CHARACTERIZATION AND MOLECULAR-CLONING OF MANNOSE-BINDING LECTINS FROM THE ORCHIDACEAE SPECIES LISTERA-OVATA, EPIPACTIS-HELLEBORINE AND CYMBIDIUM HYBRID

Mannose-binding lectins were purified from the leaves of three Orchida ceae species, namely Listera ovata (twayblade), Epipactis helleborine (broad-leaved helleborine) and Cymbidium hybrid, using affinity chroma tography on mannose-Sepharose-4B. Apparently, the Orchidaceae lectins are dimeric proteins composed of lectin subunits of 12-13 kDa. All of the isolated lectins exhibit exclusive specificity towards mannose. A cDNA library constructed from poly(A) rich RNA isolated from leaves of L. ovata was screened for cDNA clones encoding the lectin using colon y hybridization. Since N-terminal sequence analysis of the twayblade l ectin revealed some sequence similarity to the previously cloned manno se-binding lectin from Hippeastrum hybrid (amaryllis) ovaries, the ama ryllis lectin cDNA clone was used as a probe to screen the L. ovata li brary. Subsequently, the cDNA clone encoding the L. ovata lectin was u sed to screen the cDNA libraries from the taxonomically related orchid species Cymbidium hybrid and E. helleborine. Sequence analysis of the lectin cDNA clones from different Orchidaceae species revealed approx imately 50% sequence similarity both at the nucleotide and amino acid level. The Orchidaceae lectins are apparently translated from mRNAs co nsisting of approximately 800 nucleotides. The primary translation pro ducts are preproproteins which are converted into the mature lectins f ollowing post-translational modifications. Southern blot analysis of g enomic DNA has shown that the lectins are most probably encoded by a f amily of closely related genes which is in good agreement with the seq uence heterogeneity found between different lectin cDNA clones of one species.