Molecular cloning of the E-cadherin cDNAs from rabbit corneal epithelium

Abstract

Cadherins are glycoproteins that are members of the superfamily of Ca(2+)-dependent cell adhesion molecules. They are located in the adherens junctions and play an important role in cell-cell interactions that influence cell polarity, cell migration, cell sorting, and morphogenesis. To gain a better understanding of the molecules that mediate cell-cell interactions in corneal epithelium, we wished to characterize the E-cadherin from rabbit corneal epithelium. Using degenerate primers designed against an extracellular region that is highly conserved between the E-cadherin cDNA sequences of mouse and human, reverse transcription-polymerase chain reaction (RT-PCR) was performed on RNA isolated from rabbit corneal epithelial cell cultures. A 381 bp rabbit E-cadherin cDNA fragment that was amplified, was cloned and sequenced. This rabbit E-cadherin cDNA fragment was used as a probe for northern blot analysis of rabbit corneal epithelial cell cultures and subsequently, to isolate rabbit E-cadherin cDNA clones from a rabbit corneal epithelial cDNA library constructed in lambda UniZAP. A 4.3 kb transcript was detected in the northern blots of poly(A) RNA from confluent rabbit corneal epithelial cell cultures. The coding sequence of the 4.3 kb full-length rabbit E-cadherin cDNA was determined from overlapping clones. The deduced 886 amino acid sequence of the rabbit E-cadherin cDNA shows linear organization of the polypeptide into an N-terminal prosequence, five extracellular repeating domains, a transmembrane region, and a cytoplasmic domain at the C-terminus. Thus the rabbit E-cadherin from cornea is a classical member of the cadherin family and shows about 90% amino acid similarity to mouse and human E-cadherins. In the present study, in addition to the characterization of the 4.3 kb rabbit E-cadherin cDNA, a 2.3 kb E-cadherin clone was also isolated from the cDNA library. The 2.3 kb truncated cDNA was found to lack the DNA sequences encoding the transmembrane, the cytoplasmic, and the third, fourth, and fifth extracellular domains that are present in the classical rabbit E-cadherin. Transcripts corresponding to the truncated E-cadherin cDNA were detected by RT-PCR amplification of rabbit corneal epithelial cell culture RNA, but could not be detected by northern analysis, suggesting that message for this putative truncated E-cadherin may be expressed at low levels.