scholarly journals Identification and Characterization of Aminopeptidase-N as a Binding Protein for Cry3Aa in the Midgut of Monochamus alternatus (Coleoptera: Cerambycidae)

2020 ◽  
Vol 113 (5) ◽  
pp. 2259-2268
Author(s):  
Yajie Guo ◽  
Rebeca Carballar-Lejarazú ◽  
Liangjing Sheng ◽  
Yan Fang ◽  
Shaozhen Wang ◽  
...  
Keyword(s):  
Insect Pests ◽  
Binding Protein ◽  
Phosphatidyl Inositol ◽  
In Silico Analysis ◽  
Membrane Vesicles ◽  
Aminopeptidase N ◽  
Monochamus Alternatus ◽  
Cry Proteins ◽  
C Terminus

Abstract Bacillus thuringiensis Cry proteins have been widely used over the past decades for many different insect pests, which are safe for users and the environment. The coleopteran-specific Cry3Aa toxin from B. thuringiensis exhibits toxicity to the larvae of Monochamus alternatus. Receptors play a key role in the mechanisms underlying the toxic action of Cry. However, the binding receptor for Cry3Aa has yet to be identified in the midgut of M. alternatus larvae. Therefore, the aim of this study was to identify the receptor for Cry3Aa toxin in the brush border membrane vesicles (BBMVs) of M. alternatus larvae. Our results indicate that the Cry3Aa toxin binds to the BBMVs (Kd = 247 nM) of M. alternatus via a 107 kDa aminopeptidase N (APN) (Kd = 57 nM). In silico analysis of the APN protein predicted that an 18 amino acid sequence in the N-terminal acted as a signal peptide, and that the Asn residue, located at position 918 in the C-terminus is an anchored site for glycosyl phosphatidyl inositol. Further analysis showed that M. alternatus APN exhibits 75% homology to the APN from Anoplophora glabripenis. Our work, therefore, confirmed that APN, which is localized in the BBMVs in the midgut of M. alternatus larvae, acts as a binding protein for Cry3Aa toxins.

Partial purification and characterization of a membrane-associated steroid-binding protein from Pseudomonas testosteroni

1982 ◽  
Vol 60 (8) ◽  
pp. 798-803 ◽  
Author(s):  
Mike Francis ◽  
Mamoru Watanabe
Keyword(s):  
Binding Protein ◽  
Membrane Vesicles ◽  
Activity Analysis ◽  
Binding Activity ◽  
Regulatory Function ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Steroid Binding ◽  
Steroid Binding Protein

A steroid-binding protein obtained from the supernatant of the final wash from the preparation of membrane vesicles was purified severalfold to near homogeneity. The protein binds C18 and C19 steroids but has the highest affinity for androstenedione (Kd = 1.6 × 10−10 M). The molecular weight is 51 000 – 58 000. Binding activity is slightly inhibited by Cu2+, Ca2+, and Mg2+ and completely inhibited by Zn2+. The protein has no detectable steroid degradative activity. Analysis of androstenedione binding revealed negative cooperativity of binding for this ligand and may indicate a regulatory function for this protein. It is postulated that this protein binds the steroid after testosterone is converted to androstenedione.

scholarly journals Preparation and characterization of basolateral membrane vesicles from pig and human colonocytes: the mechanism of glucose transport

1993 ◽  
Vol 294 (2) ◽  
pp. 529-534 ◽  
Author(s):  
S A Pinches ◽  
S M Gribble ◽  
R B Beechey ◽  
A Ellis ◽  
J M Shaw ◽  
...  
Keyword(s):  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Independent Manner ◽  
Basolateral Membranes ◽  
Luminal Membrane ◽  
C Terminus ◽  
Normal Human

Membrane vesicles were isolated from the basolateral domains of pig and normal human colonocytes. The activity of the ouabain-sensitive K(+)-activated phosphatase, the basolateral membrane marker, was enriched 13-fold in these membrane vesicles over the original homogenate. The membranes displayed cross-reactions with antibodies to the (Na+/K+)ATPase and the RLA class I major histocompatibility antigen, both known indicators of the basolateral membrane. There was negligible contamination by other organelles and the luminal membrane, as revealed by marker-enzyme analysis and Western blotting, using an antibody to villin. The vesicles transported D-glucose in a cytochalasin B-inhibitable Na(+)-independent manner, with a Km of 28.1 +/- 0.8 mM and Vmax. of 3.1 +/- 0.4 nmol/s per mg of protein. The transport was inhibited by 2-deoxy-D-glucose and 3-O-methyl-D-glucose, but not by L-glucose or methyl-alpha-D-glucose. Probing the colonocyte basolateral membranes with an antibody against the C-terminus of the human liver GLUT 2 produced a cross-reaction at 52 kDa. These properties indicate the presence of a GLUT 2 isoform on the basolateral membranes of human and pig colonocytes.

