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Metalloid Reductase Activity Modified by a Fused Se⁰ Binding Peptide.

ABSTRACT: A selenium nanoparticle binding peptide was isolated from a phage display library and genetically fused to a metalloid reductase that reduces selenite (SeO₃^2-) to a Se⁰ nanoparticle (SeNP) form. The fusion of the Se binding peptide to the metalloid reductase regulates the size of the resulting SeNP to ∼35 nm average diameter, where without the peptide, SeNPs grow to micron sized polydisperse precipitates. The SeNP product remains associated with the enzyme/peptide fusion. The Se binding peptide fusion to the enzyme increases the enzyme's SeO₃^2- reductase activity. Size control of particles was diminished if the Se binding peptide was only added exogenously to the reaction mixture. The enzyme-peptide construct shows preference for binding smaller SeNPs. The peptide-SeNP interaction is attributed to His based ligation that results in a peptide conformational change on the basis of Raman spectroscopy.

SUBMITTER: Butz ZJ

PROVIDER: S-EPMC7478903 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThere is a growing body of nuclear contrast agents that are repurposed for fluorescence-guided surgery. New contrast agents are obtained by substituting the radioactive tag with, or adding a fluorescent cyanine to the molecular structure of antibodies or peptides. This enables intra-operative fluorescent detection of cancerous tissue, leading to more complete tumor resection. However, these fluorescent cyanines can have a remarkable influence on pharmacokinetics and tumor uptake, especially when labeled to smaller targeting vectors such as peptides. Here we demonstrate the effect of cyanine-mediated dead cell-binding of Ac-Lys0(IRDye800CW)-Tyr3-octreotate (800CW-TATE) and how this can be used as an advantage for fluorescence-guided surgery.ResultsBinding of 800CW-TATE could be blocked with DOTA0-Tyr3-octreotate (DOTA-TATE) on cultured SSTR2-positive U2OS cells and was absent in SSTR2 negative U2OS cells. However, strong binding was observed to dead cells, which could not be blocked with DOTA-TATE and was also present in dead SSTR2 negative cells. No SSTR2-mediated binding was observed in frozen tumor sections, possibly due to disruption of the cells in the process of sectioning the tissue before exposure to the contrast agent. DOTA-TATE blocking resulted in an incomplete reduction of 61.5 ± 5.8% fluorescence uptake by NCI-H69-tumors in mice. Near-infrared imaging and dead cell staining on paraffin sections from resected tumors revealed that fluorescence uptake persisted in necrotic regions upon blocking with DOTA-TATE.ConclusionThis study shows that labeling peptides with cyanines can result in dead cell binding. This does not hamper the ultimate purpose of fluorescence-guided surgery, as necrotic tissue appears in most solid tumors. Hence, the necrosis binding can increase the overall tumor uptake. Moreover, necrotic tissue should be removed as much as possible: it cannot be salvaged, causes inflammation, and is tumorigenic. However, when performing binding experiments to cells with disrupted membrane integrity, which is routinely done with nuclear probes, this dead cell-binding can resemble non-specific binding. This study will benefit the development of fluorescent contrast agents.

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Metalloid Reductase Activity Modified by a Fused Se⁰ Binding Peptide.

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