RESUMO
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.
Assuntos
Proteínas do Ovo/química , Proteínas do Ovo/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Dissulfetos/química , Dissulfetos/metabolismo , Proteínas do Ovo/genética , Proteínas do Ovo/isolamento & purificação , Expressão Gênica , Glicosilação , Humanos , Espectrometria de Massas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/isolamento & purificação , Camundongos , Dados de Sequência Molecular , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Glicoproteínas da Zona PelúcidaRESUMO
Cyclonellin (1), a new cyclic octapeptide, was isolated from an aqueous extract of the marine sponge Axinella carteri. Its structure was elucidated by interpretation of NMR spectral data of the intact compound and N-terminal Edman sequencing of linear peptide fragments obtained by partial hydrolysis of 1. The absolute configurations of the constituent amino acids were determined by acid hydrolysis, derivitization with FDAA, and LC-MS analyses.
