C.A.R. Boyd

Surface antigen CD98(4F2): not a single membrane protein, but a family of proteins with multiple functions.

CD98(4F2) belongs to a group of approximately two hundred proteins at the surface of leukocytes that are thought to be important for their function in the immune response, by specifying their migration, cell interactions and the signal transduction mechanisms that lead to lymphocyte activation. Following the discovery of CD98(4F2) as a surface antigen in lymphocytes (Haynes et al., 1981), the antigen was found to be present in all established human tissue culture cell lines tested and in most malignant human cells (Hemler & Strominger, 1982; Quackenbush et al., 1987). Although it was linked to cell proliferation and growth (Yagita et al., 1986, Gottesdiener et al., 1988) it essentially remained a protein in search of a function. Only recently, have reports linked CD98(4F2) to a number of specific cellular processes such as adhesion, fusion and amino acid transport across the cell membrane in different cell types. How these relate to its role in cell activation and proliferation, has not yet been established. The complete molecular structure of CD98(4F2) has been resolved during the last year. Early biochemical studies showed that the 4F2 cell surface antigen (which was later designated CD98; Barclay et al., 1997) was a 120-kDa disulfide-linked heterodimer composed of two subunits, a glycosylated 80 kDa heavy chain and a 40 kDa light chain (Haynes et al., 1981); the 4F2 antibody recognized an antigenic determinant on the polypeptide backbone of the heavy chain. Later, the heavy chain was described as a type II glycoprotein of 529 amino acids (Quackenbush et al., 1987), but the nature of the associated light chain remained unknown. Recently it has been shown that “the” light chain is not a single protein, but a family of homologous proteins with similar molecular weight and membrane topology (Fig. 1). The light subunits are able to associate to the heavy chain through a disulfide bond, giving rise to different heterodimers (Mastroberardino et al., 1998); they have multiple transmembrane-spanning domains and their amino acid sequences have been shown to conform to the APC superfamily (transporters for amino acids, polyamines and choline). Consistently, the heterodimers have been shown to function as amino acid transporters. In the context of this review, CD98 will be used to refer generically to the heterodimers (also known as 4F2) and the heavy chain and the light chains will be referred to as CD98hc or CD98lc, respectively. In those cases where information regarding the molecular nature of the light chain is available, the corresponding molecular form will be indicated following the denomination adopted by Kanai and collaborators (Kanai et al., 1998; Segawa et al., 1999) and Palacı ́n and collaborators (Torrents et al., 1998; Pineda et al., 1999). Although work in this area has never been more active, making this review clearly one that will quickly be overtaken by ongoing and future work, it is the very broad nature of the CD98 field, with overlap of immunology, membrane physiology, cell biology and cancer biology, that makes a current review of potential value. Correspondence to: R. Devés

Structure and function of eukaryotic peptide transporters.

Abstract. The cotransport of protons and peptides is now recognised as a major route by which dietary nitrogen is absorbed from the intestine, and filtered protein reabsorbed in the kidney. Recently, molecular biology has had a very substantial impact on the study of peptide transport, and here we review the molecular and functional information available within the framework of physiology. To this end we consider not only the mammalian peptide transporters and their tissue distribution and regulation but also those from other species (including Caenorhabditis elegans) which make up the proton-dependent oligopeptide transport superfamily. In addition, understanding the binding requirements for transported substrates may allow future design and targeted tissue delivery of peptide and peptidomimetic drugs. Finally, we aim to highlight some of the less well understood areas of peptide transport, in the hope that it will stimulate further research into this challenging yet exciting topic.

