Homolog of the maize  -glucosidase aggregating factor from sorghum is a jacalin-related GalNAc-specific lectin but lacks protein aggregating activity

Abstract

Recently, we identified the maize beta-glucosidase aggregating factor (BGAF) as a jacalin-related lectin (JRL) and showed that its lectin domain is responsible for beta-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 beta-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 Thr(293) and Gln(296) in site I to corresponding residues (Val(294) and Asp(297)) 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 beta-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.

Deletion of the N-terminal dirigent domain in maize β-glucosidase aggregating factor and its homolog sorghum lectin dramatically alters the sugar-specificities of their lectin domains

“…The above results clearly indicate that the deletion of dirigent domain in SL also alters the sugar specificity of its JRL domain. We have shown that SL is a monomer but with two sugar-binding sites [2]. Our gel-filtration data suggest that its JRL domain is also monomeric.…”

“…and they are distinct [1]. SL on the other hand, exhibits exclusive specificity for GalNAc, but it does not participate in proteineprotein interactions [2]. In this report, we show that deletion of the dirigent domain in BGAF and SL affects the sugar specificity of their lectin domains, indicating that the dirigent domains play a role in influencing the carbohydrate-binding specificity in this class of modular lectins.…”

“…Complementary cDNAs encoding wild type BGAF and SL, and deletion mutants of BGAF were constructed and were expressed as described previously [1,2]. To generate the JRL domain of SL, cDNA fragments encoding the G165-L305 region of SL were amplified using primers (sense: 5 0 -ACTCGCATATGGGAACAATGTATCCGGTCATAAAA-3 0 and antisense: 5 0 -GCTCTCGAGTGTCTAGAGGGTCCGCACATAAAC-3 0 , underlined sequences are NdeI and XhoI sites, respectively) incorporating NdeI and XhoI sites into the 5 0 and 3 0 ends of PCR products, respectively.…”

“…Maize b-glucosidase aggregating factor (BGAF) and its homolog sorghum lectin (SL) are modular proteins containing an N-terminal dirigent domain (Latin: dirigere, to align or guide) and a C-terminal jacalin-related lectin domain [1,2]. Dirigent proteins occur only in plants and are induced in response to infection [3].…”

“…A few of them have been characterized with respect to lectin activity and biological function [1,2,7,11,12]. The product of the Hessian fly resistance gene (hfr-1) in wheat is a BGAF-like protein.…”