Luminal heterodimeric amino acid transporter defective in cystinuria

Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b(0,+) type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b(0,+)AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b(0,+) amino acid transporter complex. We demonstrate its b(0,+)-type amino acid transport kinetics using a heterodimeric fusion construct and show its luminal brush border localization in kidney proximal tubule. These biochemical, transport, and localization characteristics as well as the chromosomal localization on 19q support the notion that the b(0,+)AT protein is the product of the gene defective in non-type I cystinuria.     

The toxicity of tritium: the effects of tritiated amino-acids on preimplanted mouse embryos

The effects of tritiated amino-acids, arginine, lysine, histidine and aspartic acid on the growth and development of two-cell mouse embryos, cultured in vitro, were investigated. The LD50 for the dibasic amino acids, measured on the third day of growth, ranged from 30 to 130 nCi/ml. This was compared with the DNA precursor, thymidine, for which the LD50 was 80 nCi/ml.     

Experimental cystinuria: the cycloleucine model. I. Amino acid interactions in renal and intestinal epithelia.

The injection of cycloleucine (1-aminocyclopentanecarboxylic acid (ACPC) into rats produces a hyperexcretion of dibasic amino acids and cystine, an aberration resembling cystinuria. This may constitute a model of experimental cystinuria, and the transport of amino acids involved in this disease was studied with the techniques of everted intestinal sacs (in vitro) and microinjections into renal tubules (in vivo). In verted sacs from normal rats, there was a decrease in transfer and in accumulation of L-cystine (0.03 mM) and L-valine (0.065 mM) when ACPC was on the mucosal (luminal) side. Dibasic amino acids such as L-arginine and L-lysine caused a similar inhibition of the transport of L-cystine. However, when ACPC was on the serosal (antiluminal) side, a lesser effect was noted while arginine and lysine had no effect. Intestinal sacs from treated rats (ACPC, 300 mg/kg X 3 days) transferred and accumulated as much L-cystine as those from control rats. The interaction between cycloleucine and L-cystine was competitive at the luminal and non-competitive at the antiluminal side of the intestine. Cycloleucine inhibited L-lysine transport in a non-competitive fashion at either side of the intestine. L-Lysine also interacted in a non-competitive fashion with L-cystine transport at the luminal membrane. In proximal convoluted tubules, the presence of L-arginine or ACPC caused a decrease in the transport of L-cystine and L-lysine. L-Valine exerted no effect. Furthermore, L-lysine and ACPC did not impair the reabsorption of L-valine significantly. These results suggest a functional heterogeneity between luminal and antiluminal membranes of renal and intestinal epitehlia and the existence, at both membranes, of different transport sites for cystine and dibasic amino acids.     

Diagnosis and follow-up of cystinuria: Use of proton magnetic resonance spectroscopy

Summary. Proton Nuclear Magnetic Resonance (NMR) Spectroscopy of urine (as well as of other biological fluids) is a very powerful technique enabling multi-component analysis useful in both diagnosis and follow-up of a wide range of inherited metabolic diseases. Among these pathologies, cystinuria is characterised by accumulation in urine of four dibasic amino acids, namely lysine, arginine, ornithine and cystine; the last one, being only slightly water soluble, generates urolithiasis. The mentioned aminoacids can be detected in the urine NMR spectrum of cystinuric patients, the most abundant being the lysine (5 mM and over are often detected), whose typical signals become very high; arginine and ornithine are also usually detectable, although pathologic concentrations are lower (usually below 2 mM). The proposed NMR technique is also suitable in monitoring the therapy with α-mercaptopropionylglycine (MPG), providing quantitation of several metabolites of interest in the follow-up of the pathology, like cystine, creatinine and citrate. Received May 9, 1999; Accepted September 26, 1999     

