Title : Mass spectrometry analysis of recombinant human
ZP3 expressed in glycosylation-deficient CHO cells
Abstract :
- 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