Immunocapture-based fluorometric assay for the ...

Journal of Neuroscience Methods 169 (2008) 177–181

Immunocapture-based fluorometric assay for the measurement of insulin-degrading enzyme activity in brain tissue homogenates James Scott Miners ∗ , Patrick Gavin Kehoe, Seth Love Dementia Research Group, Institute of Clinical Neurosciences, Clinical Science at North Bristol, University of Bristol, Bristol BS16 1LE, UK Received 5 November 2007; received in revised form 6 December 2007; accepted 7 December 2007

Abstract Internally quenched fluorogenic substrates are commonly used for measuring enzyme activity in biological samples and allow high sensitivity and continuous real-time measurement that is well suited for high throughput analysis. We describe the development and optimisation of an immunocapture-based assay that uses the fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)) and allows the specific measurement of insulindegrading enzyme (IDE) activity in brain tissue homogenates. This fluorogenic substrate can be cleaved by a number of enzymes including neprilysin (NEP), endothelin-converting enzyme-1 (ECE-1) and angiotensin-converting enzyme (ACE), as well as IDE, and we have previously shown that discrimination between these individual enzymes is not readily achieved in tissue homogenates, even in the presence of selective inhibitors and pH conditions. We tested a panel of IDE antibodies to isolate and capture IDE from brain tissue homogenates and found that immunocapture with antibody to the inactive domain of IDE prior to the addition of fluorogenic substrate allows sensitive (linear at 156–2500 ng/ml) and specific measurement of IDE activity and negligible cross-reactivity with NEP, ACE or ECE-1. This assay should allow the measurement of IDE enzyme levels in a variety of biological tissues and may be useful in study of diseases such as Alzheimer’s disease and insulin-dependent diabetes. © 2007 Elsevier B.V. All rights reserved. Keywords: Insulin-degrading enzyme; Fluorogenic substrate; Immunocapture; Alzheimer’s disease; Insulin-dependent diabetes

1. Introduction Insulin-degrading enzyme (IDE) (insulysin, insulinase, EC3.4.24.56) is a zinc metalloendopeptidase that is highly expressed in the liver, testes, muscle and brain (Kuo et al., 1993). It is a single polypeptide with a molecular weight of 110 kDa encoded by a gene located on chromosome 10q23–q25 (Affholter et al., 1990). IDE is expressed predominantly in the cytosol (Akiyama et al., 1988; Bennet et al., 1994; Duckworth et al., 1994), and in smaller amounts in peroxisomes (Authier et al., 1996; Morita et al., 2000), rough endoplasmic reticulum and plasma membrane (Goldfine et al., 1984; Seta et al., 1998). IDE also exists as a secreted form, present within extracellular compartments such as cerebrospinal fluid (Qiu et al., 1998). IDE degrades a wide range of substrates which all share a common amyloidogenic secondary protein structure, and include insulin (Duckworth et al., 1998; Kirschner and ∗ Corresponding author at: Clinical Science at North Bristol, University of Bristol, Frenchay Hospital, Bristol BS16 1LE, UK. Tel.: +44 117 9701212x2953; fax: +44 117 9573955. E-mail address: [email protected] (J.S. Miners).

0165-0270/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2007.12.003

Goldberg, 1983), amylin (Bennett et al., 2000), insulin-like growth factors I and II (Misbin and Almira, 1989) and amyloid␤ (A␤) (Kurochkin and Goto, 1994). Deficiencies in IDE, or expression of mutant, inactive forms of the enzyme, is associated with increased levels of insulin and amylin, both of which are implicated in the pathology of type 2 diabetes (Farris et al., 2003). Reduced IDE enzyme activity has also been reported in Alzheimer’s disease (AD) (Cook et al., 2003; Perez et al., 2000; Zhao et al., 2007) the importance of which is emphasised by additional evidence suggesting that IDE has a physiological role in the degradation and clearance of A␤, the accumulation of which is one of the hallmarks of AD (Farris et al., 2003; Leissring et al., 2003; Qiu et al., 1998; Vekrellis et al., 2000). We previously developed a fluorogenic immunocapture assay that specifically measures neprilysin (NEP) activity in brain tissue homogenates (Miners et al., 2008). The fluorogenic substrate used in the assay is cleaved by a number of enzymes. To achieve specificity, a monoclonal antibody raised against human neprilysin was used to isolate neprilysin in an active state on a solid-phase prior to the addition of the quenched fluorogenic substrate, which fluoresces upon enzymatic cleavage allowing enzyme activity to be measured using a fluorescent plate reader.

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J.S. Miners et al. / Journal of Neuroscience Methods 169 (2008) 177–181

Fig. 1. Standard curves of enzyme activity of (A) solutions of recombinant rat and human IDE and (B) immunocaptured recombinant rat and human IDE. The curves in both (A) and (B) were generated by incubation with fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). The measurements were based on substrate cleavage at pH 7.5 after incubation for 1 h at 37 ◦ C.

