In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the function...
In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.
In order to understand the renal reabsorption mechanism of neutral amino acids via amino acid transporters, we have isolated human L-type amino acid transporter 2 (hLAT2) and human T-type amino acid transporter 1 (hTAT1) in human, then, we have examined and compared the gene structures, the functional characterizations and the localization in human kidney. Northern blot analysis showed that hLAT2 mRNA was expressed at high levels in the heart, brain, placenta, kidney, spleen, prostate, testis, ovary, lymph node and the fetal liver. The hTAT1 mRNA was detected at high levels in the heart, placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus and prostate. Immunohistochemical analysis on the human kidney revealed that the hLAT2 and hTAT1 proteins coexist in the basolateral membrane of the renal proximal tubules. The hLAT2 transports all neutral amino acids and hTAT1 transports aromatic amino acids. The basolateral location of the hLAT2 and hTAT1 proteins in the renal proximal tubule as well as the amino acid transport activity of hLAT2 and hTAT1 suggests that these transporters contribute to the renal reabsorption of neutral and aromatic amino acids in the basolateral domain of epithelial proximal tubule cells, respectively. Therefore, LAT2 and TAT1 play essential roles in the reabsorption of neutral amino acids from the epithelial cells to the blood stream in the kidney. Because LAT2 and TAT1 are essential to the efficient absorption of neutral amino acids from the kidney, their defects might be involved in the pathogenesis of disorders caused by a disruption in amino acid absorption such as blue diaper syndrome.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
제안 방법
N32639). The cDNA fragment was labeled with [32P]dCTP ("Q니ickPrime, Amersham Pharmacia Biotech, Tokyo, Japan) in order to use it as a probe to screen the nondirectional cDNA library. The cDNA library was prepared from the human kidney poly(A)+RNA (Clontech, Palo 시to, CA, USA) using a superscript Choice System (Life Technologies, Grand Island, NY, USA).
The table was constructed based on the three separate experiments using different batches of oocytes. In each experiment, L-tryptophan uptake measurements for hLAT2 were performed to compare Vmax values among different exgriments. The Vmax value for each amino acid was normalized to that of hLAT2 for L- tryptophan in the same experiment.
of the uptake val 니 es (n = 6-8). In order to confirm the reproducibility of the results, three separate experiments were performed for each measurement using different batches of oocytes and in vitro transcribed cRNAs. The resuIts from the representative experiments are shown in the figures.
In this study, the hLAT2 transported the glycine, and the L-isomers of neutral amino acids including small ne니tral amino acids such as alanine, serine, threonine and cysteine, branched or aromatic chained amino acids such as asparagines, glutamine, methionine, leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan and histidine (Fig. 3A and Fig. 4A). In a previous study, hl_AT1 preferred larger neutral amino acids with bulky or branched side chains for its substrates (Yanagida et al.
The FISH signals and the DAPI banding pattern were separately recorded by taking photographs. The assignment of the FISH mapping data with the chromosomal bands was achieved by superimposing the FISH sign기s with the DAPI banded chromosomes.
Therefore, this study investigated the absorption mechanisms of neutral amino acids via the system L amino acid transporter and the system T amino acid transporter in the kidney. To accomplish this, the cDNAs of the system L amino acid transporter and the system T amino acid transporter in human were cloned and th으 g응n으 structures, the functional characterizations and the localization in the human kidney were examined and compared.
Therefore, this study investigated the absorption mechanisms of neutral amino acids via the system L amino acid transporter and the system T amino acid transporter in the kidney. To accomplish this, the cDNAs of the system L amino acid transporter and the system T amino acid transporter in human were cloned and th으 g응n으 structures, the functional characterizations and the localization in the human kidney were examined and compared.
대상 데이터
The cDNA fragment was labeled with [32P]dCTP ("Q니ickPrime, Amersham Pharmacia Biotech, Tokyo, Japan) in order to use it as a probe to screen the nondirectional cDNA library. The cDNA library was prepared from the human kidney poly(A)+RNA (Clontech, Palo 시to, CA, USA) using a superscript Choice System (Life Technologies, Grand Island, NY, USA). The synthesized cDNA was ligated to the IZipLox EcoRI arms (Life Technologies, Grand Island, NY, USA).
Therefore, the genes for these related proteins are not clustered in the human chromosomes. This study showed that the hLAT2 (SLC7A3) is constructed with 11 exons (丁汕le II). However the hU\T1 (SLC7A5) is assembled with 10 exons (Nii et al.
