Membrane anchors of alkaline phosphatase and trehalase associated with the plasma membrane of larval midgut epithelial cells of the silkworm, Bombyx mori

The larval midgut epithelial cell of the silkworm, Bombyx mori, has two forms of alkaline phosphatase and trehalase, soluble and membrane-bound. Alkaline phosphatase and trehalase of the latter form are found in the brush border membrane and the basolateral membrane, respectively. In this work we studied the membrane anchors of these membrane-bound enzymes. Alkaline phosphatase was solubilized by phosphatidyl-inositol-specific phospholipase C, but not by papain. Conversely, trehalase was released from the membrane by papain, but not by phosphatidylinositol-specific phospholipase C. Both enzymes were solubilized in an amphiphilic form with 0.5% Triton X-100 plus 0.5% sodium deoxycholate (pH 7.0). The detergent-solubilized alkaline phosphatase and trehalase were converted to hydrophilic form on incubation with phosphatidylinositol-specific phospholipase C and papain, respectively. The effects of papain on solubilization and conversion of trehalase were completely inhibited by leupeptin. These results suggest that, in the silkworm larvae, alkaline phosphatase is anchored in the brush-border membrane via a glycosyl-phosphatidylinositol, while trehalase is associated with the basolateral membrane through a hydrophobic segment of the polypeptide.     

Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis

The crystal proteins of Bacillus thuringiensis are widely used in transgenic crops and commercially available insecticides. Manduca sexta, the tobacco hornworm, is the model insect for B. thuringiensis studies. Although brush border vesicles prepared from larval M. sexta midgut have been used in numerous mode-of-action studies of B. thuringiensis toxins, their protein components are mostly unknown. Vesicles prepared from the brush border of M. sexta midgut were analyzed using one- and two-dimensional gel electrophoresis to establish a midgut brush border proteome. Sub-proteomes were also established for B. thuringiensis Cry1Ac binding proteins and glycosylphosphatidyl inositol (GPI) anchored proteins. Peptide mass fingerprints were generated for several spots identified as Cry1Ac binding proteins and GPI-anchored proteins and these fingerprints were used for database searches. Results generally did not produce matches to M. sexta proteins, but did match proteins of other Lepidoptera. Actin and alkaline phosphatase were identified as novel proteins that bind Cry1Ac in addition to the previously reported aminopeptidase N. Aminopeptidase N was the only GPI-anchored protein identified. Actin, aminopeptidase N, and membrane alkaline phosphatase were confirmed as accurate protein identifications through western blots.     

The receptor for Bacillus thuringiensis CrylA(c) delta-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N

A 120 kDa glycoprotein in the larval midgut membrane of the Iepidopteran Manduca sexta, previously identified as a putative receptor for Bacillus thuringiensis CrylA(c) δ-endotoxin, has been purified by a combination of protoxin affinity Chromatography and anion exchange chromatography. In immunoblotting experiments, the purified glycoprotein has the characteristics predicted of the receptor: it binds CrylA(c) toxin In the presence of GlcNAc but not GalNAc; it binds the lectin SBA; but it does not bind CrylB toxin. N-terminal and internal amino acid sequences obtained from the protein show a high degree of similarity with the enzyme aminopeptidase N (EC 3.4.11.2). When assayed for aminopeptidase activity, purified receptor preparations were enriched 5.3-fold compared to M. sexta brush border membrane vesicles. We propose that the receptor for CrylA(c) toxin in the brush border membrane of the lepidopteran M. sexta is the metalloprotease aminopeptidase N.     

A novel 96-kDa aminopeptidase localized on epithelial cell membranes of Bombyx mori midgut, which binds to Cry1Ac toxin of Bacillus thuringiensis

Proteins in the brush border membrane (BBM) of the midgut binding to the insecticidal Cry1Ac toxin from Bacillus thuringiensis were investigated to examine the lower sensitivity of Bombyx mori to Cry1Ac, and new aminopeptidase N that bound to Cry1Ac was discovered. DEAE chromatography of Triton X-100-soluble BBM proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4-kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to approximately 190-kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of the amino acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. K(D) of the APN96-Cry1Ac interaction was determined to be 1.83 +/- 0.95 microM. The lectin binding assay suggested that APN96 had an N-linked bi-antennal oligosaccharide or an O-linked mucin type one. The role of APN96 was discussed in relation to the insensitivity of B. mori to Cry1Ac.     

Role of specific activators of intestinal amino acid transport in Bombyx mori larval growth and nutrition.