Characterization of a Monoclonal Antibody, D73H, that Maps to a Highly Conserved Region on Fibrinogen Bβ Chain

2000 ◽  
Vol 84 (07) ◽  
pp. 43-48 ◽  
Author(s):  
B. J. Rybarczyk ◽  
M. Pereira ◽  
P. J. Simpson-Haidaris
Keyword(s):  
In Silico ◽  
Coiled Coil ◽  
In Silico Analysis ◽  
Antigenic Determinants ◽  
Human Fibrinogen ◽  
Western Blots ◽  
C Terminus ◽  
Probability Prediction ◽  
Silico Analysis

SummaryThe primary structure of fibrinogen is highly conserved across species, yet often times monoclonal antibodies produced against the fibrinogen of one species will not crossreact with the fibrinogen of another. Herein, we describe the production and characterization of murine MAb, D73H, raised against human fibrinogen. D73H crossreacts with a highly conserved epitope on the Bβ chain of fibrinogen from human, rat, bovine, guinea pig, and mouse. Western blotting revealed that D73H reacted with the Bβ chain of plasmin fragment D, localizing its epitope to Bβ134-461. A 7 kDa band was identified by D73H in Western blots of reduced fibrinogen CNBr-fragments. N-terminal sequencing mapped this fragment to Bβ243-253, further localizing the epitope to Bβ243-305. In silico analysis indicated that Bβ243-305 is predominantly hydrophilic, and surface probability prediction indicated three potential antigenic determinants corresponding to Bβ252-258, Bβ262-269, and Bβ279-286. Further in silico analysis of the crystal structure of fibrinogen fragment D-D indicated that Bβ262-269 (FGRKWDPY) is predominantly α-helical and located on the surface of the molecule adjacent to a bend imposed in the β chain at residue 260, which is near the junction between the rigid coiled-coil domain and the globular C-terminus. A synthetic peptide corresponding to Bβ261-272 competitively inhibited the binding of D73H to the Bβ chain of denatured intact fibrinogen and reduced and denatured Bβ chain in Western blots, experimentally proving the validity of these predictive algorithms. Together these data indicate that, although plasmin resistant, Bβ chain residues Bβ261-272 comprising the D73H epitope are highly conserved across species, surface exposed, and immunogenic.

scholarly journals Expression and Characterization of an Iron-Regulated Hemin-Binding Protein, HbpA, from Leptospira interrogans Serovar Lai

2007 ◽  
Vol 75 (9) ◽  
pp. 4582-4591 ◽  
Author(s):  
Swapna Asuthkar ◽  
Sridhar Velineni ◽  
Johannes Stadlmann ◽  
Friedrich Altmann ◽  
Manjula Sritharan
Keyword(s):  
Binding Protein ◽  
In Silico Analysis ◽  
Receptor Protein ◽  
Leptospira Interrogans ◽  
Outer Membrane Receptor ◽  
Regulated Expression ◽  
Surface Localization ◽  
Spectrofluorimetric Analysis ◽  
Silico Analysis

ABSTRACTIn an earlier study, based on the ferric enterobactin receptor FepA ofEscherichia coli, we identified and modeled a TonB-dependent outer membrane receptor protein (LB191) from the genome ofLeptospira interrogansserovar Lai. Based on in silico analysis, we hypothesized that this protein was an iron-dependent hemin-binding protein. In this study, we provide experimental evidence to prove that this protein, termed HbpA (hemin-bindingprotein A), is indeed an iron-regulated hemin-binding protein. We cloned and expressed the full-length 81-kDa recombinant rHbpA protein and a truncated 55-kDa protein fromL. interrogansserovar Lai, both of which bind hemin-agarose. Assay of hemin-associated peroxidase activity and spectrofluorimetric analysis provided confirmatory evidence of hemin binding by HbpA. Immunofluorescence studies by confocal microscopy and the microscopic agglutination test demonstrated the surface localization and the iron-regulated expression of HbpA inL. interrogans. Southern blot analysis confirmed our earlier observation that thehbpAgene was present only in some of the pathogenic serovars and was absent inLeptospira biflexa. Hemin-agarose affinity studies showed another hemin-binding protein with a molecular mass of approximately 44 kDa, whose expression was independent of iron levels. This protein was seen in several serovars, including nonpathogenicL. biflexa. Sequence analysis and immunoreactivity with specific antibodies showed this protein to be LipL41.

