot of recombinant cZP1-N1 and cZP1L600-R958 (panel g), as well as full-length cZP1 (panel h). Only cZP1-N1 forms a covalent homodimer like full-length cZP1. i Anti-5His immunoblot analysis shows that secreted hZP1-N1 also forms a covalent homodimer. j SEC-MALS analysis shows that purified hZP1A195-P590 produced in HEK293S cells has a molecular mass of ~50 kDa (red profile), consistent with the calculated mass of a monomeric species carrying 2 GlcNac residues (43 kDa). Calibration was carried out using BSA, whose molecular mass detection is shown in blue
