Glycobiology Advance Access originally published online on December 4, 2008
Glycobiology 2009 19(3):277-287; doi:10.1093/glycob/cwn132
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Homolog of the maize β-glucosidase aggregating factor from sorghum is a jacalin-related GalNAc-specific lectin but lacks protein aggregating activity
2 Department of Biological Sciences
3 Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA
1 To whom correspondence should be addressed: Tel: +1-540-231-5894; Fax: +1-540-231-9307; e-mail: aevatan{at}vt.edu
Received on September 3, 2008; revised on November 20, 2008; accepted on November 23, 2008
Recently, we identified the maize β-glucosidase aggregating factor (BGAF) as a jacalin-related lectin (JRL) and showed that its lectin domain is responsible for β-glucosidase aggregation. By searching for BGAF homologs in sorghum, we identified and obtained an EST clone and determined its complete sequence. The predicted protein had the same modular structure as maize BGAF, shared 67% sequence identity with it, and revealed the presence of two potential carbohydrate-binding sites (GG...ATYLQ, site I and GG...GVVLD, site II). Maize BGAF1 is the only lectin from a class of modular JRLs containing an N-terminal dirigent and a C-terminal JRL domain, whose sugar specificity and β-glucosidase aggregating activity have been studied in detail. We purified to homogeneity a BGAF homolog designated as SL (Sorghum lectin) from sorghum and expressed its recombinant version in Escherichia coli. The native protein had a molecular mass of 32 kD and was monomeric. Both native and recombinant SL-agglutinated rabbit erythrocytes, and inhibition assays indicated that SL is a GalNAc-specific lectin. Exchanging the GG...GVVLD motif in SL with that of maize BGAF1 (GG...GIAVT) had no effect on GalNAc-binding, whereas binding to Man was abolished. Substitution of Thr293 and Gln296 in site I to corresponding residues (Val294 and Asp297) of maize BGAF1 resulted in the loss of GalNAc-binding, indicating that site I is responsible for generating GalNAc specificity in SL. Gel-shift and pull-down assays after incubating SL with maize and sorghum β-glucosidases showed no evidence of interaction nor were any SL-protein complexes detected in sorghum tissue extracts, suggesting that the sorghum homolog does not participate in protein–protein interactions.
Key words: β-Glucosidase / homolog / Sorghum bicolor / sugar specificity / lectin