The zona pellucida is an extracellular matrix that mediates taxon-specific fertilization in which human sperm will not bind to mouse eggs. The mouse zona pellucida is composed of three
glycoproteins (ZP1, ZP2, ZP3). The primary structure of each has been deduced from the
cDNA nucleic acid sequence, and each has been analyzed by mass spectrometry. However, determination of the secondary structure and processing of the human
zona proteins have been hampered by the paucity of
biological material. To investigate if taxon-specific sperm-egg recognition was ascribable to structural differences in a
zona protein required for matrix formation, recombinant human ZP3 was expressed in CHO-Lec3.2.8.1 cells and compared to mouse ZP3. With nearly complete coverage, LC-QTOF mass spectrometry was used to determine the cleavage of an N-terminal
signal peptide (
amino acids 1-22) and the release of secreted ZP3 from a C-terminal transmembrane domain (
amino acids 379-424). The resultant N-terminal
glutamine was cyclized to
pyroglutamate (pyrGln(23)), and several C-terminal
peptides were detected, including one ending at Asn(350). The
disulfide bond linkages of eight
cysteine residues in the conserved
zona domain were ascertained (Cys(46)/Cys(140), Cys(78)/Cys(99), Cys(217)/Cys(282), Cys(239)/Cys(300)), but the precise linkage of two additional
disulfide bonds was indeterminate due to clustering of the remaining four
cysteine residues (Cys(319), Cys(321), Cys(322), Cys(327)). Three of the four potential N-linked
oligosaccharide binding sites (Asn(125), Asn(147), Asn(272)) were occupied, and clusters of O-
glycans were observed within two regions,
amino acids 156-173 and 260-281. Taken together, these data indicate that human and mouse ZP3
proteins are quite similar, and alternative explanations of taxon-specific sperm binding warrant exploration.