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J. Biol. Chem., Vol. 277, Issue 39, 35779-35782, September 27, 2002

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ACCELERATED PUBLICATION
A Missense Mutation in Kynurenine Aminotransferase-1 in Spontaneously Hypertensive Rats^*

John B. J. Kwok^§¶, Ranjna Kapoor^§, Takanari Gotoda^§**, Yasuhiko Iwamoto^**, Yoko Iizuka^§§, Nobuhiro Yamada^¶¶, Kim E. Isaacs, Virag V. Kushwaha, W. Bret Church, Peter R. Schofield^¶, and Vimal Kapoor

From the  Garvan Institute of Medical Research, Sydney, 2010 Australia,  Department of Pharmacology and Physiology, University of New South Wales, Sydney 2052, Australia, ^** Diabetes Center, Tokyo Women's Medical University, Tokyo 162-8666, Japan, ^§§ Department of Metabolic Diseases, University of Tokyo, Tokyo 113-8655, Japan, and ^¶¶ Department of Internal Medicine, University of Tsukuba, Tsukuba 305-8575, Japan

Received for publication, May 17, 2002, and in revised form, July 17, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Spontaneously hypertensive rats (SHR) are the most extensively used animal model for genetic hypertension, increased stroke damage, and insulin resistance syndromes; however, the identification of target genes has proved difficult. SHR show elevated sympathetic nerve activity, and stimulation of the central blood pressure control centers with glutamate or nicotine results in exaggerated blood pressure responses, effects that appear to be genetically determined. Kynurenic acid, a competitive glutamate antagonist and a non-competitive nicotinic antagonist, can be synthesized in the brain by the enzyme kynurenine aminotransferase-1 (KAT-1). We have previously shown that KAT-1 activity is significantly reduced in SHR compared with normotensive Wistar Kyoto rats (WKY). Here we show that KAT-1 contains a missense mutation, E61G, in all the strains of SHR examined but not in any of the WKY or outbred strains. Previous studies on F2 rats from a cross of stroke-prone SHR and WKY have shown a suggestive level of linkage between elevated blood pressure and the KAT-1 locus on chromosome 3. In addition, the mutant enzyme expressed in Escherichia coli displays altered kinetics. This mutation may explain the enhanced sensitivity to glutamate and nicotine seen in SHR that may be related to an underlying mechanism of hypertension and increased sensitivity to stroke.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The spontaneously hypertensive rat (SHR)¹ displays essential hypertension as well as insulin resistance and as such has become the most widely used model for the study of genetic hypertension and related disorders commonly coexisting in human patients. The complexity of these syndromes and the known heterogeneity of the SHR genotype have, however, made the identification of target genes difficult (1-3).

Increased sympathetic outflow is a key factor contributing to some types of hypertension in humans and in many animal models of hypertension (4). The major sympathetic output pathway for the tonic and reflex control of blood pressure, which uses glutamate as the transmitter, arises from the rostral ventrolateral medulla (RVLM) (5). In addition the RVLM receives numerous cholinergic and glutamatergic inputs, making this pathway especially sensitive to altered receptor activity (4, 5). Enhanced excitation of the RVLM has been implicated in human and in animal models of hypertension (4).

Recent evidence suggests that alterations of the levels of the endogenous antagonist kynurenic acid, which shows highest affinity for the "glycine site" of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors (6), can affect glutamate receptor function (7). In SHR, microinjection of glutamate or NMDA into the caudal ventrolateral medulla results in exaggerated depressor responses; however, the responses to the "NMDA glycine site" agonist D-serine are diminished (8). Kynurenic acid injection into the RVLM reduces basal blood pressure in SHR but not in normotensive WKY (9). This suggests that there is enhanced sensitivity to glutamate in SHR, possibly due to reduced levels of the endogenous NMDA glycine site antagonist kynurenic acid. Recently evidence has emerged that kynurenic acid can also block nicotinic (7) receptors in a non-competitive fashion (10). In support of the finding that kynurenic acid levels are reduced in SHR, nicotinic receptor agonists have also been demonstrated to show augmented blood pressure and other responses in SHR (11). These exaggerated responses to both glutamate and nicotinic agonists are independent of the presence of high blood pressure and appear be genetically determined (11, 12).

We have previously provided evidence that the activity of kynurenine aminotransferase-1 (KAT-1), which converts kynurenine to kynurenic acid, and kynurenic acid levels are reduced in SHR (13). Furthermore, KAT-1-immunoreactive neurons can be demonstrated in all the blood pressure control regions of the medulla and spinal cord. However, we were unable to detect a difference in the number or intensity KAT-1-immunoreactive cells between SHR and WKY (14). Using an enzymatic activity stain for KAT-1 on native (non-denaturing) gel separations, specific KAT-1 enzyme activity could be demonstrated in homogenates from WKY kidney but not in those from SHR kidney (15).