Human placental indoleamine 2,3-dioxygenase: cellular localization and characterization of an enzyme preventing fetal rejection

In order to test the hypothesis (Munn, Zhou, Attwood, Bondarev, Conway, Marshall, Brown, Mellor, Science 281 (1998) 1191-1193) that localized placental tryptophan catabolism prevents immune rejection of the mammalian fetus, the cellular localization and characteristics of human placental indoleamine 2,3-dioxygenase (EC 1.13.11.42) were studied. The localization of indoleamine 2, 3-dioxygenase activity was determined quantitatively using cell fractionation by differential and discontinuous sucrose gradient centrifugation. Enzyme activity was looked for in isolated brush border microvillous plasma membranes of placental syncytiotrophoblast. We found that this membrane preparation (which showed a 32.4-fold purification from the starting homogenate with reference to the activity of a membrane marker enzyme, alkaline phosphatase (EC 3.1.3.1)) was strongly negatively enriched with indoleamine 2,3-dioxygenase (which showed a one twenty-fifth decrease in its specific activity). Placental indoleamine 2, 3-dioxygenase is thus not expressed in the maternal facing brush border membrane of syncytiotrophoblast. 1-Methyl-DL-tryptophan which was used by Munn et al. as a key experimental tool for inhibiting indoleamine 2,3-dioxygenase in the murine model showed a competitive inhibition of human placental indoleamine 2,3-dioxygenase with L-tryptophan. The hypothesis, based on experiments performed in mouse, may therefore be applicable to avoidance of immune rejection of the fetus in human pregnancy.

Hypoxia alters expression and function of syncytin and its receptor during trophoblast cell fusion of human placental BeWo cells: implications for impaired trophoblast syncytialisation in pre-eclampsia.

The fundamental process of placental trophoblast cell fusion (syncytiotrophoblast formation or syncytialisation) which is a characteristic of this tissue is poorly understood. Pre-eclampsia is associated with placental hypoxia and suppressed syncytiotrophoblast formation. We therefore have studied the effect of low-oxygen tensions on the rate of cell fusion, relative abundance of mRNAs encoding syncytin and its receptor, amino acid transport system B(0), which are thought to be involved in trophoblast cell fusion (as well as the activity of amino acid transport through this system) in a cell model of syncytialisation (BeWo cells following forskolin treatment). Forskolin-induced cell fusion (determined by a quantitative flow cytometry assay) was reversibly suppressed in 2% oxygen compared to 20% oxygen. This was associated with suppressed secretion of human chorionic gonadotropin. Forskolin stimulated relatively less syncytin mRNA (determined by reverse transcription-polymerase chain reaction) in 2% than in 20% oxygen and there was no stimulation after 48 h in 2% oxygen. There was a spontaneous, time-dependent increase of amino acid transporter B(0) mRNA in vehicle, which was suppressed by 2% oxygen and by forskolin treatment in 20% oxygen. Forskolin-induced changes in amino acid transport system B(0) function were not seen in cells cultured for 48 h in 2% oxygen. These observations suggest that under conditions of low ambient oxygen, dysregulation of expression of syncytin and of its receptor may suppress the normal process of cell fusion necessary for syncytiotrophoblast formation and contributes to syncytiotrophoblast abnormalities characteristic of pre-eclampsia.

Characterisation of L‐tryptophan transporters in human placenta: a comparison of brush border and basal membrane vesicles

