Kazuko Nakanishi

Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin.

Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin‐binding region of each APN isoform. The results suggest that APN isoforms have a common toxin‐binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS–PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.

A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae

Aminopeptidase N (APN) and cadherin‐like protein (BtR175) from Bombyx mori larvae were examined for their roles in Cry1Aa‐ and Cry1Ac‐induced lysis of B. mori midgut epithelial cells (MECs). APNs and BtR175 were present in all areas of the midgut, were particularly abundant in the posterior region, and were found only on columnar cell microvilli and not on the lateral membrane that makes cell–cell contacts. This distribution was in accordance with the distribution of Cry1A‐susceptible MECs in the midgut. The lytic activity of Cry1Aa and Cry1Ac on collagenase‐dissociated MECs was linearly dependent on toxin concentration. Although pre‐treatment of MECs with anti‐BtR175 antibody was observed to partially inhibit the lytic activity exerted by 0.1–1 nM Cry1Aa toxin or 5 nM Cry1Ac toxin, no significant inhibition was observed when MECs were pre‐treated with anti‐APN antibody. These results suggest that BtR175 functions as a major receptor for Cry1A toxins in the midgut of B. mori larvae.

Location of the Bombyx mori Aminopeptidase N Type 1 Binding Site on Bacillus thuringiensis Cry1Aa Toxin

ABSTRACT We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508STLRVN513 and 582VFTLSAHV589. These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.

cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori

Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN.

Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N

The Bacillus thuringiensis Cry1Aa toxin‐binding region of Bombyx mori aminopeptidase N (APN) was analyzed, to better understand the molecular mechanism of susceptibility to the toxin and the development of resistance in insects. APN was digested with lysylendopeptidase and the ability of the resulting fragments to bind to Cry1Aa and 1Ac toxins was examined. The binding abilities of the two toxins to these fragments were different. The Cry1Aa toxin bound to the fragment containing 40‐Asp to 313‐Lys, suggesting that the Cry1Aa toxin‐binding site is located in the region between 40‐Asp and 313‐Lys, while Cry1Ac toxin bound exclusively to mature APN. Next, recombinant APN of various lengths was expressed in Escherichia coli cells and its ability to bind to Cry1Aa toxin was examined. The results localized the Cry1Aa toxin binding to the region between 135‐Ile and 198‐Pro.

Bacillus thuringiensis insecticidal Cry1Aa toxin binds to a highly conserved region of aminopeptidase N in the host insect leading to its evolutionary success.

Bacillus thuringiensis insecticidal protein, Cry1Aa toxin, binds to a specific receptor in insect midguts and has insecticidal activity. Therefore, the structure of the receptor molecule is probably a key factor in determining the binding affinity of the toxin and insect susceptibility. The cDNA fragment (PX frg1) encoding the Cry1Aa toxin-binding region of an aminopeptidase N (APN) or an APN family protein from diamondback moth, Plutella xylostella midgut was cloned and sequenced. A comparison between the deduced amino acid sequence of PX frg1 and other insect APN sequences shows that Cry1Aa toxin binds to a highly conserved region of APN family protein. In this paper, we propose a model to explain the mechanism that causes B. thuringiensis evolutionary success and differing insect susceptibility to Cry1Aa toxin.

Novel RNA sequences associated with late male killing in Homona magnanima

Maternally inherited female-biased sex ratios have been documented in many invertebrate species. One cause of such biased sex ratios is male killing, i.e. only males die. In most species, male killing occurs during embryonic stages (early male killing) and is associated with cytoplasmic bacteria, including Wolbachia, Spiroplasma, Rickettsia, Flavobacteria and gamma proteobacteria. However, the oriental tea tortrix, Homona magnanima, is one of the few species in which male death occurs in the larval or pupal stage, and is thus an example of late male killing. We partially purified the agent causing late male killing in H. magnanima and showed that it consists of two RNA sequences. This represents an entirely novel agent causing late male killing.

