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 Orchidaceae species, namely Listera ovata (twayblade), Epipactis helleborine (broad-leaved helleborine) and Cymbidium hybrid, using affinity chromatography 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 colony hybridization. Since N-terminal sequence analysis of the twayblade lectin revealed some sequence similarity to the previously cloned mannose-binding lectin Hippeastrum hybrid (amaryllis) ovaries, the amaryllis lectin cDNA clone was used as a probe to screen the L. ovata library. Subsequently, the cDNA clone encoding the L. ovata lectin was used 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 approximately 50% sequence similarity both at the nucleotide and amino acid level. The Orchidaceae lectins are apparently translated from mRNAs consisting of approximately 800 nucleotides. The primary translation products are preproproteins which are converted into the mature lectins following post-translational modifications. Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.
Van Damme
J M
JM
Laboratory for Phytopathology and Plant Protection, Katholieke Universiteit Leuven, Belgium.
Smeets
K
K
Torrekens
S
S
Van Leuven
F
F
Peumans
W J
WJ
D000372:Agglutination Tests; D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D037102:Lectins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010944:Plants / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012984:Software
D002352:Carrier Proteins; D018076:DNA, Complementary; D037102:Lectins; D037241:Mannose-Binding Lectins; D037121:Plant Lectins