A Novel Stereospecific Bioluminescent Assay for Detection of Endogenous d-Cysteine

Abstract

The presence of endogenous d-stereoisomers of amino acids in mammals dispels a long-standing dogma about their existence. d-Serine and d-aspartate function as novel neurotransmitters in mammals. However, the stereoisomer with the fastest, spontaneous in vitro racemization rate, d-cysteine, has not been reported. We utilized a novel, stereospecific, bioluminescent assay to identify endogenous d-cysteine in substantial amounts in the eye, brain, and pancreas of mice. d-Cysteine is enriched in mice embryonic brains at day E9.5 (4.5 mM) and decreases progressively with development (μM levels). d-Cysteine is also present in significantly higher amounts in the human brain white matter compared with gray matter. In the luciferase assay, d-cysteine conjugates with cyano hydroxy benzothiazole in the presence of a base and reducing agent to form d-luciferin. d-Luciferin, subsequently, in the presence of firefly luciferase and ATP, emits bioluminescence proportional to the concentration of d-cysteine. The assay is stereospecific and allows the quantitative estimation of endogenous d-cysteine in tissues in addition to its specificity for d-cysteine. Future efforts aimed at bioluminescent in vivo imaging of d-cysteine may allow a more noninvasive means of its detection, thereby elucidating its function.

Mammalian D‐Cysteine: A new addition to the growing family of biologically relevant D‐amino acids

“…16,17 Using a stereospecific bioluminescent luciferase assay, chiral chromatography, and SR À/À mice (serine racemase knockout), we identified endogenous D-Cysteine in mammalian brain and pancreas. 2,7 Our work shows that endogenous D-Cysteine is racemized from Lcysteine by SR. Endogenous D-Cysteine is enriched in the embryonic mouse brain (E9.5) at a concentration of 4.5 mM and decreases significantly with development to approximately 50 μM (Figure 1). D-Cysteine is also present in adult human brain (white matter) and in the cerebrospinal fluid at a concentration of approximately 40 μM, highlighting its relevance to human health.…”

“…7 Cysteine is unique among the amino acids in that there are additional isomers including side chain modifications. 2 Each L and or D stereoisomer of the different cysteine isomers can potentially serve as substrates for racemization. SR may have evolved to function in a promiscuous manner given the different isomers of cysteine.…”

“…15 Analysis and determination of cysteine enantiomers is challenging due to the high reactivity of the SH (thiol) group which is readily oxidized leading to the formation of cystine. 2 Recent improvements in chromatographic detection methods have improved the separation of these cysteine enantiomers. 16,17 Using a stereospecific bioluminescent luciferase assay, chiral chromatography, and SR À/À mice (serine racemase knockout), we identified endogenous D-Cysteine in mammalian brain and pancreas.…”

“…D-amino acids are novel molecules that can have specific biological function. [1][2][3] The myth surrounding their existence only in invertebrates has been dispelled. D-serine and D-aspartate in mammals are well-studied molecules functioning at the glycine and glutamate sites of the Nmethyl D-Aspartate (NMDA) receptor, respectively.…”

Coelenterazine-Type Bioluminescence-Induced Optical Probes for Sensing and Controlling Biological Processes

“…For example, a stereospecific bioluminescent luciferase probe has been engineered to detect endogenous D-cysteine quantitatively in mammals. D-cysteine combines with 2-cyano-6-hydroxybenzothiazole (CHBT) after the addition of base and reducing agent to form D-luciferin, which can be used as the substrate of luciferase to make it emit light, allowing a noninvasive method to monitor D-cysteine and further demonstrate its function [ 78 ]. Based on BRET, Park and co-workers created an ERα dimerization assay and subsequent ERβ dimerization assay in human cells to elucidate the ER dimerization potential of estrogenic compounds [ 79 , 80 ].…”