Distinct mechanisms of antibody‐mediated enzymatic reactivation in β‐galactosidase molecular sensors

Feliu, Jordi X.; Ramírez, Esther; Villaverde, Antonio

doi:10.1016/s0014-5793(98)01315-5

Cited by 25 publications

(19 citation statements)

References 30 publications

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“…upon monoclonal antibody binding (more than 250%), higher than that previously obtained with the equivalent protein JX795A, which contains an FMDV peptide (about 200%) (7,8), and higher than those observed in any of the enzymatic sensors constructed up to now (37). Protein SD7895, which displays a 27-amino acid peptide at positions 278 and 795, shows a moderate reactivation factor (140%) indistinguishable from that of protein S795gp.…”

Section: Engineering Regulable E Coli ␤-Galactosidases As Biosensorsmentioning

confidence: 65%

Engineering Regulable Escherichia coliβ-Galactosidases as Biosensors for Anti-HIV Antibody Detection in Human Sera

Ferrer-Miralles

Feliu

Vandevuer

et al. 2001

Journal of Biological Chemistry

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The activity of engineered, peptide-displaying enzymes is modulated by binding to specific anti-peptide antibodies. This new concept of a quantitative antibody detection system allows test kits to be set up for fast diagnosis of infectious diseases. To develop a quick and homogeneous assay for the detection of human immunodeficiency virus (HIV) infection, we have explored two acceptor sites of the bacterial Escherichia coli ␤-galactosidase for the accommodation of HIV antigenic peptides. Two overlapping epitopes (namely P1 and P2) from the gp41 envelope glycoprotein, contained in different sized peptides, were inserted in the vicinity of the enzyme active site to generate a set of hybrid, enzymatically active ␤-galactosidases. Regulable enzymes of different responsiveness to monoclonal antibody binding were generated with both acceptor sites tested. These biosensors were also sensitive to immune sera from HIVinfected patients. Modeling data provide insight into the structural modifications in the vicinity of the active site induced by peptide insertion that strongly affect the responsiveness of the engineered proteins through different parameters of their catalytic properties.Insertional fusion technologies are useful instruments for the analysis of membrane protein topology and structure-function relationship, as well as for the generation of randomized protein libraries and enzymatic biosensors (1). The ␤-galactosidase enzyme (EC 3.2.1.23), encoded by the Escherichia coli lacZ gene, hydrolyzes lactose into glucose and galactose (2) which are then metabolized for cell growth. In addition, this enzyme also hydrolyzes other substrates producing colored compounds useful to monitor a wide range of biological processes. The three-dimensional structure of ␤-galactosidase (3) permits the permissiveness of solvent-exposed loops to heterologously inserted peptides to be explored. In this context, we previously constructed some recombinant ␤-galactosidases displaying foot-and-mouth disease virus (FMDV) 1 B-cell epitope peptides accommodated in solvent-exposed surfaces (4, 5). The peptide insertion results in hybrid ␤-galactosidases with reduced enzymatic activity. However, in the presence of antipeptide monoclonal antibodies or polyclonal sera, these hybrid enzymes translate the antigen-antibody interaction into an easily measurable increase of the enzymatic activity (4 -6).Because the results obtained with these FMDV-based biosensor prototypes were very promising in the context of a fast and easy diagnosis of infectious diseases in a homogeneous assay, we were prompted to design new recombinant ␤-galactosidases as biosensors for anti-HIV human antibodies. The development of such a homogeneous colorimetric assay could be of great impact in human health and also be helpful in better understanding the mechanism of enzymatic regulation in biosensors by testing whether ␤-galactosidase enzymatic modulation is restricted to particular epitopes or specific peptide sequence, length, or conformation. Therefore, we have inserted i...

