Michiko Tamba

Cloning and Expression of a Plasma Membrane Cystine/Glutamate Exchange Transporter Composed of Two Distinct Proteins

Transport system xc − found in plasma membrane of cultured mammalian cells is an exchange agency for anionic amino acids with high specificity for anionic form of cystine and glutamate. We have isolated cDNA encoding the transporter for system xc − from mouse activated macrophages by expression in Xenopus oocytes. The expression of system xc − activity in oocytes required two cDNA transcripts, and the sequence analysis revealed that one is identical with the heavy chain of 4F2 cell surface antigen (4F2hc) and the other is a novel protein of 502 amino acids with 12 putative transmembrane domains. The latter protein, named xCT, showed a significant homology with those recently reported to mediate cationic or zwitterionic amino acid transport when co-expressed with 4F2hc. Thus xCT is a new member of a family of amino acid transporters that form heteromultimeric complex with 4F2hc, with a striking difference in substrate specificity. The expression of system xc − was highly regulated, and Northern blot analysis demonstrated that the expression of both 4F2hc and xCT was enhanced in macrophages stimulated by lipopolysaccharide or an electrophilic agent. However, the expression of xCT was more directly correlated with the system xc − activity.

Redox Imbalance in Cystine/Glutamate Transporter-deficient Mice

Cystine/glutamate transporter, designated as system x–c, mediates cystine entry in exchange for intracellular glutamate in mammalian cells. This transporter consists of two protein components, xCT and 4F2 heavy chain, and the former is predicted to mediate the transport activity. This transporter plays a pivotal role for maintaining the intracellular GSH levels and extracellular cystine/cysteine redox balance in cultured cells. To clarify the physiological roles of this transporter in vivo, we generated and characterized mice lacking xCT. The xCT–/– mice were healthy in appearance and fertile. However, cystine concentration in plasma was significantly higher in these mice, compared with that in the littermate xCT–/– mice, while there was no significant difference in plasma cysteine concentration. Plasma GSH level in xCT–/– mice was lower than that in the xCT–/– mice. The embryonic fibroblasts derived from xCT–/– mice failed to survive in routine culture medium, and 2-mercaptoethanol was required for survival and growth. When 2-mercaptoethanol was removed from the culture medium, cysteine and GSH in these cells dramatically decreased, and cells started to die within 24 h. N-Acetyl cysteine also rescued xCT–/–-derived cells and permitted growth. These results demonstrate that system x–c contributes to maintaining the plasma redox balance in vivo but is dispensable in mammalian development, although it is vitally important to cells in vitro.

A PORCINE HOMOLOG OF THE MAJOR SECRETORY PROTEIN OF HUMAN EPIDIDYMIS, HE1,SPECIFICALLY BINDS CHOLESTEROL

A porcine homolog of the major secretory protein of human epididymis, HE1, was for the first time purified from the porcine cauda epididymal fluid. The HE1 homolog was secreted into the epididymal fluid as a 19-kDa glycoprotein, whose sugar moiety was gradually processed to form a 16-kDa protein during transit through the epididymis. The HE1 homolog mRNA was detected only in the caput and corpus epididymis among the porcine tissues examined. The purified HE1 homolog specifically bound cholesterol with high affinity (Kd=2. 3 microM). The binding stoichiometry was determined to be 0.94 mol/mol, suggesting that 1 mol of cholesterol binds to 1 mol of the protein. It was also found that the HE1 homolog is a major cholesterol-binding protein in the porcine epididymal fluid. The possibility that the HE1 homolog is involved in the regulation of the lipid composition of the sperm membranes during the maturation in epididymis is discussed.

Molecular cloning and characterization of the epididymis-specific glutathione peroxidase-like protein secreted in the porcine epididymal fluid