Phospholipid turnover in the inflamed intestinal mucosa : Arachidonic acid-rich phosphatidyl/plasmenyl-ethanolamine in the mucosa in inflammatory bowel disease

Abstract: Cytosolic phospholipase A2 (PLase A2) is activated by low Ca2+ concentrations and translocates from the cytosol to the cell membrane, releasing arachidonic acid; the arachidonic acid cascade then leads to the production of many inflammatory mediators. The aim of this study, accordingly, was to investigate the role of phospholipid metabolism in the intestinal mucosa in inflammatory bowel disease (IBD). Surgically resected specimens from patients with Crohns disease (CD), ulcerative colitis (UC), and colrectal cancer (non-cancerous tissue; as a control) were submitted to phospholipid analysis and a PLase A2 assay, which measures the degradation of endogenous mucosal phospholipids. A high percentage of plasmenylethanolamine (plas.E) was detected in the glycerophospholipid fraction of CD mucosa. The arachidonic acid content of the phosphatidylethanolamine plus plas.E subfraction was higher in inflamed than in intact mucosa in CD. PLaseA2 activity, resulting in lysophosphatidyl ethanolamine production, was detected only in inflamed mucosa from CD and UC patients, but not in normal mucosa from controls. PLaseA2 activity was highest in moderately inflamed mucosa adjacent to a severely ulcerated area. The PLaseA2 that reacts with endogenous phosphatidylcholine (PC) to form lysoPC was found irrespective of the presence of inflammation. The PLaseA2 that reacts with ethanolamine-containing phospholipids is more closely related to inflammation than other PLaseA2 isoenzymes in IBD mucosa.

Bacillus thuringiensis Cry Toxins Bound Specifically to Various Proteins via Domain III, Which Had a Galactose-Binding Domain-Like Fold

Cry toxins have been reported to bind not only to receptors on insect cells but also to several unrelated proteins. In this study, we investigated the binding properties of Bacillus thuringiensis Cry toxins, focusing on domain III, a Cry toxin region with a structure that of the galactose-binding domain-like. Cry1Aa, Cry1Ac, and Cry8Ca specifically bound to several proteins unrelated to insect midgut cells. Cry1Aa binding to Cry toxin-binding proteins was inhibited by a monoclonal antibody, 2C2, indicating that Cry1Aa binds to these Cry toxin-binding proteins through domain III. Cry1Aa binding to Bombyx mori aminopeptidase N and other Cry toxin-binding proteins was inhibited by carbonic anhydrase, a Cry toxin-binding protein. The binding regions of carbonic anhydrase and Bombyx mori aminopeptidase N were narrowed to regions of less than 20 amino acids that did not have any similarity, suggesting that Cry toxin domain III has a binding pocket for multiple proteins.

Biochemical bases in differentiation of a mouse cell line GSM06 to gastric surface cells

A mouse gastric surface cell line GSM06 established from a transgenic mouse harboring temperature-sensitive simian virus 40 large T-antigen gene was subjected to the lipid and glycoprotein analysis. When GSM06 cells were cultured for a long time after formation of a confluent monolayer, they differentiated to resemble foveolar epithelial cells morphologically. Biochemical changes during culture were studied in cells harvested just when a monolayer had formed (day 0), on day 7, and on day 21. Content of total phospholipids, cholesterol, cholesterol sulfate, total sugar and sialic acid increased about 1.5-fold from day 0 to 7 and remained elevated till day 21. The fatty acid composition of phospholipids revealed increased relative levels of oleic acid in phosphatidylcholine and phosphatidylethanolamine, and an increased level of plasmenylethanolamine from day 0 to 7. The level of dolichylphosphate continued to increase in a time-dependent manner. Glycosylation of various proteins, detected with lectins, was enhanced from day 7. In addition, greater resistance to taurodeoxycholate and acetylsalicylic acid was observed on days 7 and 21 than on day 0. Thus, enhanced glycosylation of proteins and an overall increase in the area of cellular membranes were the major changes in GSM06 cells during culture, and they were accompanied by an enhancement of cytoprotective potential.