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Section: Engineering Regulable E Coli ␤-Galactosidases As Biosensorsmentioning

confidence: 65%

Engineering Regulable Escherichia coliβ-Galactosidases as Biosensors for Anti-HIV Antibody Detection in Human Sera

Ferrer-Miralles

Feliu

Vandevuer

et al. 2001

Journal of Biological Chemistry

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“…These peptides had been previously reported to allow ELISA detection of infection-specific anti-FMDV antibodies from all seven serotypes of the virus (44). Four plasmids were constructed, and each one harbored one or two FMDV 3B peptides in restriction sites (BamHI and/or ClaI) close to the active site of the ␤-galactosidase monomer (18) (Fig. 1A and B).…”

Section: Resultsmentioning

confidence: 99%

Discriminating Foot-and-Mouth Disease Virus-Infected and Vaccinated Animals by Use of β-Galactosidase Allosteric Biosensors

Sánchez-Aparicio

Rosas

Ferraz

et al. 2009

Clin Vaccine Immunol

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Recombinant ␤-galactosidases accommodating one or two different peptides from the foot-and-mouth disease virus (FMDV) nonstructural protein 3B per enzyme monomer showed a drastic enzymatic activity reduction, which mainly affected proteins with double insertions. Recombinant ␤-galactosidases were enzymatically reactivated by 3B-specific murine monoclonal and rabbit polyclonal antibodies. Interestingly, these recombinant ␤-galactosidases, particularly those including one copy of each of the two 3B sequences, were efficiently reactivated by sera from infected pigs. We found reaction conditions that allowed differentiation between sera of FMDV-infected pigs, cattle, and sheep and those of naïve and conventionally vaccinated animals. These FMDV infection-specific biosensors can provide an effective and versatile alternative for the serological distinction of FMDV-infected animals.

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“…Several b-galactosidase-based sensor prototypes have been previously developed that, by displaying immunodominant epitopes on their surface, are successfully activated by monoclonal antibodies but also by immune sera against either FMDV (Benito et al, 1996;Feliu et al, 2002) or HIV (Ferrer-Miralles et al, 2001). Antibody-mediated activation requires multiple and simultaneous monovalent contacts between this enzyme and the effector molecules (Alcalá et al, 2002) that modify the enzymatic constants presumably through alterations in the conformational architecture of the active site (Feliu et al, 1998. Since the modulation of substrate processing rate is the sensing signal itself (obtained by comparison of product formed in absence and in presence of the analyte), the specific substrate selected to determine the signal could be not irrelevant to the final sensing data.…”

Section: Discussionmentioning

confidence: 99%

“…The sensing enzymatic assays were performed in ELISA microplates according to previously described procedures (Feliu et al, 1998). Briefly, different concentrations of NF795gpC in buffer Z were mixed with the effector antibody in the same buffer and incubated for 60 min at 258C in a microtiter plate Labsystems IEMS reader, in a total volume of 80 mL.…”

Section: Enzymatic Assaysmentioning

confidence: 99%

Enhanced molecular recognition signal in allosteric biosensing by proper substrate selection

Ferraz

Arı́s

Villaverde

2006

Biotech & Bioengineering

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Among protein biosensors, those based on enzymatic responses to specific analytes offer convenient instruments for fast and ultra-fast molecular diagnosis, through the comparative analysis of the product formed in presence and in absence of the effector. We have explored here the performance of five beta-galactosidase substrates during the activation of a beta-galactosidase sensor by antibodies against the human immunodeficiency virus (HIV). Interestingly, the employed substrate determines the dynamic range of the allosteric signal and significantly influences the sensitivity of the senso-enzymatic reaction. While ortho-nitrophenyl beta-D-galactopyranoside allows the detection of a model anti-gp41 monoclonal antibody below 0.024 ng/microL, phenol red beta-D-galactopyranoside offers the most dynamic response with signal/background ratios higher than 12-fold and a detection limit around 0.071 ng/microL. The hydrolysis of both chromogenic substrates generates linear sensing responses to immune human sera and parallel time-course topologies of the allosteric reaction. Therefore, the obtained results stress the potential of chromogenic substrates versus those rendering quimioluminescent, amperometric, or fluorescent signals, for the further automatization, miniaturization, or adaptation of beta-galactosidase-based biosensing to high-throughput applications.

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Distinct mechanisms of antibody‐mediated enzymatic reactivation in β‐galactosidase molecular sensors

Cited by 25 publications

References 30 publications

Engineering Regulable Escherichia coliβ-Galactosidases as Biosensors for Anti-HIV Antibody Detection in Human Sera

Engineering Regulable Escherichia coliβ-Galactosidases as Biosensors for Anti-HIV Antibody Detection in Human Sera

Discriminating Foot-and-Mouth Disease Virus-Infected and Vaccinated Animals by Use of β-Galactosidase Allosteric Biosensors

Enhanced molecular recognition signal in allosteric biosensing by proper substrate selection

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