| Abstract
| - The mammalian zona pellucida is an egg extracellular matrix to which sperm bind. Mousezonae are composed of three glycoproteins (ZP1, ZP2, and ZP3), while rat zonae contain four (ZP1, ZP2,ZP3, and ZP4/ZPB). Mouse sperm bind to zonae comprised solely of mouse ZP2 and ZP3. In this report,we show that rat sperm also bind to these zonae, indicating that ZP2 and ZP3 contain a “minimumstructure(s)” to which rodent sperm can bind, and ZP1 and ZP4/ZPB are dispensable in these two rodents.These data are consistent with our mass spectrometric analysis of the native rat zona pellucida proteome(defined as the fraction of the total rat proteome to which the zonae glycoproteins contribute) demonstratingthat the rat zonae glycoproteins share a high degree of conservation of structural features with respect totheir mouse counterparts. The primary sequences of the rat zonae proteins have been deduced from cDNA.Each zona protein undergoes extensive co- and post-translational modification prior to its secretion andincorporation into an extracellular zona matrix. Each has a predicted N-terminal signal peptide that iscleaved off once protein translation begins and an anchoring C-terminal transmembrane domain fromwhich the mature protein is released. Mass spectrometric analysis with a limited amount of native materialallowed determination of the mature N-termini of rat ZP1 and ZP3, both of which are characterized bycyclization of glutamine to pyroglutamate; the N-terminus of ZP2 was identified by Edman degradation.The mature C-termini of ZP1 and ZP3 end two amino acids upstream of a conserved dibasic residue thatis part of, but distinct from, the consensus furin cleavage sequence, while the C-terminus of ZP2 was notdetermined. Each zona protein contains a “zona domain” with eight conserved cysteine residues that isthought to play a role in the polymerization of the zona proteins into matrix filaments. Partial disulfidebond assignment indicates that the intramolecular disulfide patterns in rat ZP1, ZP2, and ZP3 are identicalto those of their corresponding mouse counterparts. Last, nearly all potential N-glycosylation sites areoccupied in the rat zonae glycoproteins (three of three for ZP1, six or seven of seven for ZP2, and fouror five of six for ZP3). In comparison, potential O-glycosylation sites are numerous (59−83 Ser/Thrresidues), but only two regions were observed to carry O-glycans in rat ZP3.
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