Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y ؉ LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.
Mutations of the glycoprotein rBAT cause cystinuria type I, an autosomal recessive failure of dibasic amino acid transport (b0,+ type) across luminal membranes of intestine and kidney cells. Here we identify the permease-like protein b0,+AT as the catalytic subunit that associates by a disulfide bond with rBAT to form a hetero-oligomeric b0,+amino acid transporter complex. We demonstrate its b0,+-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 b0,+AT protein is the product of the gene defective in non-type I cystinuria.
Abstract. Humans and other mammals continue to be exposed to various forms of mercury in the environment. The kidneys, specifically the epithelial cells lining the proximal tubules, are the primary targets where mercuric ions accumulate and exert their toxic effects. Although the actual mechanisms involved in the transport of mercuric ions along the proximal tubule have not been defined, current evidence implicates mercuric conjugates of cysteine, primarily 2-amino-3-(2-amino-2-carboxyethylsulfanylmercuricsulfanyl)propionic acid (Cys-S-Hg-S-Cys), as the most likely transportable species of inorganic mercury (Hg 2ϩ ). Because Cys-S-Hg-S-Cys and the amino acid cystine (Cys-S-S-Cys) are structurally similar, it was hypothesized that Cys-S-Hg-S-Cys might act as a molecular mimic of cystine at one or more of the amino acid transporters involved in the luminal absorption of this amino acid. One such candidate is the Na ϩ -independent heterodimeric transporter system b 0,ϩ . Therefore, the transport of Cys-S-Hg-S-Cys and cystine was studied in MDCK II cells that were or were not stably transfected with b 0,ϩ AT-rBAT. Transport of Cys-S-Hg-S-Cys and cystine across the luminal plasma membrane was similar in the transfected cells, indicating that Cys-S-Hg-S-Cys can behave as a molecular mimic of cystine at the site of system b 0,ϩ . Moreover, only the b 0,ϩ AT-rBAT transfectants became selectively intoxicated during exposure to Cys-S-Hg-S-Cys. These findings indicate that system b 0,ϩ likely contributes to the nephropathy induced by Hg 2ϩ in vivo. These data represent the first direct molecular evidence for the participation of a specific transporter in the luminal uptake of a large divalent metal cation in proximal tubular cells.A large body of evidence indicates that the epithelial cells lining the convoluted and straight segments of the proximal tubule are the primary sites where inorganic mercury (Hg 2ϩ ) is taken up and accumulated in vivo (1,2). Although the actual mechanisms by which mercuric ions are taken up by these cells are not well defined, a number of recent in vivo findings indicate that the uptake of Hg 2ϩ at the luminal membrane of proximal tubular cells is dependent on the activities of the brush border enzymes ␥-glutamyltransferase (3-8) and cysteinylglycinase (8). It seems that mercuric conjugates of GSH, while in the lumen of the proximal tubule, are degraded sequentially by these enzymes to yield a cysteine-S-conjugate of mercury, primarily 2-amino-3-(2-amino-2-carboxyethylsulfanylmercuricsulfanyl)propionic acid (Cys-S-Hg-S-Cys) (2). This conjugate is thought to be the principal species of Hg 2ϩ taken up at the luminal plasma membrane of proximal tubular epithelial cells (3-8).Recent studies using brush border membrane vesicles (9) and isolated perfused proximal tubular segments (8,10) indicate that Cys-S-Hg-S-Cys is indeed transported across the luminal membrane of proximal tubular epithelial cells. Moreover, competitive inhibition experiments using isolated perfused proximal tubular segments have i...
The immunodominant central portion of the circumsporozoite (CS) surface protein of the malaria parasite Plasmodium falciparum contains a tetrapeptide motif, Asn-Pro-Asn-Ala (NPNA), tandemly repeated almost 40 times. The three-dimensional structure of the CS protein, including the central repeat region, is presently unknown. We have investigated an approach to stabilize β-turns in a single NPNA motif, by its incorporation into a template-bound cyclic peptide comprising the sequence ANPNAA. The template was designed to stabilize β-turns in the peptide loop and to allow its conjugation to T-cell epitopes in a multiple-antigen-peptide. NMR studies and MD simulations with time-averaged NOE-derived upper distance restraints support the formation of a stable β-I turn conformation in the NPNA motif of this template-bound antigen. Balb/c mice immunized with a multiple-antigen-peptide containing four copies of the template-bound loop conjugated to a single universal T-cell epitope produced antibodies that bound P. falciparum sporozoites in immunofluorescence assays. These results provide further support for the immunological relevance of a type-I β-turn conformation based on the NPNA cadence in the repeat region of the CS protein and illustrate the use of a novel template for the evaluation of conformationally constrained peptide immunogens.
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