1 The mechanisms responsible for l‐tryptophan transport at both the maternal‐ and fetal‐facing surfaces of the term placenta have been determined in isolated membrane vesicles as part of a study on placental indoleamine 2,3‐dioxygenase, the l‐tryptophan‐catabolising enzyme recently shown to regulate feto‐maternal immunology. 2 Brush border vesicle uptake of l‐tryptophan is substantially into an osmotically active space. It is sodium independent and N‐ethylmaleimide sensitive. Uptake of l‐tryptophan, which is markedly stereospecific, has a Km of 26.3 μm and Vmax of 1.72 pmol (mg protein)−1 s−1 and is completely abolished by the L‐system‐specific substrate 2‐aminobicyclo‐(2,2,1)‐heptane‐2‐carboxylic acid (BCH). These findings are in keeping with l‐tryptophan transport being exclusively via system L (induced by the heterodimeric heavy chain of CD98 and system L‐amino acid transporter‐1 (LAT‐1)). 1‐Methyl‐tryptophan (which is a known competitive inhibitor of indoleamine 2,3‐dioxygenase) is a competitive inhibitor of l‐tryptophan flux through this transport system (Ki= 113μm). 3 Basal membrane transport of l‐tryptophan is more complex. Uptake is slower than at the brush border and although, as in the brush border, uptake is sodium independent, it is less sensitive to N‐ethylmaleimide. There is clear evidence that two systems contribute to basal membrane transport since BCH is (in sodium‐free media) only a partial inhibitor whereas l‐histidine and l‐cysteine are fully effective. The simplest explanation of these and other findings is that the basal membrane possesses two systems, one of which is similar to that induced by the heavy chain of CD98 and system L‐amino acid transporter‐2 (LAT‐2). The other appears to be system y+L since in the presence of BCH inhibition by l‐leucine but not by l‐lysine is sodium dependent. 4 These findings suggest the existence of non‐identical carrier‐mediated transport systems for l‐tryptophan in brush border and basal membranes. This asymmetry may explain net transplacental transfer of this amino acid.

Peptide Mimics as Substrates for the Intestinal Peptide Transporter

4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated. 4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAAtrans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and inXenopus oocytes expressing PepT1, 4-APAA producedtrans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter. These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.

Tryptophan degradation by human placental indoleamine 2,3‐dioxygenase regulates lymphocyte proliferation

1 The physiological importance of human placental indoleamine 2,3‐dioxygenase (EC 1.13.11.42), the first and rate‐limiting enzyme in tryptophan metabolism, in regulating feto‐maternal immunology has been studied. 2 Concentrations were measured in placental villous explant conditioned media of 14 amino acids that are known to be required for lymphocyte proliferation. In the absence of interferon‐γ only tryptophan and threonine were significantly lowered; in the presence of interferon‐γ (known to stimulate indoleamine 2,3‐dioxygenase) tryptophan but not threonine depletion was much greater. 3 Peripheral blood mononuclear cell proliferation determined by measuring thymidine incorporation into DNA following culture in the medium previously conditioned by culture of villous explants was markedly reduced when placental indoleamine 2,3‐dioxygenase was stimulated with interferon‐γ. Inhibition of placental indoleamine 2,3‐dioxygenase by 1‐methyl‐tryptophan prevented inhibition of thymidine incorporation. Supplementation of the conditioned medium with tryptophan but no other amino acid completely reversed the inhibition of thymidine incorporation. 4 Flow cytometric analysis showed that CD4‐positive T lymphocyte division was specifically suppressed by indoleamine 2,3‐dioxygenase‐mediated tryptophan depletion. This inhibition of T cell proliferation was due to arrest of cell cycle progression. 5 To study the mechanism of tryptophan sensing we examined the ability of 11 L‐tryptophan analogues to support lymphocyte proliferation. Only L‐tryptophan methyl and ethyl esters were able to stimulate proliferation in tryptophan‐free media. Since both of these molecules are readily degraded to tryptophan by intracellular esterases this suggests that the tryptophan sensor is intracellular. 6 Our results show that mechanisms are present in the human placenta which are able to regulate cellular proliferation of the maternal immune system. This mechanism is dependent both on placental indoleamine 2,3‐dioxygenase‐mediated tryptophan degradation and on tryptophan sensing systems within lymphocytes.

