Maize β-Glucosidase-aggregating Factor Is a Polyspecific Jacalin-related Chimeric Lectin, and Its Lectin Domain Is Responsible for β-Glucosidase Aggregation

Abstract

In certain maize genotypes, called "null," ␤-glucosidase does not enter gels and therefore cannot be detected on zymograms after electrophoresis. Such genotypes were originally thought to be homozygous for a null allele at the glu1 gene and thus devoid of enzyme. We have shown that a ␤-glucosidase-aggregating factor (BGAF) is responsible for the "null" phenotype. BGAF is a chimeric protein consisting of two distinct domains: the disease response or "dirigent" domain and the jacalin-related lectin (JRL) domain. First, it was not known whether the lectin domain in BGAF is functional. Second, it was not known which of the two BGAF domains is involved in ␤-glucosidase binding and aggregation. To this end, we purified BGAF to homogeneity from a maize null inbred line called H95. The purified protein gave a single band on SDS-PAGE, and the native protein was a homodimer of 32-kDa monomers. Native and recombinant BGAF (produced in Escherichia coli) agglutinated rabbit erythrocytes, and various carbohydrates and glycoproteins inhibited their hemagglutination activity. Sugars did not have any effect on the binding of BGAF to the ␤-glucosidase isozyme 1 (Glu1), and the BGAF-Glu1 complex could still bind lactosyl-agarose, indicating that the sugar-binding site of BGAF is distinct from the ␤-glucosidase-binding site. Neither the dirigent nor the JRL domains alone (produced separately in E. coli) produced aggregates of Glu1 based on results from pull-down assays. However, gel shift and competitive binding assays indicated that the JRL domain binds ␤-glucosidase without causing it to aggregate. These results with those from deletion mutagenesis and replacement of the JRL domain of a BGAF homolog from sorghum, which does not bind Glu1, with that from maize allowed us to conclude that the JRL domain of BGAF is responsible for its lectin and ␤-glucosidase binding and aggregating activities.␤-Glucosidases (␤-D-glucoside glucohydrolase; EC 3.2.1.21) hydrolyze ␤-glycosidic linkage(s) in alkyl and aryl ␤-D-glucosides, glycoproteins, and glycolipids and that between two glucose residues in ␤-linked oligosaccharides. They are found in all three (Archaea, Eubacteria, and Eukarya) domains of liv-

Functional Characterization of HFR1, a High-MannoseN-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae

“…It would be interesting to evaluate in the future whether the N terminus or the C terminus or both is responsible for the anti-insect activity. However, in a related chimeric protein in maize, b-glucosidase-aggregating factor, the JRL domain (C terminus) and not the dirigent domain (N terminus) is responsible for lectin and b-glucosidase-binding and aggregating activities (Kittur et al, 2007).…”

Immobilized Subpopulations of Leaf Epidermal Mitochondria Mediate PENETRATION2-Dependent Pathogen Entry Control in Arabidopsis

“…Pathogen Attack Induces Formation of PEN2 Dimers and Oligomers of Higher Order PEN2 orthologs tend to form homo-or hetero-oligomers and large heteromeric protein aggregates (Kittur et al, 2007;Yamada et al, 2011). Oligomerization and aggregate formation have been shown to control hydrolytic activity (Kwak et al, 2009;Yamada et al, 2011).…”

β-Glucosidases

“…Upon cell disruption, plant defensive b-glucosidases and b-thioglucosidases often bind to cytoplasmic aggregating factors, which are thought to help localize the otherwise soluble b-glucosidases and b-thioglucosidases at the site of injury, ensuring a maximal release of defense compounds [53,[59][60][61][62][63]. The functional significance of the interactions of the various defensive b-glucosidases from different cellular compartments and their multiple aggregating factors is an area of active investigation.…”