To extend these observations and to clarify the discrepancy between the immunochemistry data and the physiological and biochemical studies, we hypothesized that the KAT-1 gene in SHR may be mutated. In this report we present evidence for a missense mutation in the KAT-1 gene in SHR. The mutation, which could be demonstrated in all strains of SHR examined, appears to account for the reduced activity of KAT-1 observed in SHR.

	EXPERIMENTAL PROCEDURES

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Animals-- SHR, stroke-prone SHR, WKY, and Wistar rats were provided by the Flinders Medical Centre, Adelaide, Australia and the Baker Medical Research Institute, Melbourne, Australia. Sources of DNA from other SHR and WKY substrains have been reported previously (2).

Native Gel Separation and Activity Stains-- Native gel enzyme activity stain protocols have been published previously (15). Western blots were carried out on the native gels using a polyclonal antibody directed against rat kidney KAT-1 kindly provided to us by Prof. R. Kido (Wakayama University, Japan).

cDNA Sequencing of rKAT-1-- Whole brains of the SHR, stroke-prone SHR, WKY, and Wistar rats were used for isolation of total RNA, which was reverse-transcribed using the SuperScript II RT kit (Roche Molecular Biochemicals). The RIN 2A cell line, a subclone of the rat pancreatic cell line RINm5F, served as an internal control. The KAT-1/GTK gene (GenBank^TM accession number Z49696) was screened for nucleotide substitutions using five overlapping pairs of PCR primers covering the entire coding sequence of the gene. Following PCR amplification and purification, the cDNA was sequenced directly using the ABI Prism Big Dye Terminator Cycle Sequencing kit (PE Applied Biosystems).

Genomic rKAT-1 Sequencing-- A 267-bp fragment comprising exon 3 of the rat KAT-1 gene, using the primer pair rKat-ex3F (5'-ggggctgactttctacttgttg-3') and rKat-ex3R (5'-ggatgctaatctgtgctctgtc-3') (GenBank^TM accession number AF100154, nucleotides 13103-13369), was amplified from leukocyte DNA by PCR, and the resulting DNA was sequenced.

Expression of KAT-1-- SHR and WKY KAT-1 cDNA from the sequencing work was ligated into a pET vector that was then used to transfect competent B21 Escherichia coli cells. The resulting ampicillin-resistant colonies were grown to log phase in the presence of ampicillin, and the expression of KAT-1 was induced using isopropyl--D-thiogalactopyranoside overnight (16 h). Expression was verified by Western slot-blot (as above). Analysis of enzyme activity in partially purified extracts was carried out as described previously using kynurenine (0.25-2 mM) and pyruvate (1.0-7.5 mM) as substrates (8).

The chromosomal localization of the rat KAT-1 gene was determined by radiation hybrid mapping analysis using a whole genome rat/hamster radiation hybrid panel (Research Genetics). The result was analyzed by two-point placement mapping (MultiMap) with a radiation hybrid mapping server (ratmap.ims.u-tokyo.ac.jp).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Using partially purified kidney homogenates, KAT-1 activity stains in native gel separations were carried out as described previously (15). Omission of each substrate in turn allowed us to demonstrate that there was only one band specific for KAT-1 activity (Fig. 1), the other band being ascribed to amino acid oxidase activity (data not presented) (17). The specific KAT-1 activity band was apparent only in the homogenate from WKY kidney. Western blots of the same native gels, however, showed KAT-1-immunoreactive bands in homogenates from both rat strains (Fig. 1).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Native gel activity stains and Western blots for KAT-1 from SHR and WKY. Partially purified kidney homogenates were separated by native gel electrophoresis and either used for a KAT-1 activity stain (left) or probed by Western blot for KAT-1-like immunoreactivity (right) using methods as previously described (15). Specific KAT-1 activity was observed only in WKY homogenates. KAT-1-like immunoreactive bands (right) were present in homogenates from both WKY and SHR kidney homogenates, but only the WKY band coincided with the specific KAT-1 enzyme activity stain.

Only the immunoreactive band from WKY corresponded to the KAT-1 activity seen in the gels from these rats. The immunoreactive band from the SHR homogenates migrated more slowly under native conditions than that from the WKY, although under denaturing conditions (SDS-PAGE Western blots) the immunoreactive bands were coincident (data not shown). This helped reconcile our previous data that, compared with WKY, KAT-1 in SHR shows reduced enzyme activity but similar immunohistochemistry (13, 14) and led us to predict the presence of a mutation in SHR KAT-1 that would alter the charge and activity of the protein without greatly affecting its molecular weight.