The molecular basis of cystinuria: the role of the rBAT gene

Summary The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b^0,+ -like and y⁺L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.The b^0,+-like system expressed in oocytes by rBAT cRNA transports L-cystine, L-dibasic and L-neutral amino acids with high-affinity. This transport system shows exchange of amino acids through the plasma membrane ofXenopus oocytes, suggesting a tertiary active transport mechanism. The rBAT gene is mainly expressed in the outer stripe of the outer medulla of the kidney and in the mucosa of the small intestine. The protein localizes to the microvilli of the proximal straight tubules (S3 segment) of the nephron and the mucosa of the small intestine. All this suggested the participation of rBAT in a high-affinity reabsorption system of cystine and dibasic amino acids in kidney and intestine, and indicated rBAT (named SLC3A1 in Gene Data Bank) as a good candidate gene for cystinuria. This is an inherited aminoaciduria due to defective renal and intestinal reabsorption of cystine and dibasic amino acids. The poor solubility of cystine causes the formation of renal cystine calculi. Mutational analysis of the rBAT gene of patients with cystinuria is revealing a growing number (~20) of cystinuria-specific mutations, including missense, nonsense, deletions and insertions. Mutations M467T (substitution of methionine 467 residue for threonine) and R270X (stop codon at arginine residue 270) represent approximately half of the cystinuric chromosomes where mutations have been found. Mutation M467T reduces transport activity of rBAT in oocytes. All this demonstrates that mutations in the rBAT gene cause cystinuria.Three types of cystinuria (types, I, II and III) have been described on the basis of the genetic, biochemical and clinical manifestations of the disease. Type I cystinuria has a complete recessive inheritance; type I heterozygotes are totally silent. In contrast, type II and III heterozygotes show, respectively, high or moderate hyperaminoaciduria of cystine and dibasic amino acids. Type III homozygotes show moderate, if any, alteration of intestinal absorption of cystine and dibasic amino acids; type II homozygotes clearly show defective intestinal absorption of these amino acids. To date, all the rBAT cystinuria-specific mutations we have found are associated with type I cystinuria (~70% of the chromosomes studied) but not to types II or III. This strongly suggests genetic heterogeneity for cystinuria. Genetic linkage analysis with markers of the genomic region of rBAT in chromosome 2 (G band 2p16.3) and intragenic markers of rBAT have demonstrated genetic heterogeneity for cystinuria; the rBAT gene is linked to type I cystinuria, but not to type III. Biochemical, genetic and clinical studies are needed to identify the additional cystinuria genes; a low-affinity cystine reabsortion system and the putative light subunit of rBAT are additional candidate genes for cystinuria.     

Association between M467T and 114 C→A variants within the SLC3A1 gene and some phenotypical traits in cystinuria patients from Spain

Cystinuria is an inherited metabolic disease characterized by an abnormal urinary excretion of cystine and dibasic amino acids. Formation of renal calculi, recurrent infections and renal failure are the main complications of this disease. The SLC3A1 gene, which codes for a dibasic amino acid transporter protein, is involved in the pathogenesis of cystinuria. We investigated the possible association between molecular variants (M467T, E483X, T216 M and 114 C-->A) within the SLC3A1 gene and some phenotypical traits in a Spanish area. The study population consisted of 45 cystinuria patients, 42 cystinuria relatives and 81 healthy control subjects. Only the M467T mutation was found in chromosomes of cystinuria patients and relatives. However, the 114 C-->A polymorphism was detected in cystinuria patients, in relatives and in control subjects but with different prevalences. Moreover, a statistically significant association between this polymorphism and urinary amino acid levels was found in cystinuria patients (P<0.05). Subjects with the C/C genotype showed significantly higher urinary levels of cystine, arginine and their sum as compared with carriers of the A allele (P<0.05). When multiple linear regression analysis was performed in cystinuria patients, the 114 C-->A polymorphism remained significantly associated (P=0.047) with cystine levels even after controlling for age, gender and the M467T mutation. Furthermore, we also found a statistically significant interaction term (P=0.028) between M467T and 114 C-->A in determining urinary cystine levels. According to our results, the 114 C-->A polymorphism might be a marker of a functional variant in the SLC3A1 gene or in other genes related to urinary amino acid excretion in cystinuria patients.     