이론/모형
The Km and Vmax of the amino acid s니bstrates were determined using the Eadie-Hofstee equation based on the hLAT2-mediated amino acid uptakes measured at 0.003, 0.01, 0.03, 0.1, 0.3, 1, and 3 mM. The Km and Vmax of the amino acid s니bstrates were determined using the Eadie-Hofstee equation based on the hTAT1 -mediated amino acid uptakes measured at 0.
3, 1, and 3 mM. The Km and Vmax of the amino acid s니bstrates were determined using the Eadie-Hofstee equation based on the hTAT1 -mediated amino acid uptakes measured at 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 mM. The hLAT2 and h77\T1 mediated amino acid uptakes were calculated as the differences between the means of the uptakes into the oocytes, which had been injected with the cRNA for either hLAT2 or hTAT1 and those of the control oocytes injected with water.
The cDNAs in the positive IZipLox phages were rescued into plasmid pZL1 by in vivo excision according to the mar心facturer's instruction (Life T&이】nologies, Grand Island, NY, USA). The cDNA was sequenced in both directions by the dye terminator cycle sequencing method (Perkin-Elmer, Norwalk, CT, USA and Applied Biosystems, Foster City, CA, USA). The transmembrane regions of proteins were predicted based on the amino acid sequence using the SOSUI algorithm (Hirokawa et al.
The cDNA was sequenced in both directions by the dye terminator cycle sequencing method (Perkin-Elmer, Norwalk, CT, USA and Applied Biosystems, Foster City, CA, USA). The transmembrane regions of proteins were predicted based on the amino acid sequence using the SOSUI algorithm (Hirokawa et al., 1998).
성능/효과
Overall, these results suggest that the hLAT2 plays important roles in the reabsorption of all neutral amino acids including aromatic amino acids, and the hTAT1 plays essential roles in the reabsorption of aromatic amino acids in the human kidney. In conclusion, the neutral amino acids are absorbed from the luminal fluid of the renal proximal tubule via the system B° amino acid transporter located on the apical membrane of the epithelial cells, and are reabsorped from the epithelial cells to the blood stream via the system L amino acid transporter and system T amino acid transporter. Although the specific diseases are, at the moment, not mapped to a particular region of the human chromosome where the hLAt2 (SLC7A8) and h7AT1 (SLC16A10) are located, the defect of these amino acid transporters might be involved in the pathogenesis of disorders caused by a disruption of amino acid absorption such as blue diaper syndrome (Rosenberg et al.
In order to understand the r이e of hLAT2 and hTAT1 in the human kidney, immunohistochemical analyses were performed on human kidney. The results showed that the hLAT2 and hTAT1 proteins co-exist in the basolateral membrane of the renal proximal tubule (Fig. 2). To our knowledge, this is the first study to report the expression of the hl_AT2 and hTZVTI proteins in the basolateral membrane of the epithelial cells from human tissues.
, 2001). This study showed that the hLAT2 and hTAT1 proteins co-exist in the basolateral membrane of the epithelial cells of the renal proximal tubule. In addition, it is known that the LAT2 and TZVT1 are the facilitated amino acid transporters (Pineda et al.
참고문헌 (44)
Altman, A., Cardenas, J. M., Houghten, R. A., Dixon, F. J., and Theofilopoulos, A. N., Antibodies of predetermined specificity against chemically synthesized peptides of human interleukin 2. Proc. Natl. Acad. Sci. U.S.A., 81, 2176-2180 (1984)
Blondeau, J. P., Beslin, A., Chantoux, F., and Francon, J., Triiodothyronine is a high-affinity inhibitor of amino acid transport system L1 in cultured astrocytes. J. Neurochem., 60, 1407-1413 (1993)
Broer, A., Klingel, K., Kowalczuk, S., Rasko, J. E., Cavanaugh, J., and Broer, S., Molecular cloning of mouse amino acid transport system $B^0$ , a neutral amino acid transporter related to Hartnup disorder. J. Biol. Chem., 279, 24467-24476 (2004)