To investigate this further, RNA was isolated from SHR, stroke-prone SHR, WKY, and Wistar rat brains, and KAT-1 cDNA was generated for DNA sequence analysis. The DNA sequence analysis of KAT-1 cDNA from WKY and Wistar rats matches the published sequence (18). However, in the SHR and stroke-prone SHR sequences we found an A to G point mutation corresponding to amino acid 61 (numbered from the predicted amino acid sequence from GenBank^TM accession number Z49696). This would result in the substitution of glutamate 61 to glycine (GAG to GGG), which could affect the surface charge of the protein (Fig. 2).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Initial KAT-1 DNA sequence analysis from SHR and other strains. Sequence analysis of KAT-1 transcripts reveal an A to G nucleotide substitution (arrow) as the only difference between WKY and SHR (including stroke-prone SHR). This results in an amino acid difference, E61G, between the WKY and SHR strains. RIN 2A, a rat pancreatic cell line, served as an internal control. SP, stroke-prone.

Since the genetic heterogeneity of the SHR strain is well documented, we also examined the KAT-1 genomic DNA from other sources. The original SHR/lzm (Kyoto, Japan), the SHR NCrj (Charles River), and the SHR/Tac (Taconic Farms) strains and their genetic controls, the WKY strains, were used. The mutation was not present in any of the WKY strains examined but was confirmed in all the SHR strains examined, including the original SHR/lzm strain, indicating that the E61G mutation is an authentic mutation common to SHR strains.

Using standard protein expression protocols, full-length KAT-1 cDNA was cloned into an E. coli expression vector. Low levels of KAT-1 were found in the soluble fraction, which were verified by Western slot-blots, and partially purified homogenates used for enzyme analysis. Kinetic analysis of the mutant SHR and wild type WKY forms of the enzyme expressed in E. coli was carried out using different concentrations of both substrates (kynurenine and pyruvate) for this "ping-pong bi-bi" reaction. The apparent K_m values for kynurenine (1.4 mM) and pyruvate (1.2 mM) for the WKY KAT-1 were found to be similar to values reported in the literature (18). KAT-1 from SHR, however, showed much more variability and a markedly lower K_m for kynurenine of 0.2 mM (Fig. 3). The K_m for pyruvate for KAT-1 from SHR was also lower (0.1 mM), and the initial velocity (at 1 h) was between 40-60% lower at all the concentrations of substrate tested.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Kinetic properties of the wild type WKY and the mutant SHR KAT-1 enzymes expressed in E. coli. A secondary plot of the reciprocal of the calculated apparent Vmax (measured at 7.5-1.0 mM pyruvate) versus the reciprocal of kynurenine concentration was used to calculate the apparent Km for kynurenine.

The chromosomal localization of the rat KAT-1 gene was determined by mapping with a radiation hybrid panel (Research Genetics) and showed that the gene was located on the proximal end of rat chromosome 3 with D3Rat54 as the closest microsatellite marker. Initial -BLAST analysis (www.ncbi.nlm.nih.gov/BLAST/) of the KAT-1 amino acid sequence revealed KAT-1 to be an unusual aminotransferase, possibly a subset of the 1 family, showing a close relationship with HB8 aspartate aminotransferase (AAT) from Thermus thermophilus. The structure of HB8 AAT (Protein Data Bank code 1BKG) was used as a template to model KAT-1 using the Homology module in Insight-II (Molecular Simulations Inc.) and also using SwissModel (www.expasy.ch/swissmod/SWISS-MODEL.html). As shown in Fig. 4, both theoretical models placed the mutation site (Glu-61) at the hinge of the first -helix of the small domain (N-terminal -helix), the movement and flexibility of which is critical to the mechanism of action of HB8 AAT (19, 20).

View larger version (59K): [in this window] [in a new window]   Fig. 4.   Homology model of kynurenine aminotransferase-1. Molscript view of the WKY (wild type) homology model built using aspartate aminotransferase from T. thermophilus as the template. The model shows the location of the mutation site, Glu-61, the large and small domains, and the substrate binding pocket.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Selective breeding of rat strains for high blood pressure has led to the identification of at least 24 quantitative trait loci associated with hypertension and highlighting the genetic complexity of the disease (21). Our demonstration of a novel missense mutation (E61G) in the KAT-1 gene of all the SHR strains examined, but not in any of the control WKY and outbred normotensive strains, is among the most extensive characterization of a mutation in SHR to date. This study provides evidence that the KAT-1 mutation may underlie, at least in part, the development of hypertension and other abnormalities seen in SHR.