SLC7A9 cDNA cloning and mutational analysis of SLC3A1 and SLC7A9 in canine cystinuria

Cystinuria is a genetic disorder in the domestic dog that leads to recurrent urolith formation. The genetic basis of the disorder is best characterized in humans and is caused by mutations in one of the amino acid transporter genes SLC3A1 or SLC7A9, which results in hyperexcretion of cystine and the dibasic amino acids in the urine and subsequent precipitation of cystine due to its low solubility in urine. In this study we describe the cloning of the canine SLC7A9 cDNA and present a thorough mutation analysis of the coding SLC3A1 and SLC7A9 regions in cystinuric dogs of different breeds. Mutation analysis of the two cystinuria disease genes revealed one SLC7A9 mutation (A217T) and two SLC3A1 mutations (I192V and S698G) in French and English Bulldogs that affect nonconserved amino acid residues, arguing against functional impact on the proteins. The absence of deleterious mutations linked to cystinuria in the remainder of our panel of cystinuric dogs is surprising because SLC3A1 or SLC7A9 mutations explain approximately 70% of all human cystinuria cases studied. The present study, along with previous investigations of canine and human cystinuria, implies that regulatory parts of the SLC3A1 and SLC7A9 genes as well as other unknown genes may harbor mutations causing cystinuria.     

Mutation analysis of SLC7A9 in cystinuria patients in Sweden

Cystinuria is an autosomal recessive disorder characterized by increased urinary excretion of cystine and dibasic amino acids, which cause recurrent stone formation in affected individuals. Three subtypes of cystinuria have been described (type I, II, and III): type I is caused by mutations in the SLC3A1 gene, whereas nontype I (II and III) has been associated with SLC7A9 mutations. Of the 53 patients reported in our previous work, patients that showed SLC7A9 mutations in single-strand conformation polymorphism (SSCP) screening and/or either lacked or showed heterozygosity for SLC3A1 mutations were included in the present study. The entire coding region and the exon/intron boundaries of the SLC7A9 gene were analyzed by means of both SSCP and DNA sequencing in 16 patients, all but one of which were clinically diagnosed as homozygous cystinurics. Three novel SLC7A9 mutations were identified in the patient group: two missense mutations (P261L and V330M), and one single base-pair deletion (1009 delA). We also detected the previously reported A182T and nine novel polymorphisms in the patients. Mutations V330M and 1009delA occurred on different alleles in one individual, and we suggest that these mutations cause cystinuria in this patient. One patient that was homozygously mutated in the SLC3A1 gene carried the third novel mutation (P261L). We conclude that SLC3A1 is still the major disease gene among Swedish cystinuria patients, with only a minor contribution of SLC7A9 mutations as the genetic basis of cystinuria. The absence of SLC3A1 and SLC7A9 mutations in a substantial proportion of the patients implies that mutations in parts of the genes that were not analyzed may be present, as well as large deletions that escape detection by the methods used. However, our results raise the question of whether other, as yet unknown genes, may also be involved in cystinuria.     

Liquid chromatographic—mass spectrometric analysis for screening of patients with cystinuria, and identification of cystine stone

Analyses of amino acids in the urine of a normal human and of patients with heterozygous and homozygous cystinuria have been carried out, using liquid chromatography—mass spectrometry with an atmospheric pressure ionization interface system. A kidney cystine stone was also analysed by this system. Very intense quasi-molecular ions ([M + H]⁺) of standard cystine, arginine, lysine and ornithine were observed on mass chromatograms as base peaks. Mass chromatograms of the urine samples from a normal human and from patients with heterozygous and homozygous cystinuria were easily distinguishable. The retention times in the mass chromatogram and mass spectrum of kidney stone cystine was almost the same as that of authentic cystine.     