A novel role for carbonic anhydrase: cytoplasmic pH gradient dissipation in mouse small intestinal enterocytes

1 The spatial and temporal distribution of intracellular H+ ions in response to activation of a proton‐coupled dipeptide transporter localized at the apical pole of mouse small intestinal isolated enterocytes was investigated using intracellular carboxy‐SNARF‐1 fluorescence in combination with whole‐cell microspectrofluorimetry or confocal microscopy. 2 In Hepes‐buffered Tyrode solution, application of the dipeptide Phe‐Ala (10 mM) to a single enterocyte reduced pHi locally in the apical submembranous space. After a short delay (8 s), a fall of pHi occurred more slowly at the basal pole. 3 In the presence of CO2/HCO3−‐buffered Tyrode solution, the apical and basal rates of acidification were not significantly different and the time delay was reduced to 1 s or less. 4 Following application of the carbonic anhydrase inhibitor acetazolamide (100 μM) in the presence of CO2/HCO3− buffer, addition of Phe‐Ala once again produced a localized apical acidification that took 5 s to reach the basal pole. Basal acidification was slower than at the apical pole. 5 We conclude that acid influx due to proton‐coupled dipeptide transport can lead to intracellular pH gradients and that intracellular carbonic anhydrase activity, by facilitating cytoplasmic H+ mobility, limits their magnitude and duration.

Membrane potential dependence of the kinetics of cationic amino acid transport systems in human placenta.

1. Mediated influx of L‐lysine into human placental brush‐border membrane vesicles occurs through two systems, one of lower affinity but high capacity, the other of very high affinity but low capacity. These transporters have features characteristic of systems y+ (the classical system) and y+L (recently described in the erythrocyte), respectively. 2. In solutions containing sodium the entry of lysine through the high‐affinity system (y+L) is inhibited by the neutral amino acids L‐leucine, L‐methionine and L‐glutamine with comparable high affinity. The removal of sodium reduces the affinity but not the maximal extent of this inhibition. Leucine and methionine, but apparently not glutamine, inhibit lysine entry through system y+ with a much lower affinity. 3. The influx of lysine through system y+ changes markedly in response to alterations of membrane potential. In the presence of an inwardly directed negative diffusion potential created by an inwardly directed thiocyanate (SCN‐) gradient, the influx of lysine through this route is accelerated; with an inwardly directed positive potassium diffusion potential, lysine influx through this route is reduced. The influx of lysine through system y+L is much less sensitive to such alterations of potential. 4. Analysis of the kinetic constants characterizing system y+ shows that with a change of potential from zero to negative (approximately ‐60 mV) the maximum velocity (Vmax) is roughly doubled and the half‐saturation constant (Km) halved leading to a 4‐fold increase in permeability. For system y+L smaller changes are seen and Km does not change; the resulting increase in y+L permeability is 1.5‐fold. 5. These findings are discussed with respect both to the mechanism of membrane transport and placental epithelial function.

The role of l-tryptophan transport in l-tryptophan degradation by indoleamine 2,3-dioxygenase in human placental explants

1 The physiological importance of l‐tryptophan transport for placental indoleamine 2,3‐dioxygenase‐mediated degradation of l‐tryptophan has been studied using human placental chorionic villous explants. 2 l‐Tryptophan influx into villous explants is supported exclusively by transport system L and is substantially inhibited by the L‐system‐specific substrate 2‐aminobicyclo‐(2,2,1)‐heptane‐2‐carboxylic acid (BCH) and also by 1‐methyl‐tryptophan which is also an inhibitor of indoleamine 2,3‐dioxygenase. l‐Tryptophan influx is enhanced 2.3‐fold following in vitro culture of the villous explant. Interferon‐γ, which increases villous explant indoleamine 2,3‐dioxygenase expression, has no effect on l‐tryptophan influx. 3 In explants both BCH and 1‐methyl‐tryptophan inhibit indoleamine 2,3‐dioxygenase‐mediated l‐tryptophan degradation. This also applies when l‐tryptophan degradation has been stimulated by interferon‐γ. 4 These findings show transport of l‐tryptophan into the trophoblast to be a rate‐limiting step for indoleamine 2,3‐dioxygenase‐mediated l‐tryptophan degradation and therefore for the normal physiology of mammalian pregnancy.