Nutrient absorption and its modulation are critical for animal growth. In this paper, we demonstrate that leucine methyl ester (Leu-OMe) can greatly increase the activity of the transport system responsible for the absorption of most essential amino acids in the larval midgut of the silkworm Bombyx mori. We investigated leucine uptake activation by Leu-OMe in brush border membrane vesicles and in the apical membrane of epithelial cells in the midgut incubated in vitro. Moreover, the addition of this strong activator of amino acid absorption to diet significantly affected larval growth. Silkworms fed on artificial diet supplemented with Leu-OMe reached maximum body weight 12-18 h before control larvae, and produced cocoon shells up to 20% heavier than those of controls. The activation of amino acid absorption plays an essential role in larval development so that larval growth and cocoon production similar to controls reared on an artificial diet with 25% of dry mulberry leaf powder were observed in silkworms fed on an artificial diet with only 5% of mulberry powder. Arch.     

Selective transport of the mulberry leaf urease from the midgut into the larval hemolymph of the silkworm,Bombyx mori

Just before spinning, larvae of the silkworm, Bombyx mori, absorb intact urease of the host plant (mulberry leaf) from the midgut lumen into the hemolymph. In order to investigate whether the transport of the mulberry leaf urease is selective, crude proteins extracted from the mulberry leaves were labeled with biotin and orally administered to the fifth instar larvae. The biotinylated proteins transported into the hemolymph were detected by ligand blotting using streptavidin. When the biotinylated proteins were administered to 5-day-old fifth instar larvae, a strong signal of a biotinylated protein was detected in the hemolymph 2 days after the administration. In contrast, when the biotinylated mulberry leaf proteins were administered to 3-day-old fifth instar larvae, no signal derived from the biotinylated proteins was detected in the hemolymph. The signal weakened when the biotinylated proteins had been immunoprecipitated before administering to the larvae, indicating that the signal came from the mulberry leaf urease. These results show that the transport of the mulberry leaf urease from the midgut into the hemolymph is selective and larval-stage specific. Subsequently, binding assays were carried out to test the binding ability of the mulberry leaf urease to the brush border membrane in the epithelial cells of larval midgut. The urease was not bound to the brush border membrane vesicles (BBMV) from the midgut of 3-day-old fifth instar larvae, while more than 60% of the total amount of incubated urease was bound to the BBMV from the midgut of 6-day-old fifth instar larvae. The urease binding ability of BBMV correlated with the uptake of the mulberry leaf urease. This suggests that a urease binding molecule(s) exists in the BBM of the midgut epithelium, which is involved in the uptake of the mulberry leaf urease. In addition, the uptake of the mulberry leaf urease into the hemolymph was induced by 20-hydroxyecdysone.     

Expression of a glycosylphosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells.

Aminopeptidase N (APN; EC 3.4.11.2) is an exopeptidase that is attached to cell membranes by a hydrophobic amino-terminal stalk in vertebrates or a glycosylphosphatidylinositol (GPI) anchor in insects. In this study, we report the cloning, expression, and characterization of an aminopeptidase N from Manduca sexta midgut. The full-length aminopeptidase N cDNA (APN1a) encodes a 995-amino-acid protein. The predicted amino acid sequence differs by 8 amino acids from M. sexta APN1. These different amino acids do not modify any putative glycosylation or glycosylphosphatidylinositol anchor sites. The full-length cDNA was cloned into an expression plasmid, pHSP-HR5, and transiently expressed in an insect cell line derived from Spodoptera frugiperda (Sf21 cells). Immunoblot analysis with anti-APN antiserum showed that APN1a expressed in Sf21 cells is the same size (120 kDa) as APN found in midgut brush border membranes. After treatment with phosphatidylinositol-specific phospholipase C (PIPLC), anti-cross-reacting determinant antibody specific for PIPLC cleavage products recognized the expressed 120-kDa APN1a, but not endogenous Sf21 proteins, indicating that APN1a has an intact glycosylphosphatidylinositol anchor. These results are evidence that Sf21 cells synthesize few, if any, endogenous GPI-linked proteins. Immunofluorescence staining showed that the expressed APN1a was located on the surface of Sf21 cells.     

昆虫中肠Bt晶体蛋白受体的研究进展

苏云金芽孢杆菌Ｂａｃｉｌｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ杀虫作用的主要成份是胞内产生的伴孢晶体,晶体蛋白经昆虫吞食,在肠道降解为激活的毒性肽。普遍认为毒性肽的作用机制主要有两个步骤：１）与中肠表面的受体专一结合;２）在细胞膜上形成跨膜通道。杀虫晶体蛋白的专一性与中肠细胞膜表面的受体蛋白紧密相连,晶体蛋白的杀虫作用是通过昆虫中肠细胞的专一性受体而起作用。本文通过说明受体蛋白的生物学特性、分子本质及与昆虫抗性的关系,概述了近年来中肠受体蛋白的研究进展。１　昆虫中肠受体蛋白的生物学特性１１　受体蛋白…     

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori

• 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
• 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
• 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.