Founder mutations in Tunisia: implications for diagnosis in North Africa and Middle East

Cystinuria (OMIM 220100) is an inborn congenital disorder characterised by a defective cystine metabolism resulting in the formation of cystine stones. Among the heterogeneous group of kidney stone diseases, cystinuria is the only disorder which is exclusively caused by gene mutations. So far, two genes responsible for cystinuria have been identified: SLC3A1 (chromosome 2p21) encodes the heavy subunit rBAT of a renal b^0,+ transporter while SLC7A9 (chromosome 19q12) encodes its interacting light subunit b^0,+AT. Mutations in SLC3A1 are generally associated with an autosomal-recessive mode of inheritance whereas SLC7A9 variants result in a broad clinical variability even within the same family. The detection rate for mutations in these genes is larger than 85%, but it is influenced by the ethnic origin of a patient and the pathophysiological significance of the mutations. In addition to isolated cystinuria, patients suffering from the hypotonia-cystinuria syndrome have been reported carrying deletions including at least the SLC3A1 and the PREPL genes in 2p21. By extensive molecular screening studies in large cohort of patients a broad spectrum of mutations could be identified, several of these variants were functionally analysed and thereby allowed insights in the pathology of the disease as well as in the renal trafficking of cystine and the dibasic amino acids. In our review we will summarize the current knowledge on the physiological and the genetic basis of cystinuria as an inborn cause of kidney stones, and the application of this knowledge in genetic testing strategies.     

An Animal Model of Type A Cystinuria Due to Spontaneous Mutation in 129S2/SvPasCrl Mice

Cystinuria is an autosomal recessive disease caused by the mutation of either SLC3A1 gene encoding for rBAT (type A cystinuria) or SLC7A9 gene encoding for b^0,+AT (type B cystinuria). Here, we evidenced in a commonly used congenic 129S2/SvPasCrl mouse substrain a dramatically high frequency of kidney stones that were similar to those of patients with cystinuria. Most of 129S2/SvPasCrl exhibited pathognomonic cystine crystals in urine and an aminoaciduria profile similar to that of patients with cystinuria. In addition, we observed a heterogeneous inflammatory infiltrate and cystine tubular casts in the kidney of cystinuric mice. As compared to another classical mouse strain, C57BL/6J mice, 129S2/SvPasCrl mice had an increased mortality associated with bilateral obstructive hydronephrosis. In 129S2/SvPasCrl mice, the heavy subunit rBAT of the tetrameric transporter of dibasic amino acids was absent in proximal tubules and we identified a single pathogenic mutation in a highly conserved region of the Slc3a1 gene. This novel mouse model mimicking human disease would allow us further pathophysiological studies and may be useful to analyse the crystal/tissue interactions in cystinuria.     

A micellar prodrug of paclitaxel conjugated to cyclotriphosphazene

A novel water soluble and biodegradable cyclotriphosphazene-paclitaxel conjugate was prepared by reacting 2'-succinyl paclitaxel with cyclotriphosphazenes bearing equimolar glycyl-L-lysine and methoxy poly(ethylene glycol) as side groups. The macromolecular conjugate was found to self-assemble in aqueous solution to form stable micelles with a mean hydrodynamic diameter of 24.7 nm and a low critical micelle concentration of 10 mg/L. The present conjugate exhibited lower than free paclitaxel but reasonably high in vitro cytotoxicity against selected human tumor cells due to their hydrolytic degradation in PBS solution.     

Free Amino-acids in the Endosperm of the Developing Coconut (Cocos nucifera)

The free amino-acids in the sap flowing from the incised inflorescenceof the coconut palm (Cocos nucifera) and in the liquid and solidendosperm of the coconut at various stages of development (4–12months from the time of opening of the spathe) have been studiedby paper and ion-exchange chromatography. The chief amino-acids in the sap were in decreasing order ofmagnitude: glutamic acid, aspartic acid, glutamine, serine,and asparagine. Smaller amounts of threonine, alanine, -aminobutyricacid, proline, valine, arginine, and tryptophan were found,along with traces of tyrosine, ß-alanine, lysine,histidine, leucine, isoleucine, and methionine. The chief amino-acids in the liquid endosperm of the nut until6 months of age were alanine, glutamic acid, serine, valine,and proline with smaller amounts of asparagine, aspartic acid,glycine, threonine, leucine, isoleucine, and glutamine. Thealanine, glutamic acid, proline, and -aminobutyric acid contentsof the liquid endosperm increased greatly as the solid endospermwas laid down, ultimately forming about 75 per cent. of thetotal free amino-acids. Nineteen amino-acids were detected atthe 8–10-month stage. -Aminobutyric acid appears in the liquid endosperm soon afterthe solid endosperm commences to form, and increases strikinglythereafter. It was also found in the free amino-acids of thesolid endosperm at all stages of development. This amino-acidwas not found in the acid hydrolysate of coconut globulin. The free amino-acids in the solid endosperm were found to reflectqualitatively the free amino-acid composition of the correspondingliquid endosperm. In addition, ß-alanine, -aminobutyricacid, phenylalanine, and tryptophan were present in small amounts.Alanine, serine, glutamic acid, and -aminobutyric acid, increasedwith age of the nut while the others increased up to 6 monthsand then decreased, only traces being found at maturity exceptin the case of aspartic acid, proline, and threonine. Twenty-oneamino-acids were detected in the free amino-acids of the solidendosperm. The source of the free amino-acids in the liquid endosperm andthe possible use of these amino-acids for protein synthesisin the coconut are briefly discussed.     

