CLONING AND EXPRESSION OF A XENOPUS-LAEVIS OOCYTE LECTIN AND CHARACTERIZATION OF ITS MESSENGER-RNA LEVELS DURING EARLY DEVELOPMENT

cDNA clones encoding a soluble, calcium-dependent, melibiose-binding l ectin from Xenopus laevis oocytes have been isolated, characterized, a nd expressed in bacteria. This lectin has been shown by others to be l ocalized in oocyte cortical granules where it ultimately is released a nd participates in the formation of the fertilization envelope. A lect in 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 an d 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 i n the oocyte. After obtaining internal peptide sequences, a PCR-based cloning approach allowed the isolation of full length cDNAs from an ov ary lambda gt11 library encoding a protein of 313 amino acids with thr ee potential N-linked oligosaccharide sites. Although this lectin, ter med XL35, requires calcium ions for oligosaccharide binding, its seque nce 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 purif ied on a Ni2+-NTA column from bacterial extracts. The recombinant lect in was active using an agglutination assay, and this activity was inhi bitable by EDTA and melibiose, properties exhibited by the native lect in. Southern blot analysis revealed a single hybridizing band, arguing against the existence of a multigene family. Northern blot analysis d emonstrated that the lectin mRNA is expressed in oocytes and remains a t relatively high levels through late gastrulae, continuing until tadp ole stages. The persistence of the lectin mRNA, coupled with results f rom earlier studies, strongly suggests that XL35 is zygotically expres sed and functions during morphogenesis.