Chillaron, J., Estevez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpi, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacin, M., Obligatory amino acid exchange via systems $b^{o,+}$ -like and $y^+L-like$ . A tertiary active transport mechanism for renal reabsorption of cystine and dibasic amino acids. J. Biol. Chem., 271, 17761-17770 (1996)
Goldenberg, G. J., Lam, H. Y., Begleiter, A., Active carriermediated transport of melphalan by two separate amino acid transport systems in LPC-1 plasmacytoma cells in vitro. J. Biol. Chem., 254, 1057-1064 (1979)
Gomes, P. and Soares-da-Silva, P., L-DOPA transport properties in an immortalized cell line of rat capillary cerebral endothelial cells, RBE 4. Brain Res., 829, 143-150 (1999)
Heng, H. H. Q., Squire, J., and Tsui, L. -C., High resolution mapping of mammalian genes by in situ hybridization to free chromosome. Proc. Natl. Acad. Sci. U.S.A., 89, 9509？9513 (1992)
Hisano, S., Haga, H., Miyamoto, K., Takeda, E., and Fukui, Y., The basic amino acid transporter (rBAT)-like immunoreactivity in paraventricular and supraoptic magnocellular neurons of the rat hypothalamus. Brain Res., 710, 299-302 (1996)
Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H., Expression cloning and characterization of a transporter for large neutral amino acids activated by the heavy chain of 4F2 antigen (CD98). J. Biol. Chem., 273, 23629-23632 (1998)
Kanai, Y. and Endou, H., Heterodimeric amino acid transporters: molecular biology and pathological and pharmacological relevance. Curr. Drug Metab., 2, 339-354 (2001)
Kim, D. K., Kanai, Y., Chairoungdua, A., Matsuo, H., Cha, S. H., and Endou, H., Expression cloning of a $Na^+-independent$ aromatic amino acid transporter with structural similarity to $H^+$ /monocarboxylate transporters. J. Biol. Chem., 276, 17221-17228 (2001)
Kim, D. K., Kanai, Y., Choi, H. W., Tangtrongsup, S., Chairoungdua, A., Babu, E., Tachampa, K., Anzai, N., Iribe, Y., and Endou, H., Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells. Biochim. Biophys. Acta, 1565, 112-121 (2002a)
Kim, D. K., Kanai, Y., Matsuo, H., Kim, J. Y., Chairoungdua, A., Kobayashi, Y., Enomoto, A., Cha, S. H., Goya, T., and Endou, H., The human T-type amino acid transporter-1: characterization, gene organization, and chromosomal location. Genomics, 79, 95-103 (2002b)
Lakshmanan, M., Goncalves, E., Lessly, G., Foti, D., and Robbins, J., The transport of thyroxine into mouse neuroblastoma cells, NB41A3: the effect of L-system amino acids. Endocrinology, 126, 3245-3250 (1990)
Lee, S. H., Chae, K. S., Nan, J. X., and Sohn, D. H., The increment of purine specific sodium nucleoside cotransporter mRNA in experimental fibrotic liver induced by bile duct ligation and scission. Arch. Pharm. Res., 23, 613-619 (2000)
Lee, S. H., Chae, K. S., and Sohn, D. H., Identification of expressed sequence tags of genes expressed highly in the activated hepatic stellate cell. Arch. Pharm. Res., 27, 422- 428 (2004)
Mannion, B. A., Kolesnikova, T. V., Lin, S. -H., Thompson, N. L., and Hemler, M. E., The light chain of CD98 is identified as E16/TA1 protein. J. Biol. Chem., 273, 33127-33129 (1998)
Mizoguchi, K., Cha, S. H., Chairoungdua, A., Kim, D. K., Shigeta, Y., Matsuo, H., Fukushima, J., Awa, Y., Akakura, K., Goya, T., Ito, H., Endou, H., and Kanai, Y., Human cystinuriarelated transporter: localization and functional characterization. Kidney Int., 59, 1821-1833 (2001)
Nakamura, E., Sato, M., Yang, H., Miyagawa, F., Harasaki, M., Tomita, K., Matsuoka, S., Noma, A., Iwai, K., and Minato, N., 4F2 (CD98) heavy chain is associated covalently with an amino acid transporter and controls intracellular trafficking and membrane topology of 4F2 heterodimer. J. Biol. Chem., 274, 3009-3016 (1999)
Nii, T., Segawa, H., Taketani, Y., Tani, Y., Ohkido, M., Kishida, S., Ito, M., Endou, H., Kanai, Y., Takeda, E., and Miyamoto, K., Molecular events involved in up-regulating human $Na^+$ - independent neutral amino acid transporter LAT1 during Tcell activation. Biochem. J., 358, 693-704 (2001)