We have previously shown that KAT-1 activity and associated glutamine transaminase K activities are reduced in SHR rats (15). The present study extends these findings to show that the electrophoretic movement of KAT-1 from SHR tissue is slower under non-denaturing conditions (Fig. 1), suggesting that the SHR enzyme may be altered. Sequence analysis of the KAT-1 gene from WKY and outbred Wistar rats confirmed previously published data (18) and revealed a single missense mutation in the codon for position 61, E61G, of the cDNA from SHR. Since the genetic variability of the SHR is well known, KAT-1 genomic DNA from several different strains of SHR was also sequenced. The same missense mutation, E61G, was confirmed in all strains of SHR examined but was not found in any of the respective WKY controls. Recently genotype analysis by single strand conformation polymorphism of SHR, stroke-prone SHR, and their WKY controls (lzm, NCrj, and Tac strains) has confirmed the presence of the mutation in all SHR strains but not in any of the WKY strains (data not presented).

WKY-derived KAT-1 expressed in E. coli showed apparent K_m and V_max values that are similar to published results (18). Kinetic analysis of the SHR mutant enzyme, however, showed markedly altered kinetics and reduced activity. This is consistent with literature reports that the binding of the dicarboxylic acid induces a rotational movement of the small (N-terminal) domain of aminotransferases, alteration of which leads to a large reduction of substrate K_m and V_max in related enzymes with a similar kinetic mechanism (19, 20).

A critical difference between aminotransferases, like HB8 AAT, that belong to the 1b classification of Nakai et al. (19) and other aminotransferases is that in this group substrate binding leads to movement only of the N-terminal region of the small domain (shaded red in Fig. 4) toward the substrate binding site. The presence and location of glycine residues in the hinge region of the N terminus is likely to be critical to the movement, and hence the activity of this subclass of enzymes (classified as 1 according to Jensen et al. (22)).

As shown in Fig. 4, homology models of KAT-1 built with HB8 AAT as the template placed the mutation site (Glu-61) at the hinge of the first -helix of the small domain (N-terminal -helix), the movement and flexibility of which is central to the mechanism of action of HB8 AAT (19). The location and accessibility of the mutation site indicate that the E61G mutation has the potential to alter both the surface charge and the activity of the enzyme. Consistent with these observations, since a Glu to Gly substitution is likely to result in increased flexibility, N-terminal deletion mutants of related aminotransferases result in similar changes in enzyme kinetics (20).

The KAT-1 gene was mapped to the proximal end of chromosome 3 in the rat (homology with mouse chromosome 2). Quantitative trait loci for hypertension in stroke-prone SHR have previously been mapped on the proximal end of rat chromosome 3 (suggestive logarithm of the odds score of about 3) and thus very close to the KAT-1 locus at D3Rat54 (near D3Mgh16) (16, 23, 24). Furthermore, our preliminary studies on F2 crosses between SHR and WKY show a significant correlation of the inheritance of the SHR KAT-1 allele not only to elevated blood pressures but also to reduced body weights and altered adipocyte metabolism.²

The human homologue of the rat KAT-1 gene (89% protein identity), which codes for the cysteine conjugate -lyase enzyme (CCBL1), maps to chromosome 9q34 (25). Examining whether alleles of the KAT-1/CCBL1 gene confer susceptibility for human hypertension and insulin resistance is now a high priority. The identification of a KAT-1 mutation and its role in enhancing sensitivity to glutamate and nicotine highlights a possible mechanism of genetic predisposition to hypertension associated with insulin resistance and identifies a novel therapeutic target for the treatment of this major health problem.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ These authors contributed equally to this work.

^¶ Supported by Australian National Health and Medical Research Council Block Grant 993050.

Supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture by the Organization for Pharmaceutical Safety and Research (OPSR) and health sciences research grants from the Ministry of Health and Welfare of Japan.

Supported by The Rebecca Cooper Foundation, Australia. To whom correspondence should be addressed. Tel.: 61-2-9385-3741; Fax: 61-2-9385-1099; E-mail: V.Kapoor@unsw.edu.au.

Published, JBC Papers in Press, July 26, 2002, DOI 10.1074/jbc.C200303200

² Y. Iizuka, T. Gotoda, and N. Yamada, unpublished results.

	ABBREVIATIONS

The abbreviations used are: SHR, spontaneously hypertensive rat(s); WKY, Wistar Kyoto rat(s); KAT-1, kynurenine aminotransferase-1; RVLM, rostral ventrolateral medulla; NMDA, N-methyl-D-aspartate; AAT, aspartate aminotransferase; CCBL, cysteine conjugate -lyase.

	REFERENCES

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This Article Abstract Full Text (PDF) All Versions of this Article: 277/39/35779    most recent C200303200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kwok, J. B. J. Articles by Kapoor, V. Search for Related Content PubMed PubMed Citation Articles by Kwok, J. B. J. Articles by Kapoor, V. Social Bookmarking                      What's this?