The assay provides high specificity and allows a high-throughout analysis of a large number of samples in a short time period. We have now tested a number of IDE-specific capture antibodies (raised against the N- and C- termini, inactive domain and full-length protein) and, using the same fluorogenic peptide substrate, have adapted the assay to measure IDE activity in brain tissue homogenates.

Glasgow, UK) in 0.5% triton-X-100, 20 mM Tris pH 7.4, 10% sucrose (w/v), containing the protease inhibitors aprotinin (1 ␮g/ml; Sigma, UK) and PMSF (10 ␮M; Sigma, UK). The homogenates were centrifuged at 13,000 rpm for 15 min at 4 ◦ C, and the supernatants aliquoted and stored at −80 ◦ C until used. 2.2. Fluorogenic assay of enzyme activity

2. Materials and methods 2.1. Materials The internally quenched fluorogenic substrate (McaRPPGFSAFK(Dnp)) and recombinant human IDE, angiotensinconverting enzyme (ACE), endothelin-converting enzyme-1 (ECE-1) and NEP standards were purchased from R&D systems (Abingdon, UK). Rat recombinant IDE was purchased from Calbiochem (California, U.S.A). Nunc FluorNunc MaxiSorp 96-well plates were purchased from Fisher Scientific (Loughborough, UK). We used the following IDE antibodies: goat polyclonal anti-human IDE raised against full-length recombinant human IDE (R&D systems, Abingdon, UK); polyclonal rabbit antihuman IDE raised against a synthetic N-terminal peptide (Calbiochem, California, U.S.A); rabbit polyclonal antihuman IDE raised against a synthetic C-terminal peptide of IDE (Abcam, Cambridge, UK); and rabbit anti-human IDE raised against a synthetic peptide corresponding to part of the inactive domain of the enzyme (Abcam, Cambridge, UK). Brain tissue was obtained from the South West Dementia Brain Bank, University of Bristol, with local Research Ethics Committee approval. Crude brain tissue homogenates were prepared from dissected samples of unfixed frozen cortex from the left mid-frontal region (Brodmann area 6). 200 mg tissue samples were homogenised for 30 s in a Precellys 24 automated tissue homogeniser (Stretton Scientific, Derbyshire, UK) with 2.3 mm silica beads (Biospec, Thistle Scientific,

Fluorogenic peptide substrate (10 ␮M) was added to serial dilutions of recombinant human and rat IDE (2500–20 ng/ml) in 100 mM Tris–HCl pH 7.5, 50 mM NaCl, 10 ␮M ZnCl2 and incubated in the dark for 1 h at 37 ◦ C to produce standard curves of enzyme activity. Fluorescence was measured with excitation at 320 nm and emission at 405 nm, in a fluorescent plate reader (FLUOstar, BMG Labtech, UK). 2.3. Immunocapture assay using the fluorogenic peptide substrate 2.3.1. Immunocapture-based IDE-specific enzyme activity assay Nunc MaxiSorp 96-well plates were coated with capture IDE antibodies (100 ␮g/ml) diluted in PBS (pH 7.4), and left for 18 h at room temperature. The plates were then washed six times in PBS containing 0.5% tween-20 (Sigma Aldrich, Dorset, UK). Serial dilutions of recombinant rat and human IDE (2500–20 ng/ml) or crude brain tissue homogenates (50–0.05 ␮g total protein) diluted in PBS (pH 7.4), were incubated at room temperature for 1.5 h with continuous shaking. After a further six washes, the fluorogenic peptide (10 ␮M) diluted in 100 mM Tris–HCl pH 7.5 containing 50 mM NaCl and 10 ␮M ZnCl2 was added and incubated at 37 ◦ C in the dark. Fluorescent readings were taken every hour from 1 to 5 h and then after overnight incubation. Control wells included in each plate contained PBS alone.

J.S. Miners et al. / Journal of Neuroscience Methods 169 (2008) 177–181

Fig. 2. Standard curves of recombinant rat IDE enzyme activity after immunocapture of IDE with antibodies to epitopes in different IDE domains. Measurements were based on substrate cleavage at pH 7.5 after incubation for 1 h at 37 ◦ C.

3. Results 3.1. Measurements of enzyme activity of recombinant human and rat IDE standards before and after immunocapture Serial dilutions of recombinant rat and human IDE were incubated with the internally quenched fluorogenic substrate (Mca-RPPGFSAFK(Dnp)), producing standard curves of enzyme activity. Enzyme activity, measured by substrate cleavage, was linear in the range 20–150 ng/ml for both human and rat IDE after 1 h incubation at 37 ◦ C (Fig. 1A). No further increase in enzyme activity was observed after 1 h incubation (data not shown). Serial dilutions of recombinant human and rat IDE were then respectively captured on 96-well microplates pre-coated with anti-human IDE antibody (Calbiochem) prior to the addition of the fluorogenic substrate. Enzyme activity, measured by substrate cleavage after 1 h incubation at 37 ◦ C, was linear in the range 156–2500 ng/ml for both human and rat IDE (Fig. 1B) with no further increase occurring after longer incubation times. Rat IDE enzyme activity, measured by fluorogenic substrate cleavage following initial immunocapture, was next assessed with a panel of anti-human IDE capture antibodies. Standard curves of enzyme activity were generated for all of the IDE capture antibodies. Enzyme activity was highest when immunocapture was mediated by antibody raised to full-length IDE > N-terminus > inactive domain > C-terminus (Fig. 2).

