Elsevier

Gene

Volume 256, Issues 1–2, 3 October 2000, Pages 183-188
Gene

Human serine racemase: moleular cloning, genomic organization and functional analysis

https://doi.org/10.1016/S0378-1119(00)00356-5Get rights and content

Abstract

High levels of d-serine are found in mammalian brain, where it is an endogenous agonist of the strichinine-insensitive site of N-methyl d-aspartate type of glutamate receptors. d-serine is enriched in protoplasmic astrocytes that occur in gray matter areas of the brain and was shown to be synthesized from l-serine . We now report cloning and expression of human serine racemase, an enzyme that catalyses the synthesis of d-serine from l-serine. The enzyme displays a high homology to the murine serine racemase. It contains a pyridoxal 5′-phosphate attachment sequence similar to bacterial biosynthetic threonine dehydratase. Northern-blot analysis show high levels of human serine racemase in areas known to contain large amounts of endogenous d-serine, such as hippocampus and corpus callosum. Robust synthesis of d-serine was detected in cells transfected with human serine racemase, demonstrating the conservation of d-amino acid metabolism in humans. Serine racemase may be a therapeutic target in psychiatric diseases as supplementation of d-serine greatly improves schizophrenia symptoms. We identify the human serine racemase genomic structure and show that the gene encompasses seven exons and localizes to chromosome 17q13.3. Identification of the intron–exon boundaries of the human serine racemase gene will be useful to search for mutations in neuropsychiatric disorders.

Introduction

Large amounts of d-serine have been found in mammalian brain, where it is an endogenous ligand of the ‘glycine site’ of NMDA receptors (Hashimoto et al., 1992, Hashimoto et al., 1993, Mothet et al., 2000, Schell et al., 1995). Since amino acid racemases were thought to be restricted to bacteria, the origin of d-serine in brain has been puzzling. We recently showed that d-serine is synthesized from l-serine by a serine racemase enzyme, a process dependent on pyridoxal-5′-phosphate (PLP). We purified, cloned and localized mouse serine racemase (Wolosker et al., 1999a, Wolosker et al., 1999b). The cloned mouse serine racemase has a PLP binding consensus region, and mutations of the lysine 56, which is predicted to bind PLP, abolished the activity (Wolosker et al., 1999a). Serine racemase is highly enriched in the brain and co-localizes with d-serine by immunohistochemical analysis. Both d-serine and serine racemase occur in murine astrocytes, in regions enriched in N-methyl d-aspartate (NMDA) receptors, suggesting that serine racemase physiologically synthesizes d-serine to regulate NMDA receptors (Wolosker et al., 1999a).

As an endogenous co-agonist of NMDA receptors, d-serine may play a role in several pathological conditions related to NMDA receptor dysfunction. Massive stimulation of NMDA receptors by glutamate takes place during stroke, leading to excitotoxicity and cell death (Choi and Rothman, 1990). d-Serine is released into cerebroventricular fluid in animal models of stroke (Lo et al., 1998), and drugs that block the ‘glycine site’ of NMDA receptors prevent stroke damage (Danysz and Parsons, 1998, Gill et al., 1995, Warner et al., 1995).

Low NMDA receptor activity also results in pathology. Thus, NMDA receptor hypofunction is implicated in the pathology of schizophrenia (Mohn et al., 1999). d-Serine associated with neuroleptics greatly improved positive, negative and cognitive symptoms in schizophrenic patients (Tsai et al., 1998), demonstrating the importance of the co-agonist site of NMDA receptor in the pathology of neuropsychiatric disorders. However, little is known about d-serine disposition in humans, and human serine racemase has not been identified yet. Cloning and localization of human serine racemase in humans would help clarify the neurobiological role of d-serine.

In this report, we cloned the human serine racemase and functionally characterized the gene product, showing that it catalyses robust formation of d-serine in living cells. We identified the intron–exon structure of the human serine racemase, which will be useful to search for mutations associated with diseases.

Section snippets

Cloning human serine racemase

Based on overlapping human ESTs (Accession Nos H73097, H86748, A1525507, AW294103 and AI832063), we designed primers with SalI/NotI restriction sites (forward primer, 5′ ACG CGT CGA CCA CCA TGT GTG CTC AGT ATT GCA TCT CC 3′; reverse primer, 5′ATA AGA ATG CGG CCG CTT AAA CAG AAA CAG ACT GAT AAG A 3′). We amplified a 1.1 kb fragment by PCR using human brain cDNA as template (Clontech). The fragment, sequenced at both strands, contained the full-length human serine racemase sequence that was

Results

We identified in the GenBank several overlapping humand ESTs of an unknown gene, which contained a sequence with a very high homology to the mouse serine racemase. Based on the 5′ and 3′ ends of the ESTs, we designed specific primers and cloned the full-length human serine racemase by PCR from human brain cDNA. The human serine racemase cDNA encodes for a protein of 340 amino acids. The sequence for initiation of translation of human serine racemase is in agreement with the Kozak consensus rule

Discussion

d-Serine has been detected in the human brain (Fisher et al., 1994, Kumashiro et al., 1995, Nagata et al., 1995) and urine (Huang et al., 1998), but the origin of d-serine has been elusive. In the present paper, we report the occurrence of a human serine racemase catalysing robust synthesis of d-serine in living cells. The presence of this enzyme in humans implies an endogenous origin for d-serine.

Analysis of human serine racemase distribution should help clarify several aspects of d-serine

Acknowledgements

We thank Dr Vivaldo Moura-Neto (Department of Anatomy, UFRJ) for encouragement and continuous support. This study was supported by grants from Theodor and Vada Stanley Foundation and FAPERJ to S.E. and H.W., and FUJB (Proc. 8998-2) to H.W.

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