     

Intestinal uptake of dipeptides and beta-lactam antibiotics. I. The intestinal uptake system for dipeptides and beta-lactam antibiotics is not part of a brush border membrane peptidase

The uptake of beta-lactam antibiotics into small intestinal enterocytes occurs by the transport system for small peptides. The role of membrane-bound peptidases in the brush border membrane of enterocytes from rabbit and pig small intestine for the uptake of small peptides and beta-lactam antibiotics was investigated using brush border membrane vesicles. The enzymatic activity of aminopeptidase N was inhibited by beta-lactam antibiotics in a non-competitive manner whereas dipeptidylpeptidase IV was not affected. The peptidase inhibitor bestatin led to a strong competitive inhibition of aminopeptidase N whereas the uptake of cephalexin into brush border membrane vesicles was only slightly inhibited at high bestatin concentrations (greater than 1 mM). Modification of brush border membrane vesicles with the histidine-modifying reagent diethyl pyrocarbonate led to a strong irreversible inhibition of cephalexin uptake whereas the activity of aminopeptidase N remained unchanged. A modification of serine residues with diisopropyl fluorophosphate completely inactivated dipeptidylpeptidase IV whereas the transport activity for cephalexin and the enzymatic activity of aminopeptidase N were not influenced. With polyclonal antibodies raised against aminopeptidase N from pig renal microsomes the aminopeptidase N from solubilized brush border membranes from pig small intestine could be completely precipitated; the binding protein for beta-lactam antibiotics and oligopeptides of apparent Mr 127,000 identified by direct photoaffinity labeling with [3H]benzylpenicillin showed no crossreactivity with the aminopeptidase N anti serum and was not precipitated by the anti serum. These results clearly demonstrate that peptidases of the brush border membrane like aminopeptidase N and dipeptidylpeptidase IV are not directly involved in the intestinal uptake process for small peptides and beta-lactam antibiotics and are not a constituent of this transport system. This suggests that a membrane protein of Mr 127,000 is (a part of) the uptake system for beta-lactam antibiotics and small peptides in the brush border membrane of small intestinal enterocytes.     

EFFECT OF CRY1A δ-ENDOTOXINS OF BACILLUS THURINGIENSIS ON Mg+-ATPASE IN THE COLUMNAR CELLS OF BOMBYX MORI MIDGUT

Abstract  The effects of Cry1A δendotoxins from Bacillus thringiensis subsp. kurstaki HD-1 on the activity of M²+-dependent ATPase in the columnar cells (CC) of Sombyx mori were examined. Cytochemical localization showed a strong M$ ' -ATPase activity on the apical brush border of CC and even on the peritrophic membrane, but not on the goblet cells except slight activity on their interdigitated valves. Although overdose toxins were imposed to the larvae and resulted in serious cellular pathological changes in the treated midgut in vim , no obvious reduction of the M²+-ATPase activity on the CC membrane was observed. This may exclude the M²+ ATPase on the CC brush border membrane to be the potential natural channel modulated by B-endotoxins of B. thuringiensis or a target of the toxin during larval intoxication.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae).

The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.     

A novel regulatory mechanism for amino acid absorption in lepidopteran larval midgut

A number of methyl and ethyl esters of naturally occurring amino acids exert a potent stimulatory effect on the cotransport system responsible for the absorption of most essential amino acids along the midgut of the silkworm Bombyx mori. L-Leucine methyl ester (Leu-OMe), one of the most effective activators, induces a large increase of the initial rate of leucine uptake in midgut brush border membrane vesicles (BBMV) from the anterior-middle (AM) region, and a small effect in BBMV from the posterior (P) region. Nonetheless, the methyl ester causes in both regions a relevant K(+)-, Deltapsi- and pH-independent increase of the intravesicular accumulation of the amino acid. The activation by Leu-OMe proves that amino acid absorption can be modulated all along the B. mori larval midgut and that the AM region, where the ability to transport and concentrate the substrate is very low, is more susceptible than the P region. Leucine uptake in AM-BMMV can be activated by amino acid methyl esters with definite structural requisites, with the following order of potency: L-leucine>L-phenylglycine>L-methionine>L-phenylalanine>L-norleucinez.Gt;L-isoleucine. The activation is stereospecific and occurs also with some ethyl esters (e.g. leucine and phenylalanine). No activation was observed with esters of amino acids with short hydrophobic or polar side-chains. The activation mechanism here described plays a fundamental role in larval growth since silkworms reared on artificial diets supplemented with leucine or methionine methyl esters reach maximum body weight 12-18 h before control larvae and spin cocoons with a larger shell weight. This novel regulatory mechanism of an amino acid transport protein appears to be widespread among lepidopteran larvae.     