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to increase over incubation for several hours at 37 ◦ C in the dark. Enzyme activity reached a maximum level after 5 h (Fig. 3A) without further increase after 18 h (data not shown). IDE enzyme activity in crude brain tissue homogenates was next compared after immunocapture with the panel of anti-IDE capture antibodies and incubation with the fluorogenic substrate for 5 h at 37 ◦ C in the dark. When immunocapture was performed with antibodies to the inactive domain or N-terminal region of IDE, measured enzyme activity was directly related to the total amount of protein added to the well. In contrast, after immunocapture with antibodies to the C-terminal region of IDE or to recombinant full-length enzyme, the fluorescent signal was not consistently related to the amount of protein added (Fig. 3B). Maximum fluorescent signal was achieved after immunocapture with rabbit anti-human IDE raised against peptide corresponding to part of the inactive domain of IDE (Fig. 3B). We then compared the fluorescent signal generated by adding homogenates containing 50 ␮g of protein from four separate brains, to plates coated with either N-terminal or inactive domain IDE antibodies, and confirmed that immunocapture with inactive domain IDE antibodies consistently yielded the highest signal (data not shown).

3.2. Immunocapture-based measurement of IDE enzyme activity in crude tissue homogenates Serial dilutions of crude brain tissue homogenates (total protein 0.05–50 ␮g) were added to microplate wells previously coated with a saturating concentration of antibodies to the N-terminal region of IDE before being incubated with the fluorogenic substrate. Standard curves of enzyme activity in brain homogenates, as measured by fluorogenic substrate cleavage, were linear in the range (0.05–50 ␮g total protein) (Fig. 3A). The amount of cleaved substrate continued

Fig. 3. (A) IDE enzyme activity measurements in crude brain homogenates after immunocapture with antibodies to the N-terminal region of IDE and incubation with the fluorogenic substrate for up to 5 h at pH 7.5 and 37 ◦ C. (B) The bars indicate IDE enzyme activity measurements ± standard errors, in crude tissue homogenates, after immunocapture with different IDE antibodies. The measurements were based on substrate cleavage at pH 7.5 after incubation at 37 ◦ C for 5 h.

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The measurement of IDE enzyme activity levels in tissue samples, in isolation from other closely related enzymes, should provide further insights into the role of IDE in the pathogenesis of a range of diseases, including Alzheimer’s disease and insulindependent diabetes. Acknowledgements Supported by BRACE (Bristol Research into Alzheimer’s and Care of the Elderly), the James Tudor Foundation, the Sigmund Gestetner Foundation and the Alzheimer’s Research Trust (ART). References Fig. 4. Standard curves of recombinant human IDE activity measured by immunocapture assay. The measurements were based on incubation with the fluorogenic substrate at pH 7.5 for 1 h at 37 ◦ C. Negligible cross-reactivity was observed with NEP, ACE or ECE-1.

To determine whether the inactive domain IDE antibodies bound any of the other enzymes that are known to cleave the Mca-RPPGFSAFK(Dnp) substrate, we measured the fluorescent signal generated after adding recombinant human IDE, ACE, NEP and ECE-1 to the antibody-coated wells. Negligible signal was detected after the addition of recombinant ACE, NEP or ECE-1 (Fig. 4). 4. Discussion We report the development and optimisation of a solid-phase immunocapture assay that allows the specific measurement of IDE enzyme activity in brain tissue homogenates. The fluorogenic peptide substrate used in this assay is normally cleaved by a number of related enzymes, such as NEP, ACE and ECE1, which coexist in various tissue samples, thereby making the accurate measurement of individual enzymes problematic (Miners et al., 2008). We have demonstrated that immunocapture of IDE from brain tissue homogenates prior to the addition of the fluorogenic substrate allows accurate measurement of IDE activity in brain tissue samples. This fluorogenic assay is not only specific and highly sensitive but is readily amenable to automation and high-throughput analysis. We compared the utility of a panel of anti-IDE antibodies for immunocapture that does not interfere with IDE enzyme activity. IDE enzyme activity following immunocapture was found to be similar whether measured using recombinant human or rat IDE. The choice of IDE antibodies for immunocapture was, however, critical: antibodies raised against the N-terminal and inactive domain of the recombinant protein performed best. This is presumably because these two antibodies interfered least with the interaction between the IDE catalytic domain and the fluorogenic substrate. Measured enzyme activities were highest when we used the IDE inactive domain capture antibody, and immunocapture-based assay with this antibody yielded consistent standard curves and negligible cross-reactivity with the closely related enzymes NEP, ACE or ECE-1.

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