Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An aminopeptidase N (APN) isozyme having the molecular weight of 90 kDa, was released by phosphatidylinositol-specific phospholipase C (PI-PLC) and purified homogeneously, from the brush border membrane of Bombyx mori. From the result of cDNA cloning, the primary structure of 90 kDa APN proved to consist of 948 amino acid residues, containing a typical metalloprotease-specific zinc-binding motif in the deduced sequence. Moreover, the primary sequence contained two hydrophobic segments on N- and C-termini. The N-terminal one showed characteristics of leader peptide for secretion and the C-terminal one contained a possible glycosylphosphatidylinositol (GPI) anchoring site, suggesting that the APN encoded by the cDNA is not only a zinc-binding enzyme, but also a GPI-anchored protein. The primary sequence is significantly homologous with those of insect and mammalian APNs, and contains four conserved segments around the zinc-binding motif, two potential N-glycosylation sites and four conserved Cys residues. The deduced primary sequence had 30.7% identity with that of B. mori 110 kDa APN, and did not contain the N-terminal and internal amino acid sequences of B. mori 100 kDa APN, revealing B. mori 90 kDa APN to be the third isozyme on the midgut brush border membrane. On the other hand, the primary sequence of 90 kDa APN showed high homology with Manduca sexta APN2 (65.1% identity) and Plutella xylostella APN2 (63.8% identity). It appears that the B. mori 90 kDa APN should be classified in the insect apn2 cluster and differentiated from insect apn1 and mammalian apn clusters by phylogenetic analysis. These results suggest that 90 kDa APN isozyme encoded by the cDNA is a product of B. mori apn2 gene.
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PMID:Cloning and sequence analysis of the aminopeptidase N isozyme (APN2) from Bombyx mori midgut. 997 96

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.
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PMID:Expression of a glycosylphosphatidylinositol-linked Manduca sexta aminopeptidase N in insect cells. 1049 76

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.
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PMID:Interaction of gene-cloned and insect cell-expressed aminopeptidase N of Spodoptera litura with insecticidal crystal protein Cry1C. 1220 Mar 17

Bacillus thuringiensis Cry1A toxins, in contrast to other pore-forming toxins, bind two putative receptor molecules, aminopeptidase N (APN) and cadherin-like proteins. Here we show that Cry1Ab toxin binding to these two receptors depends on the toxins' oligomeric structure. Toxin monomeric structure binds to Bt-R1, a cadherin-like protein, that induces proteolytic processing and oligomerization of the toxin (Gomez, I., Sanchez, J., Miranda, R., Bravo A., Soberon, M., FEBS Lett. (2002) 513, 242-246), while the oligomeric structure binds APN, which drives the toxin into the detergent-resistant membrane (DRM) microdomains causing pore formation. Cleavage of APN by phospholipase C prevented the location of Cry1Ab oligomer and Bt-R1 in the DRM microdomains and also attenuates toxin insertion into membranes despite the presence of Bt-R1. Immunoprecipitation experiments demonstrated that initial Cry1Ab toxin binding to Bt-R1 is followed by binding to APN. Also, immunoprecipitation of Cry1Ab toxin-binding proteins using pure oligomeric or monomeric structures showed that APN was more efficiently detected in samples immunoprecipitated with the oligomeric structure, while Bt-R1 was preferentially detected in samples immunoprecipitated with the monomeric Cry1Ab. These data agrees with the 200-fold higher apparent affinity of the oligomer than that of the monomer to an APN enriched protein extract. Our data suggest that the two receptors interact sequentially with different structural species of the toxin leading to its efficient membrane insertion.
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PMID:Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. 1553 4