The epididymis-specific glutathione peroxidase was purified from the porcine cauda epididymal fluid in order to analyze its enzymatic activity and roles in the epididymis. The purified protein was found to consist of four identical 23 kDa subunits. The complementary DNA encoding the 23 kDa subunit was cloned from the cDNA library of the porcine proximal caput epididymis, only where the 23 kDa subunit is expressed. Although the selenocysteine codon (TGA) is contained in the cDNA of the other cytosolic type of glutathione peroxidases, it is replaced by cysteine codon (TGT) in the 23 kDa subunit cDNA, similarly to the results previously obtained for cDNAs encoding the epididymis-specific form of the secreted glutathione peroxidases of mouse, rat and monkey. By the direct analysis of the selenium, the purified protein was proved to contain no selenium atom in the molecule. The activities of the purified epididymis-specific glutathione peroxidase toward hydrogen peroxide or organic hydroperoxides were by far lower than the activity of cytosolic selenium-dependent glutathione peroxidase (less than 0.1%). In addition, the concentration of glutathione in the porcine epididymal fluids was about 20 microM, which is much lower than the optimal concentration for the glutathione peroxidase activity of the purified protein. These results strongly suggest that this protein is enzymatically quiescent at least in the porcine epididymal fluid. An immunocytochemical study showed that this protein was found to bind to the acrosomal region of the epididymal sperm and to disappear during the acrosome reaction. Furthermore, this protein significantly retarded the acrosome reaction induced in vitro. The possibilities have been discussed that it protects sperm from the premature acrosome reaction and maintains sperm fertilizing ability in the epididymis.

Organ-specific Sulfation Patterns of Heparan Sulfate Generated by Extracellular Sulfatases Sulf1 and Sulf2 in Mice

Background: Extracellular endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate. Results: Disaccharide analysis showed that 2-O-, 6-O-, and N-trisulfated disaccharide units in heparan sulfate were increased to different degrees in different organs in Sulf1 and Sulf2 knock-out mice. Conclusion: Sulfs generate organ-specific sulfation patterns of heparan sulfate. Significance: This may indicate differences in activity between Sulf1 and Sulf2 in vivo. Heparan sulfate endosulfatases Sulf1 and Sulf2 hydrolyze 6-O-sulfate in heparan sulfate, thereby regulating cellular signaling. Previous studies have revealed that Sulfs act predominantly on UA2S-GlcNS6S disaccharides and weakly on UA-GlcNS6S disaccharides. However, the specificity of Sulfs and their role in sulfation patterning of heparan sulfate in vivo remained unknown. Here, we performed disaccharide analysis of heparan sulfate in Sulf1 and Sulf2 knock-out mice. Significant increases in ΔUA2S-GlcNS6S were observed in the brain, small intestine, lung, spleen, testis, and skeletal muscle of adult Sulf1−/− mice and in the brain, liver, kidney, spleen, and testis of adult Sulf2−/− mice. In addition, increases in ΔUA-GlcNS6S were seen in the Sulf1−/− lung and small intestine. In contrast, the disaccharide compositions of chondroitin sulfate were not primarily altered, indicating specificity of Sulfs for heparan sulfate. For Sulf1, but not for Sulf2, mRNA expression levels in eight organs of wild-type mice were highly correlated with increases in ΔUA2S-GlcNS6S in the corresponding organs of knock-out mice. Moreover, overall changes in heparan sulfate compositions were greater in Sulf1−/− mice than in Sulf2−/− mice despite lower levels of Sulf1 mRNA expression, suggesting predominant roles of Sulf1 in heparan sulfate desulfation and distinct regulation of Sulf activities in vivo. Sulf1 and Sulf2 mRNAs were differentially expressed in restricted types of cells in organs, and consequently, the sulfation patterns of heparan sulfate were locally and distinctly altered in Sulf1 and Sulf2 knock-out mice. These findings indicate that Sulf1 and Sulf2 differentially contribute to the generation of organ-specific sulfation patterns of heparan sulfate.

Expression and function of cystine/glutamate transporter in neutrophils

Reactive oxygen species (ROS) produced by neutrophils are essential in the host defense against infections but may be harmful to neutrophils themselves. Glutathione (GSH) plays a pivotal role in protecting cells against ROS‐mediated oxidant injury. Cystine/glutamate transporter, designated as system xc– and consisting of two proteins, xCT and 4F2hc, is important to maintain GSH levels in mammalian‐cultured cells. In the present paper, we have investigated system xc– in neutrophils. In human peripheral blood neutrophils, neither the activity of system xc– nor xCT mRNA was detected. The activity was induced, and xCT mRNA was expressed when they were cultured in vitro. The mRNA expression was much enhanced in the presence of opsonized zymosan or PMA. In contrast, mouse peritoneal exudate neutrophils, immediately after preparation, exhibited system xc– activity and expressed xCT mRNA. The activity and the expression were heightened further when they were cultured. Peritoneal exudate cells (mostly neutrophils) from xCT‐deficient (xCT−/−) mice had lower cysteine content than those from the wild‐type mice. GSH levels in the xCT−/−cells decreased rapidly when they were cultured, whereas those in the wild‐type cells were maintained during the culture. Apoptosis induced in culture was enhanced in the xCT−/−cells compared with the wild‐type cells. These results suggest that system xc– plays an important role in neutrophils when they are activated, and their GSH consumption is accelerated.

