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.
authors.affiliation: Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Ming
authors.initials: M
authors.lastname: Zhao
authors.forename: Emily S
authors.initials: ES
authors.lastname: Boja
authors.forename: Tanya
authors.initials: T
authors.lastname: Hoodbhoy
authors.forename: Joseph
authors.initials: J
authors.lastname: Nawrocki
authors.forename: Jeanne B
authors.initials: JB
authors.lastname: Kaufman
authors.forename: Nicole
authors.initials: N
authors.lastname: Kresge
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
authors.forename: Lewis
authors.initials: L
authors.lastname: Pannell
authors.forename: Rodney L
authors.initials: RL
authors.lastname: Levine
authors.forename: Henry M
authors.initials: HM
authors.lastname: Fales
authors.forename: Jurrien
authors.initials: J
authors.lastname: Dean
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004527:Egg Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D000072216:Zona Pellucida Glycoproteins
chemical_list: D004220:Disulfides; D004527:Egg Proteins; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C000606514:ZP1 protein, human; C000606515:ZP2 protein, human; C000606517:ZP3 protein, human; D000072216:Zona Pellucida Glycoproteins; C000606519:Zp1 protein, mouse; C000606520:Zp2 protein, mouse; C000606521:Zp3 protein, mouse