Genetic heterogeneity of cystinuria. Study of 12 families

The authors attempted to predict the genotypes of 15 cystinuric children, from the results of oral lysine loads on the propositus and the urinary excretion rates of cystine, lysine, ornithine and arginine of their parents and siblings. Type I cystinuria is more common, as well in the homozygous state, as in combination to type II or III (compound heterozygous genotypes).     

Change in the amino-acid content during male gametophyte formation of Datura metel in Situ

Summary Determination of the free and bound aminoacids during microsporogenesis of Datura metel showed that the principal amino-acids were proline, glutamic acid, aspartic acid, threonine-serine and alanine. Of these, only proline showed a consistent increment during pollen development. In contrast, aspartic acid and lysine decreased in the later stages of microsporogenesis. The amounts of other amino-acids did not show any consistent pattern. Four amino-acids, namely proline, glutamic acid and threonine-serine constituted nearly 85% of the free amino-acid pool in the developed anther (stage IV). Proline accumulation, relative to the total free amino-acid pool in mature anthers, was correlated with the water-content. The results were discussed in view of possible relationships between metabolic activity and free and bound amino-acid concentrations.Abbreviations ABA -Aminobutyric acid - mol/g micromoles of each amino-acid per gram of dry material     

The Metabolism of Labelled Lysine and Pipecolic Acid by Acacia Phyllodes

C¹⁴-lysine and C¹⁴- and H³-pipecolic acids have been used tostudy the metabolism of the lysine family of amino-acids inAcacia, which contains ₄-hydroxypipecolic acid as a characteristiccomponent of the soluble-nitrogen fraction. Degradation of C¹⁴-lysinewas rapid and was far more extensive than that observed earlierin higher plants. Pipecolic acid was the major radioactive productin short-term experiments. After longer metabolic periods, radioactivitywas distributed over a wide range of amino-acids, organic acids,and sugars. A tentative metabolic scheme is produced to explainthese observations concerning lysine degradation. The distributionof radioactivity in the amino-acids of the protein present inthe phyllodes was determined 24 hours after supplying C¹⁴-lysine.Specific activities of free and bound amino-acids are comparedat this time. Hydroxyproline forms a notable component of thephyllode protein. Pipecolic acid degradation has been demonstrated for the firsttime in a biological system. The breakdown pathway was studiedin Acacia phyllodes using H²-pipecolic acid. Substances tentativelyidentified as '-piperdine-2-carboxylic acid and -amino--hydroxycaproicacid were amongst the early degradation products. Ultimately,lysine and -aminoadipic acid became labelled. In contrast tothe experiments with C organic acids and sugars did not becomeradioactive. The explanation of this finding is probably tobe found in the ease with which H³ atoms present in certainchemical groupings may undergo exchange with normal hydrogenatoms of water molecules. The biosynthesis of 4-hydroxypipecolic acid probably involvesa direct hydroxylation of the parent imino-acid.     

