Cloning and expression of a Xenopus laevis oocyte lectin and characterization of its mRNA levels during early development

Jin Kyu Lee, Phillip Buckhaults, Christopher Wilkes, Meredith Teilhet, Mary Lou King, Kelley W. Moremen, Michael Pierce

Research output: Contribution to journalArticlepeer-review

49 Scopus citations

Abstract

cDNA clones encoding a soluble, calcium-dependent, melibiose-binding lectin from Xenopus laevis oocytes have been isolated, characterized, and expressed in bacteria. This lectin has been shown by others to be localized in oocyte cortical granules where it ultimately is released and participates in the formation of the fertilization envelope. A lectin with similar specificity has been purified by others from blastula and immunolocalized to specific locations in developing embryos, which suggests it may also function after fertilization in regulating cell adhesion and migration. We have used melibiose affinity chromatography to isolate the oocyte lectin (monomer molecular masses of about 45 and 43 kDa) and shown that after exhaustive treatment with N-glycanase, only one major protein band at 35 kDa was observed, suggesting that a single polypeptide with variable N-linked glycosylation is expressed in the oocyte. After obtaining internal peptide sequences, a PCR-based cloning approach allowed the isolation of full length cDNAs from an ovary λgt11 library encoding a protein of 313 amino acids with three potential N-linked oligosaccharide sites. Although this lectin, termed XL35, requires calcium ions for oligosaccharide binding, its sequence does not contain the sequence motif defined for 'C-type' lectins. A 6-His-tagged form of the lectin was expressed in E. coli and purified on a Ni2+-NTA column from bacterial extracts. The recombinant lectin was active using an agglutination assay, and this activity was inhibitable by EDTA and melibiose, properties exhibited by the native lectin. Southern blot analysis revealed a single hybridizing band, arguing against the existence of a multigene family. Northern blot analysis demonstrated that the lectin mRNA is expressed in oocytes and remains at relatively high levels through late gastrulae, continuing until tadpole stages. The persistence of the lectin mRNA, coupled with results from earlier studies, strongly suggests that XL35 is zygotically expressed and functions during morphogenesis.

Original languageEnglish (US)
Pages (from-to)367-372
Number of pages6
JournalGlycobiology
Volume7
Issue number3
DOIs
StatePublished - Apr 1997

Keywords

  • Carbohydrate binding protein
  • Fertilization
  • Lectin
  • Morphogenesis

ASJC Scopus subject areas

  • Biochemistry

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