scholarly journals Cloning and Characterization of a Gene,pbpF, Encoding a New Penicillin-Binding Protein, PBP2B, inStaphylococcus aureus

1999 ◽  
Vol 43 (7) ◽  
pp. 1578-1583 ◽  
Author(s):  
Hitoshi Komatsuzawa ◽  
Gil H. Choi ◽  
Kouji Ohta ◽  
Motoyuki Sugai ◽  
Monique T. Tran ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Binding Protein ◽  
Protein Band ◽  
Binding Activity ◽  
Amino Acid Residues ◽  
Penicillin Binding Protein ◽  
Putative Signal Peptide ◽  
C Terminus ◽  
Histidine Tag

ABSTRACT A previously unrecognized penicillin binding protein (PBP) gene,pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and β-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed inEscherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region ofpbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.

Purification and characterization of a membrane-associated testosterone-binding protein from Pseudomonas testosteroni

1983 ◽  
Vol 61 (5) ◽  
pp. 307-312 ◽  
Author(s):  
Mike McD. Francis ◽  
Mamoru Watanabe
Keyword(s):  
Binding Protein ◽  
Divalent Cations ◽  
Membrane Surface ◽  
Membrane Vesicles ◽  
Affinity Constant ◽  
Steroid Binding Proteins ◽  
Steroid Binding ◽  
Steroid Binding Protein ◽  
Pseudomonas Testosteroni

A steroid-binding protein, identified in the supernatant generated when membrane vesicles of Pseudomonas testosteroni are produced and harvested by centrifugation, has been purified 49-fold to homogeneity. It has a molecular weight of 30 000 – 35 000 and it specifically binds the C19 steroids dihydrotestosterone, testosterone, and androstenedione. It is a basic protein with an isoelectric point at pH 7.3. Binding of testosterone exhibited normal saturation kinetics with an affinity constant, Kd, of 3.9 × 10−8 M. Binding was inhibited by divalent cations, but the sulfhydryl reagents dithiothreitol and mercaptoethanol did not affect activity. It is suggested that this and other membrane-associated steroid-binding proteins concentrate the steroid at the membrane surface before it is transported into the cytoplasm of P. testosteroni.

scholarly journals Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens

2020 ◽  
Author(s):  
Daniel Pinos ◽  
Maissa Chakroun ◽  
Anabel Millán-Leiva ◽  
Juan Luis Jurat-Fuentes ◽  
Denis J. Wright ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Heliothis Virescens ◽  
Insect Pests ◽  
Resistance Management ◽  
Membrane Vesicles ◽  
Membrane Receptors ◽  
Insecticidal Protein ◽  
Down Regulation ◽  
Cry Proteins ◽  
Membrane Bound

ABSTRACTThe Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests, thus understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, a laboratory-selected colony of Heliothis virescens (Vip-Sel) highly resistant to the Vip3Aa protein was used to test whether an alteration of membrane receptors in the insect midgut might explain the resistance phenotype. Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae show dramatically reduced levels of alkaline phosphatase activity, which was further confirmed by a strong down-regulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, its involvement as a receptor for the Vip3Aa protein was not supported by ligand blotting and viability assays with insect cells expressing HvmALP1. These data support that reduced alkaline phosphatase, previously observed in insect colonies resistant to Cry proteins from Bt, may also serve as an indirect marker that is not mechanistically involved in resistance to Vip3Aa.IMPORTANCEThe Vip3Aa insecticidal protein remains the only lepidopteran-specific trait in transgenic Bt crops with no cases of field-evolved resistance. While laboratory-selected resistance to Vip3A has been reported elsewhere, the mechanism for resistance is unknown. Results in this work show lack of significant Vip3Aa binding alterations in resistant and reference colonies of H. virescens. These observations are in contrast to most cases of high levels of resistance to insecticidal Bt proteins for which decreased binding is commonly detected. In addition, this study provides the first evidence of down-regulation of membrane bound alkaline phosphatase (mALP) associated with Vip3Aa resistance, a phenomenon commonly associated with resistance to Cry proteins from Bt. Results from this work suggest that mALP down-regulation may be a useful biomarker yet reject its direct participation in resistance to Vip3Aa.

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