Effects of midgut-protein-preparative and ligand binding procedures on the toxin binding characteristics of BT-R1, a common high-affinity receptor in Manduca sexta for Cry1A Bacillus thuringiensis toxins.

Parasporal crystalline inclusions of Bacillus thuringiensis subspecies are among the most promising bacterial biopesticides available for use today. As a whole, these proteins (B. thuringiensis toxins) demonstrate great specificity toward certain orders of insects and, to date, have shown no known side effects for nontarget animals. Currently, one of the major drawbacks to the use of B. thuringiensis toxins as externally applied biopesticides is their lack of persistence in the field due to factors such as rain washout and degradation by UV irradiation. In part, these problems are being addressed by the production of transgenic food and textile crops expressing B. thuringiensis toxin genes in their own tissues. Agricultural biotechnology companies are pursuing these transgenic methodologies in the hope of producing food and textile crops which will be resistant to major insect pests without the need for externally applied pesticides. To date, field trials of such plants have resulted in mixed success due, in part, to one of the most attractive advantages of the toxins as externally applied pesticides, i.e., a narrow spectrum of toxicity. This situation can be addressed by the engineering of transgenic plants expressing more than one toxin or by the use of novel B. thuringiensis subspecies producing toxins with multiple specificities. As the use of transgenic crops increases, however, insect resistance may become a problem. A few species of insects have already demonstrated increased tolerance for B. thuringiensis toxins, either in the field or in the laboratory. As suggested in these studies, decreased susceptibility to B. thuringiensis toxins may be due to a variety of factors, including alterations in insect gut physiology (14, 30, 37) or alterations of the ligand binding characteristics of the toxin receptor(s) involved (8, 32, 47). To understand the development of receptor-mediated resistance, investigators first need to identify and characterize the physiologically important receptor molecules for each class of B. thuringiensis toxins from a background of low-affinity toxin binding proteins. That specific protein receptors are involved in Cry toxin killing of target insects has been known since the mid-1980s. Studies of the binding of radiolabeled Cry toxins in suspensions of insect midgut proteins isolated by various procedures have generated a rather extensive list of putative receptor molecules (4, 7, 15, 26, 41, 45, 46) without simultaneously identifying the binding protein(s) in question by use of midgut protein sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) blots. SDS-PAGE blots, when incubated with radiolabeled toxins, at least permit a visual estimation of both the number of proteins involved and their molecular masses. Ligand blots of Manduca sexta midgut proteins have been used successfully to identify and partially characterize for this particular insect binding proteins for the Cry1A lepidopteran-specific toxins (3, 10, 11, 19, 23, 24, 33, 42, 43). Cry1Aa, Cry1Ab, and Cry1Ac demonstrate 82 to 90% amino acid identity to one another and, when compared directly, exhibit indistinguishable toxicities toward M. sexta larvae (16, 17, 45). Cry1Aa and Cry1Ab recognize a single midgut protein in M. sexta, the 210-kDa cadherin-like glycoprotein BT-R1 (42, 43). For Cry1Ac, however, at least two populations of receptor protein have been identified by SDS-PAGE ligand blot analyses of whole midgut protein preparations, including BT-R1 (10, 19, 28, 33) and other proteins with molecular masses ranging from 85 to 120 kDa (5, 11, 19, 21, 33, 44). BT-R1 and at least two aminopeptidases of approximately 120 kDa have now been subjected to partial purification, and their ligand binding characteristics have been described in some detail (10, 12, 13, 29, 34, 39). Both BT-R1 and a 120-kDa aminopeptidase from M. sexta have also been cloned, and their complementary DNA sequences have been reported (22, 43). BT-R1 has been shown to specifically bind with high affinity the three tested Cry1A toxins (Cry1Aa, Cry1Ab, and Cry1Ac) both in M. sexta midgut protein preparations (19, 33) and in heterologous cell cultures expressing the BT-R1 cDNA (19). BT-R1 is also the only Cry1A binding protein that has been shown to have Cry1A-specific ligand binding characteristics when expressed in mammalian and insect cell cultures. This glycoprotein binds Cry1Aa, Cry1Ab, and Cry1Ac with extremely high and virtually equal affinities and specificities in both heterologous and homologous competition binding experiments with membrane proteins prepared from M. sexta larval midguts and transiently transfected Sf21 insect cells (19). The experiments in that report were the first to show a positive correlation between the binding affinities of M. sexta midgut protein suspensions and the identity of a single binding protein from whole midgut protein preparations immobilized on polyvinylidene difluoride (PVDF) filters. This correlation is of paramount importance for understanding the conflicting reports that appear when binding data are indirectly compared with the identification of a given binding protein on ligand blots, as pointed out recently by Lee and Dean (27). To resolve the confusion surrounding the identification of the relevant M. sexta midgut protein receptor(s) which binds the Cry1A toxins of B. thuringiensis, including Cry1Ac, we have designed experiments with different ligand binding protocols to determine whether various procedures affect toxin binding by M. sexta midgut proteins. The results of our work clearly demonstrate that BT-R1 is a common high-affinity receptor for the Cry1A B. thuringiensis toxins and that the binding properties of BT-R1 are not affected by any procedures commonly accepted and used to study ligand-receptor properties of lepidopteran insects.