Purification, characterization, molecular cloning, and expression of novel members of jacalin-related lectins from rhizomes of the true fern Phlebodium aureum (L) J. Smith (Polypodiaceae).
A lectin was purified from rhizomes of the fern Phlebodium aureum by affinity chromatography on mannose-Sepharose. The lectin, designated P. aureum lectin (PAL), is composed of two identical subunits of approximately 15 kDa associated by noncovalent bonds. From a cDNA library and synthetic oligonucleotide probes based on a partial amino acid sequence, 5'- and 3'-rapid amplification of cDNA ends allowed the generation of two similar full-length cDNAs, termed PALa and PALb, each of which had an open reading frame of 438 bp encoding 146 amino acid residues. The two proteins share 88% sequence identity and showed structural similarity to jacalin-related lectins. PALa contained peptide sequences exactly matching those found in the isolated lectin. PALa and PALb were expressed in Escherichia coli using pET-22b(+) vector and purified by one-step affinity chromatography. Native and recombinant forms of PAL agglutinated rabbit erythrocytes and precipitated with yeast mannan, dextran, and the high mannose-containing glycoprotein invertase. The detailed carbohydrate-binding properties of the native and recombinant lectins were elucidated by agglutination inhibition assay, and native lectin was also studied by isothermal titration calorimetry. Based on the results of these assays, we conclude that this primitive vascular plant, like many higher plants, contains significant quantities of a mannose/glucose-binding protein in its storage tissue, whose binding specificity differs in detail from either legume mannose/glucose-binding lectins or monocot mannose-specific lectins. The identification of a jacalin-related lectin in a true fern reveals for the first time the widespread distribution and molecular evolution of this lectin family in the plant kingdom.
Tateno
Hiroaki
H
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606, USA.
Winter
Harry C
HC
Petryniak
Jerzy
J
Goldstein
Irwin J
IJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004912:Erythrocytes / Q000187:drug effects; D006386:Hemagglutination Tests; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D010936:Plant Extracts / Q000494:pharmacology; D037121:Plant Lectins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D029621:Polypodiaceae / Q000737:chemistry*; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D010936:Plant Extracts; D037121:Plant Lectins; D011994:Recombinant Proteins; C043696:jacalin