Oxender, D. L. and Christensen, H. N., Evidence for two types of mediation of neutral amino acid transport in Ehrlich cells. Nature, 197, 765-767 (1963)
Palacin, M., Estevez, R., Bertran, J., and Zorzano, A., Molecular biology of mammalian plasma membrane amino acid transporters. Physiol. Rev., 78, 969-1054 (1998)
Peghini, P., Janzen, J., and Stoffel, W., Glutamate transporter EAAC-1-deficient mice develop dicarboxylic aminoaciduria and behavioral abnormalities but no neurodegeneration. EMBO J., 16, 3822-3832 (1997)
Pineda, M., Fernandez, E., Torrents, D., Estevez, R., Lopez, C., Camps, M., Lloberas, J., Zorzano, A., and Palacin, M., Identification of a membrane protein, LAT-2, that Co-expresses with 4F2 heavy chain, an L-type amino acid transport activity with broad specificity for small and large zwitterionic amino acids. J. Biol. Chem., 274, 19738-19744 (1999)
Prasad, P. D., Wang, H., Huang, H., Kekuda, R., Rajan, D. P., Leibach, F. H., and Ganapathy, V., Human LAT1, a subunit of system L amino acid transporter: molecular cloning and transport function. Biochem. Biophys. Res. Commun., 255, 283-288 (1999)
Rossier, G., Meier, C., Bauch, C., Summa, V., Sordat, B., Verrey, F., and Kuhn, L. C., LAT2, a new basolateral 4F2hc/ CD98-associated amino acid transporter of kidney and intestine. J. Biol. Chem., 274, 34948-34954 (1999)
Sang, J., Lim, Y. -P., Panzia, M., Finch, P., and Thompson, N. L., TA1, a highly conserved oncofetal complementary DNA from rat hepatoma, encodes an integral membrane protein associated with liver development, carcinogenesis, and cell activation. Cancer Res., 55, 1152-1159 (1995)
Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y., Identification and functional characterization of a $Na^+$ -independent neutral amino acid transporter with broad substrate selectivity. J. Biol. Chem., 274, 19745- 19751 (1999)
Shayakul, C., Kanai, Y., Lee, W. S., Brown, D., Rothstein, J. D., and Hediger, M. A., Localization of the high-affinity glutamate transporter EAAC1 in rat kidney. Am. J. Physiol., 273, F1023- F1029 (1997)
Silbernagl, S., Renal transport of amino acids. Klin. Wochenschr., 57, 1009-1019, (1979)
Stevens, B. R., Kaunitz, J. D., and Wright, E. M., Intestinal transport of amino acids and sugars: advances using membrane vesicles. Annu. Rev. Physiol., 46, 417-433 (1984)
Su, T. Z., Lunney, E., Campbell, G., and Oxender, D. L., Transport of gabapentin, a gamma-amino acid drug, by system l alpha-amino acid transporters: a comparative study in astrocytes, synaptosomes and CHO cells. J. Neurochem., 64, 2125-2131 (1995)
Utsunomiya-Tate, N., Endou, H., and Kanai, Y., Cloning and functional characterization of a system ASC-like $Na^+$ - dependent neutral amino acid transporter. J. Biol. Chem., 271, 14883-14890 (1996)
van Winkle, L. J., Mann, D. F., Campione, A. L., and Farrington, B. H., Transport of benzenoid amino acids by system T and four broad scope systems in preimplantation mouse conceptuses. Biochim. Biophys. Acta, 1027, 268？277 (1990)
Wolf, D. A., Wang, S., Panzia, M. A., Bassily, N. H., and Thompson, N. L., Expression of a highly conserved oncofetal gene, TA1/E16, in human colon carcinoma and other primary cancers: homology to Schistosoma mansoni amino acid permease and Caenorhabditis elegans gene products. Cancer Res., 56, 5012-5022 (1996)
Yanagida, O., Kanai, Y., Chairoungdua, A., Kim, D. K., Segawa, H., Nii, T., Cha, S. H., Matsuo, H., Fukushima, J., Fukasawa, Y., Tani, Y., Taketani, Y., Uchino, H., Kim, J. Y., Inatomi, J., Okayasu, I., Miyamoto, K., Takeda, E., Goya, T., and Endou, H., Human L-type amino acid transporter 1 (LAT1): characterization of function and expression in tumor cell lines. Biochim. Biophys. Acta, 1514, 291-302 (2001)
Yoon, J. H., Kim, Y. B., Kim, M. S., Park, J. C., Kook, J. K., Jung, H. M., Kim, S. G., Yoo, H., Ko, Y. M., Lee, S. H., Kim, B. Y., Chun, H. S., Kanai, Y., Endou, H., and Kim, D. K., Expression and functional characterization of the system L amino acid transporter in KB human oral epidermoid carcinoma cells. Cancer Lett., 205, 215-226 (2004)
※ AI-Helper는 부적절한 답변을 할 수 있습니다.