The molecular bases of cystinuria and lysinuric protein intolerance

Cystinuria and lysinuric protein intolerance are inherited aminoacidurias caused by defective amino-acid transport activities linked to a family of heteromeric amino-acid transporters (HATs). HATs comprise two subunits: co-expression of subunits 4F2hc and y(+)LAT-1 induces the efflux of dibasic amino acids from cells, whereas co-expression of subunits rBAT and b(o,+)AT induces the renal reabsorption and intestinal absorption of cystine and dibasic amino acids at the brush border of epithelial cells. Recently, the role of b(o,+)AT (SLC7A9) in cystinuria (non Type I) and the role of y(+)LAT-1 (SLC7A7) in lysinuric protein intolerance have been demonstrated.     

A comprehensive library of blocked dipeptides reveals intrinsic backbone conformational propensities of unfolded proteins

Despite prolonged scientific efforts to elucidate the intrinsic peptide backbone preferences of amino-acids based on understanding of intermolecular forces, many open questions remain, particularly concerning neighboring peptide interaction effects on the backbone conformational distribution of short peptides and unfolded proteins. Here, we show that spectroscopic studies of a complete library of 400 dipeptides reveal that, irrespective of side-chain properties, the backbone conformation distribution is narrow and they adopt polyproline II and β-strand, indicating the importance of backbone peptide solvation and electronic effects. By directly comparing the dipeptide circular dichroism and NMR results with those of unfolded proteins, the comprehensive dipeptides form a complete set of structural motifs of unfolded proteins. We thus anticipate that the present dipeptide library with spectroscopic data can serve as a useful database for understanding the nature of unfolded protein structures and for further refinements of molecular mechanical parameters.     

The Uptake of Amino-Acids and their Incorporation into the Proteins of Excised Barley Embryos

Methods are described by which barley embryos may be excisedand grown under sterile conditions on a medium containing sucrose,minerals, and a complete mixture of amino-acids. Growth underthese conditions was comparable with that of intact seedlingsand the uptake of sugar and amino-acid could be studied withoutdisturbing the metabolic steady state. Purified preparationsof the embryo proteins have been made and the constituent amino-acidsseparated. ¹⁴C labelling in these amino-acids was determinedby a new gas-scintillation method. In an isotopic competitionexperiment embryos were grown in ¹⁴C-sucrose with nitrate oran amino-acid mixture as nitrogen source. The presence of exogenousamino-acids suppressed the incorporation of carbon from carbohydrateinto amino-acid residues of the embryo protein. The degree ofsuppression varied, being undetectable for glutamic acid butalmost complete for lysine and leucine; it appeared to be relatedto the length of the synthetic pathway from carbohydrate tothe amino-acid. The evidence suggested that amino-acids areprotein precursors, and this conclusion was confirmed in furtherexperiments in which ¹⁴C-aspartic acid, -glutamine, -proline,-leucine, or -lysine were supplied singly in a complete mixtureof amino-acids. The ¹⁴C was found predominantly in the amino-acidresidue of protein corresponding to the ¹⁴C-amino-acid supplied,with smaller amounts in other amino-acids of the same or relatedfamilies. Aspartic acid and glutamine yielded appreciable quantitiesof respiratory carbon dioxide, although the contribution wassmall compared to that of sucrose. Little carbon was lost ascarbon dioxide from leucine or lysine. The results are discussedin relation to the role of amino-acids in protein synthesis,and to the existence of feedback control in the amino-acid metabolismof higher plants.     

Model system studies of staining procedures for lysine and arginine residues.

Using polyacrylamide films containg poly-lysine, polyarginine and DNA as test models, a variety of reportedly specific staining procedures have been examine. Contrary to published observations, mixtures of fast green and eosin Y show no specific staining of either lysine or arginine. Both amino-acids bind eosin from the mixture more strongly than fast green. Arginine apparently has a greater affinity for this eosin than has lysine which contradicts previous reports that lysine will be stained by eosin arginine will stain with fast green, if proteins containing both amino-acids are stained with dye mixture. In films containing lysine and/or arginine picric acid is shown to bind specifically to the arginine. The picric acidarginine complex resists disruption in 0.004 M borate buffer which is a solvent used for subsequent staining of lysine residues with bromophenol blue. Picric acid may also be used as a hydrolysant and substitute for hydrocholoric acid in a Feulgen-like procedure which stains DNA to the same level as the classiclal hydrochloric acid based procedure while also staining arginine present.