A new fungal lectin recognizing alpha(1-6)-linked fucose in the N-glycan.
In this report, we describe a new lectin from the fungus Rhizopus stolonifer that agglutinates rabbit red blood cells. Agglutinating activity was detected in the extract of mycelium-forming spores cultured on agar plates but not in the mycelium-forming no spores from liquid medium. This lectin, which we designated R. stolonifer lectin (RSL), was isolated by affinity chromatography with porcine stomach mucin-Sepharose. SDS-polyacrylamide gel electrophoresis and mass spectral analysis showed that RSL is approximately 4.5 kDa, whereas gel filtration indicated a mass of 28 kDa. This indicates that the lectin is a hexamer of noncovalently associated RSL monomers. RSL activity was very stable, since it was insensitive to heat treatment at 70 degrees C for 10 min. Analysis of RSL binding specificity by both microtiter plate and precipitation assays showed that N-glycans with l-fucose linked to the reducing terminal GlcNAc residues are the most potent inhibitors of RSL binding, whereas N-glycans without alpha(1-6)-linked fucose residues are approximately 100-fold weaker inhibitors of binding. Oligosaccharides with alpha(1-2, -3, and -4) linkages showed no inhibition of binding in these assays. In a mirror resonance biosensor assay, high affinity binding was observed between RSL and the glycopeptide of bovine gamma-globulin, which has N-glycans with alpha(1-6)-linked fucose residues. However, RSL showed only a weak interaction with the glycopeptide of quail ovomucoid, which lacks fucose residues. Finally, capillary affinity electrophoresis studies indicated that RSL binds strongly to N-glycans with alpha(1-6)-linked fucose residues. Together, these results show that RSL recognizes the core structure of N-glycans with alpha(1-6)-linked l-fucose residues. This specificity could make RSL a valuable tool for glycobiological studies.
Oda
Yasuo
Y
Faculty of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka, Japan 577-8502. y_oda@phar.kindai.ac.jp
Senaha
Tsutomu
T
Matsuno
Yuuki
Y
Nakajima
Kazuki
K
Naka
Ryousuke
R
Kinoshita
Mitsuhiro
M
Honda
Eiko
E
Furuta
Itaru
I
Kakehi
Kazuaki
K
D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D005456:Fluorescent Dyes; D005643:Fucose / Q000378:metabolism*; D007700:Kinetics; D037102:Lectins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D012233:Rhizopus / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D037102:Lectins; D011134:Polysaccharides; D005643:Fucose