Direct evidence for the elevated synthesis and secretion of procathepsin L in the distal caput epididymis of boar.

The proteins which are secreted from the restricted part of the epididymis are suggested to sustain sperm maturation. In porcine species, as the potential abilities of sperm for movement and fertilization greatly increase in the corpus epididymis, the secretions in both the caput and corpus epididymis seem to be very important for the sperm maturation. In this study, we have directed our attention to the 40 kDa protein which is detected in the fluid of the distal caput epididymis of boar. It was purified from the porcine cauda epididymal fluid and its cDNA was cloned from the cDNA library of the distal caput epididymis. According to the deduced amino acid sequence, the 40 kDa protein has been identified as procathepsin L. Northern blot analysis showed that the procathepsin L mRNA was most abundant in the distal caput epididymis among the tissues as examined. Consistent with the distribution of the procathepsin L mRNA in the epididymis, the activity of procathepsin L was absent in the fluid of the proximal and mid caput epididymis and first appeared in the distal caput epididymal fluid, whose contents gradually decreased with the passage through the epididymis. These results first appeared in the first distal caput epididymis expresses very high levels of procathepsin L and unusually secretes it into the luminal fluid instead of targeting it to lysosomes. It has been also found that the mRNA of PDGF, which is known to enhance cathepsin L expression in the culture cells, is very high in the mid caput epididymis, which just precedes the site of procathepsin L secretion. This result indicates that PDGF directly regulates the locally restricted expression and secretion of procathepsin L in the epididymis, which is one of the possible mechanisms involved in the functional differentiation in the epididymis.

Induction of cystine/glutamate transporter in bacterial lipopolysaccharide induced endotoxemia in mice

BackgroundCystine/glutamate transporter, system xc-, contributes to the maintenance of intracellular glutathione levels and the redox balance in the extracellular space. The main component of the transporter, xCT, is known to be strongly induced by various stimuli like oxidative stress in mammalian cultured cells. We examined the expression of xCT mRNA in vivo in the experimental endotoxemia.MethodsNorthern blot analysis and in situ hybridization were used to investigate the expression of xCT mRNA in the tissues of the mice exposed to bacterial lipopolysaccharide (LPS).ResultsNorthern blot analysis revealed that xCT mRNA was constitutively expressed in the brain, thymus, and spleen, and that the expression of xCT mRNA was strongly up-regulated in thymus and spleen by the administration of a sublethal dose of LPS. In addition to brain, thymus, and spleen, xCT mRNA was detected also in the bronchiolar epithelium of the lung by the administration of the lethal dose of LPS.ConclusionxCT is induced in some specific tissues by the administration of LPS. The results suggest that cystine/glutamate transporter plays an important role under the inflammatory conditions.

Expression of the activity of cystine/glutamate exchange transporter, system x(c)(-), by xCT and rBAT.

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.

CABS1 Is a Novel Calcium-Binding Protein Specifically Expressed in Elongate Spermatids of Mice

Abstract Single intraperitoneal injection of busulfan at 20 mg/kg body weight to mature male mice induced the deletion of the spermatogenic cells, followed by the restoration of the spermatogenesis by the surviving undifferentiated spermatogonia. The changes of the protein contents in testis during these processes were analyzed by two-dimensional gel electrophoresis in order to identify the proteins expressed at the specific stages of spermatogenesis. An acidic protein that disappeared and recovered in the same time course as spermatids after the busulfan treatment was identified as CABS1 by mass spectrometry. It was found that CABS1 was specifically expressed in the elongate spermatids at steps 13 to 16 in stages I to VIII of the seminiferous epithelium cycle of the mouse, and then it localized to the principal piece of flagellum of the mature sperm in the cauda epididymis. We have found for the first time that CABS1 is a calcium-binding protein that binds calcium during the maturation in the epididymis.