The Use of RNA-Seq to Identify a Transcript Encoding an N-Acetylglucosamine-Binding Lectin from the Edible Kurokawa (Boletopsis) Mushroom

Lectins are a ubiquitous class of proteins that bind to specific sugar moieties in a non-catalytic and reversible manner.  They play important roles in both biology and biotechnology.  Lectins are involved in a diverse array of important biological processes such as cell adhesion, viral infection, glycoprotein synthesis and trafficking, and immune response to pathogens.  Because of their ability to bind specific epitopes within oligosaccharides, lectins have been adopted for use in techniques such as glycoprotein affinity purification, glycan microarrays and imaging.  Most lectins used in molecular techniques are derived from plants where they are abundantly produced and readily purified.  However, there is a need for identification of novel lectin specificities to improve the sensitivity and selectivity of these methods.  In recent years, more attention has been paid to identifying and characterizing lectins from yeasts and fungi. One interesting fungal lectin (BLL) derives from the Japanese edible mushroom “Kurokawa” (Boletopsis leukomelaena).  BLL is small (~15 kDa), aglycosylated, and selectively binds terminal N-acetylglycosamine (GlcNAc) on partially or fully agalactosylated biantennary complex-type N-glycans (1,2).  However, despite its useful specificity, the gene encoding BLL has not been previously identified and B. leukomelaena is difficult to source for the isolation of native BLL.  In the present study, we have utilized RNA-Seq to identify a transcript encoding an ortholog of BLL from B. grisae (termed BGL), a close North American relative of B. leukomelaena. A strand specific transcript library was constructed, sequenced on an Illumina platform, and raw sequence data was assembled using Trinity software.  Over 43,000 transcript sequences were obtained and queried via blastx against signature peptides derived from the purified BLL and BGL proteins.  A transcript harboring a putative BGL-encoding sequence was identified and verified through heterologous protein expression and determination of the ability of recombinant BGL to bind GlcNAc.

1. Koyama, Y. et al. (2002) Biosci. Biotechnol. Biochem. 66: 784-789.

2. Nakamura-Tsuruta et al. (2006) J. Biochem. 140: 285-291.