Evidence for the transit of aminopeptidase N through the basolateral membrane before it reaches the brush border of enterocytes

Summary In vivo pulse-chase labeling of rabbit jejunum loops was used in conjunction with subcellular fractionation and quantitative immunoprecipitation to determine whether or not the newly synthesized aminopeptidase N transits through the basolateral membrane before it reaches the apical brush border, its final localization. The kinetics of the arrival of the newly synthesized enzyme in the Golgi complex, basolateral and brush border membrane fractions strongly suggest that on leaving the Golgi aminopeptidase N is transiently integrated into the basolateral domain before reaching the brush border.     

一种家蚕类浓核病毒的组织病理学特征

本文从家蚕病蚕中分离到一种家蚕类浓核病毒（BmDNV-Like）,对它的组织病理学研究表明:该病毒首先寄生家蚕中肠柱状细胞,继而引起其细胞核的膨大和破裂;组织原位杂交结果表明该病毒既能在家蚕中肠柱状细胞中增殖,也能在中肠的杯形细胞中增殖,甚至在感染后期能在家蚕幼虫的大部分组织细胞中感染和增殖。     

Intestinal amino acid absorption in tobacco hornworm larvae is stimulated by potassium and sodium but not rubidium or lithium

We used rapid filtration assays to determine the ion selectivity of ion gradientdriven phenylalanine uptake by brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm (Manduca sexta). Phenylalanine uptake by these vesicles is stimulated by both potassium and sodium. Phenylalanine uptake by larval M. sexta midgut brush border membrane vesicles is voltage sensitive and shows little selectivity for potassium over sodium. However, phenylalanine uptake by these vesicles is stimulated by neither rubidium nor lithium.     

Identification of amino acid residues of Bacillus thuringiensis delta-endotoxin CryIAa associated with membrane binding and toxicity to Bombyx mori.

Alanine substitution (A3) or deletion (D3) of residues 365 to 371 of Bacillus thuringiensis CryIAa insect toxin removed nearly all toxicity for Bombyx mori (> 1,000-fold less active than the wild type). The loss of larvicidal activity in the mutants was not caused by increased sensitivity to larval gut enzymes but could be attributed to significantly reduced binding to B. mori brush border membrane vesicles. Some or all of the affected amino acid residues may interact directly or indirectly with the B. mori membrane receptor(s). Such receptor binding appears to be directly correlated with insect toxicity.     

Brush border membrane and amino acid transport

Fundamental differences in midgut structure, physiology, brush border proteins, and transporters among Leptinotarsa decemlineata, lepidopteran caterpillars, other insect taxa, and vertebrates are reviewed. The effects of dietary protein concentration on Manduca sexta midgut amino acid transport and brush border membrane proteins are reported. M. sexta fed diet with reduced protein had elevated levels of leucine aminopeptidase in the brush border membrane. No changes in amino acid transport or alkaline phosphatase activity due to dietary differences were detected. Changes in brush border proteins could affect the toxicity and pathogenicity of microbial agents. © 1996 Wiley-Liss, Inc.     

家蚕幼虫5龄期中肠蛋白质组学

利用双向电泳技术对家蚕幼虫5龄期第2天、第5天和第7天的中肠蛋白质进行分离, 并利用ImageMaster 2D Platinum对所分离得到的蛋白图谱进行比较, 并对一些蛋白质斑点进行了质谱鉴定。研究发现, 家蚕中肠蛋白质具有区别于家蚕其他组织的明显特征: 蛋白质大多分布在PI值4-8、分子量20~70 kD的区域, 且分布不均匀, 主要集中在酸性一侧, 这一特点在家蚕5龄期第7天的图谱尤为明显。5龄期家蚕第2天的蛋白质斑点数目为869个, 而到第5天增加到966个, 新增蛋白数目97个, 进一步观察发现增加的蛋白主要分布在PI值6-9, 分子量20~40 kD区间; 随着进入幼虫成熟期, 蛋白质斑点数目明显减少, 第7天斑点数仅为420个, 比第5天减少了56.5%。这些结果说明家蚕中肠组织蛋白质组成在5龄早、中、晚期经历了较大变化, 暗示这可能与中肠的功能相适应。从MALDI-TOF-MS鉴定的斑点发现了构成家蚕中肠组织的一些新的部分结构蛋白和一些可能与消化、吸收相关的蛋白, 还发现一些能够抵御外界微生物入侵的相关蛋白。这些结果为进一步认识家蚕中肠提供了重要的理论基础。