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dc.contributor.authorTracy, Alexander Standish
dc.date.accessioned2014-03-05T16:03:03Z
dc.date.available2014-03-05T16:03:03Z
dc.date.issued2002-05
dc.identifier.othertracy_alexander_s_200205_phd
dc.identifier.urihttp://purl.galileo.usg.edu/uga_etd/tracy_alexander_s_200205_phd
dc.identifier.urihttp://hdl.handle.net/10724/29446
dc.description.abstractThe Xenopus laevis cortical granule lectin, termed XL35, is an oligomeric, Ca 2+ -dependent lectin that is exocytosed from the oocyte at fertilization and forms the permanent block to polyspermy by crosslinking its mucin-like ligand present in the jellycoat of Xenopus eggs. Structural characterization studies have been performed using dynamic light scattering and sedimentation equilibrium measurements, and the results suggest that XL35 as purified from oocytes consists of a homo-oligomer composed of 12 monomers. The region of the polypeptide responsible for the formation of oligomers most likely resides in a hydrophobic domain at the C-terminus of the lectin. SDS-PAGE analysis of denatured XL35 in the presence or absence of reducing agents indicates that XL35 oligomers are formed through a combination of hydrophobic contacts and intermolecular disulfide bonds. MALDI-TOF mass spectrometry analysis of reduced and carboxymethylated (RCM) XL35 treated with N-glycanase indicates that only a limited amount of secreted XL35 is processed by signal peptidase: the majority of XL35 is secreted as the full-length polypeptide. The limited processing of the XL35 putative signal sequence and the localization of XL35 in the cortical granules suggests that XL35 transits the intracellular regulated secretion pathway as it becomes packaged in the cortical granules. Molecular mass determinations before and after N-glycanase treatment suggest that at least 2 of the 3 potential N-linked glycosylation sites are utilized. Based on the chemical modification of selected amino acids, tryptophan and a carboxylic acid moiety appear to be involved in ligand binding. XL35 binds to a wide variety of galactose-terminating oligosaccharides and recognizes both the a- and b-anomers of galactose, although a-anomers are preferred. XL35 binds poorly or not at all to sugars other than galactose. The monovalent dissociation constants that XL35 exhibits for monovalent ligands were determined to be in the millimolar range, but XL35 creates a highly stable complex with multivalent ligands, both native and synthetic, that exhibits little, if any, dissociation, as assessed by surface plasmon resonance measurements.|The ability of XL35 to efficiently form the block to polyspermy is intimately linked to its intrinsically low monovalent affinity along with the high-avidity multivalent interactions that are a result of its oligomeric structure. XL35 has also shown Ca 2+ -dependent binding to Cryptococcus neoformans and Candida albicans, and its localization to the epidermis in the Xenopus tailbud-stage embryos suggests that it may have a role in innate immunity.
dc.languageLow affinity, high-avidity lectin-carbohydrate interactions : structure-function studies on the cortical granule lectin from Xenopus laevis
dc.publisheruga
dc.rightspublic
dc.subjectCa2+-dependent lectin
dc.subjectfertilization
dc.subjectXenopus laevis
dc.subjectmultivalent interaction
dc.subjectinhibition assay
dc.subjectsurface plasmon resonance
dc.titleLow affinity, high-avidity lectin-carbohydrate interactions : structure-function studies on the cortical granule lectin from Xenopus laevis
dc.typeDissertation
dc.description.degreePhD
dc.description.departmentBiochemistry and Molecular Biology
dc.description.majorBiochemistry and Molecular Biology
dc.description.advisorMichael Pierce
dc.description.committeeMichael Pierce
dc.description.committeeKojo Mensa-Wilmot
dc.description.committeeJames Prestegard
dc.description.committeeJ. David Puett
dc.description.committeeRobert Woods


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