Bacillus thuringiensis Cry1Ac toxin bound to a 120-kDa protein isolated from the brush border membranes of both susceptible and resistant larvae of Plutella xylostella, the diamondback moth. The 120-kDa protein was purified by Cry1Ac toxin affinity chromatography. Like Cry1Ac-binding aminopeptidase N (EC 3.4.11.2) from other insects, this protein was eluted from the affinity column with 200 mM N-acetylgalactosamine. The purified protein had aminopeptidase activity and bound Cry1Ac toxin on ligand blots. Purified aminopeptidase was recognized by antibodies to the cross-reacting determinant found on phosphatidylinositol-specific phospholipase C-solubilized proteins. The results show that the presence of Cry1Ac-binding aminopeptidase in the brush border membrane is not sufficient to confer susceptibility to Cry1Ac. Furthermore, the results do not support the hypothesis that resistance to Cry1Ac was caused by lack of a Cry1Ac-binding aminopeptidase.
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PMID:Binding of Bacillus thuringiensis Cry1Ac Toxin to Aminopeptidase in Susceptible and Resistant Diamondback Moths (Plutella xylostella). 1653 36

The insecticidal Cry proteins produced by Bacillus thuringiensis strains are pore-forming toxins (PFTs) that bind to the midgut brush border membrane and cause extensive damage to the midgut epithelial cells of susceptible insect larvae. Force-feeding B. thuringiensis PFTs to Lymantria dispar larvae elicited rapid and massive shedding of a glycosylphosphatidylinositol (GPI)-anchored aminopeptidase N (APN) from midgut epithelial cells into the luminal fluid, and depletion of the membrane-anchored enzyme on the midgut epithelial cells. The amount of APN released into the luminal fluid of intoxicated larvae was dose- and time-dependent, and directly related to insecticidal potency of the PFTs. The induction of toxin-induced shedding of APN was inhibited by cyclic AMP and MAPK kinase (MEK) inhibitors PD98059 and U0126, indicating that signal transduction in the MEK/ERK pathway is involved in the regulation of the shedding process. APN released from epithelial cells appears to be generated by the action of a phosphatidylinositol-specific phospholipase C (PI-PLC) cleavage of the GPI anchor based upon detection of a cross-reacting determinant (CRD) on the protein shed into the luminal fluid. Alkaline phosphatase was also released from the gut epithelial cells, supporting the conclusion that other GPI-anchored proteins are released as a consequence of the activation PI-PLC. These observations are the basis of a novel and highly sensitive tool for evaluating the insecticidal activity of new Cry proteins obtained though discovery or protein engineering.
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PMID:Bacillus thuringiensis pore-forming toxins trigger massive shedding of GPI-anchored aminopeptidase N from gypsy moth midgut epithelial cells. 1851 Sep 72

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.
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PMID:A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae. 1882 60

Transintestinal cholesterol efflux (TICE) has been proposed to represent a non-hepatobiliary route of cholesterol secretion directly "from blood to gut" and to play a physiologically significant role in excretion of neutral sterols, but so far little is known about the proteins involved in the process. We have previously observed that apolipoprotein A-1 (apoA-1) synthesized by enterocytes of the small intestine is mainly secreted apically into the gut lumen during fasting where its assembly into chylomicrons and basolateral discharge is at a minimal level. In the present work we showed, both by immunomicroscopy and subcellular fractionation, that a fraction of the apically secreted apoA-1 in porcine small intestine was not released from the cell surface but instead deposited in the brush border. Cholesterol was detected in immunoisolated microvillar apoA-1, and it was partially associated with detergent resistant membranes (DRMs), indicative of localization in lipid raft microdomains. The apolipoprotein was not readily released from microvillar vesicles by high salt or by incubation with phosphatidylcholine-specific phospholipase C or trypsin, indicating a relatively firm attachment to the membrane bilayer. However, whole bile or taurocholate efficiently released apoA-1 at low concentrations that did not solubilize the transmembrane microvillar protein aminopeptidase N. Based on these findings and the well known role played by apoA-1 in extrahepatic cellular cholesterol removal and reverse cholesterol transport (RCT), we propose that brush border-deposited apoA-1 in the small intestine acts in TICE by mediating cholesterol efflux into the gut lumen.
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PMID:Apolipoprotein A-1 (apoA-1) deposition in, and release from, the enterocyte brush border: a possible role in transintestinal cholesterol efflux (TICE)? 2211 76


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