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.
Endogenous D-stereoisomers are being recognized as functionally important molecules in mammals. Here we report the first identification of endogenous D-cysteine in mammalian pancreas. Serine Racemase (SR) is the biosynthetic enzyme for D- cysteine. D-cysteine is present in substantial amounts in the eyes, brain and pancreas of mice. To characterize endogenous D-cysteine, we used SR deficient mice lacking racemizing ability and show 3.5 fold reduction in pancreatic D-cysteine. SR-/- mice produce 6-10 fold higher levels of insulin in the pancreas and plasma and are stored as amyloid aggregates in secretory vesicles and exosomes respectively. Lack of SR and endogenous D-cysteine globally decreased levels of nucleotides and cAMP, followed by reduced phosphorylation of CREB (S133) including lower expression of DNA methyltransferase (DNMT) 1, 3A and 3B, and reduced DNMT enzymatic and promoter activities in the pancreas. This results in decreased DNA methylation globally and specifically of the Ins1 promoter. D-cysteine is efficiently metabolized by D-amino acid oxidase and transported by ASCT2 and Asc1 transporters in cells. Dietary supplementation with methyl donors rescues the high insulin levels and low DNMT activity in SR-/- mice. Our data show that SR racemizes cysteine in the pancreas and is a physiologic down regulator of insulin promoter methylation.
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