Structure-Function Studies of the SLC17 Transporter Sialin Identify Crucial Residues and Substrate-induced Conformational Changes

Salla disease and infantile sialic acid storage disorder are human diseases caused by loss of function of sialin, a lysosomal transporter that mediates H⁺-coupled symport of acidic sugars N-acetylneuraminic acid and glucuronic acid out of lysosomes. Along with the closely related vesicular glutamate transporters, sialin belongs to the SLC17 transporter family. Despite their critical role in health and disease, these proteins remain poorly understood both structurally and mechanistically. Here, we use substituted cysteine accessibility screening and radiotracer flux assays to evaluate experimentally a computationally generated three-dimensional structure model of sialin. According to this model, sialin consists of 12 transmembrane helices (TMs) with an overall architecture similar to that of the distantly related glycerol 3-phosphate transporter GlpT. We show that TM4 in sialin lines a large aqueous cavity that forms a part of the substrate permeation pathway and demonstrate substrate-induced alterations in accessibility of substituted cysteine residues in TM4. In addition, we demonstrate that one mutant, F179C, has a dramatically different effect on the apparent affinity and transport rate for N-acetylneuraminic acid and glucuronic acid, suggesting that it may be directly involved in substrate recognition and/or translocation. These findings offer a basis for further defining the transport mechanism of sialin and other SLC17 family members.     

Functional characterization of vesicular excitatory amino acid transport by human sialin

Sialin, the protein coded by SLC17A5, is responsible for membrane potential (Δψ)-driven aspartate and glutamate transport into synaptic vesicles in addition to H+/sialic acid co-transport in lysosomes. Rodent sialin mutants harboring the mutations associated with Salla disease in humans did not transport aspartate and glutamate whereas H+/sialic acid co-transport activity was about one-third of the wild-type protein. In this study, we investigate the effects of various mutations on the transport activities of human sialin. Proteoliposomes containing purified heterologously expressed human sialin exhibited both Δψ-driven aspartate and glutamate transport activity and H+/sialic acid co-transport activity. Aspartate and glutamate transport was not detected in the R39C and K136E mutant forms of SLC17A5 protein associated with Salla disease, whereas H+/sialic acid co-transport activity corresponded to 30-50% of the recombinant wild-type protein. In contrast, SLC17A5 protein harboring the mutations associated with infantile sialic acid storage disease, H183R and Δ268SSLRN272 still showed normal levels of Δψ-driven aspartate and glutamate transport even though H+/sialic acid co-transport activity was absent. Human sialin carrying the G328E mutation that causes both phenotypes, and P334R and G378V mutations that cause infantile sialic acid storage disease showed no transport activity. These results support the idea that people suffering from Salla disease have been defective in aspartergic and glutamatergic neurotransmissions.     

Diversity of function and mechanism in a family of organic anion transporters

Originally identified as transporters for inorganic phosphate, solute carrier 17 (SLC17) family proteins subserve diverse physiological roles. The vesicular glutamate transporters (VGLUTs) package the principal excitatory neurotransmitter glutamate into synaptic vesicles (SVs). In contrast, the closely related sialic acid transporter sialin mediates the flux of sialic acid in the opposite direction, from lysosomes to the cytoplasm. The two proteins couple in different ways to the H⁺ electrochemical gradient driving force, and high-resolution structures of the Escherichia coli homolog d-galactonate transporter (DgoT) and more recently rat VGLUT2 now begin to suggest the mechanisms involved as well as the basis for substrate specificity.     

Functional characterization of wild-type and mutant human sialin

The modification of cell surface lipids or proteins with sialic acid is essential for many biological processes and several diseases are caused by defective sialic acid metabolism. Sialic acids cleaved off from degraded sialoglycoconjugates are exported from lysosomes by a membrane transporter, named sialin, which is defective in two allelic inherited diseases: infantile sialic acid storage disease (ISSD) and Salla disease. To develop a functional assay of human sialin, we redirected the protein to the plasma membrane by mutating a dileucine-based internalization motif. Cells expressing the plasmalemmal construct accumulated neuraminic acid at acidic pH by a process equivalent to lysosomal efflux. The assay was used to determine how pathogenic mutations affect transport. Interestingly, while two missense mutations and one small, in-frame deletion associated with ISSD abolished transport, the mutation causing Salla disease (R39C) slowed down, but did not stop, the transport cycle, thus explaining why the latter disorder is less severe. Since neurological symptoms predominate in Salla disease, our results suggest that sialin is rate-limiting to specific sialic acid-dependent processes of the nervous system.     

Varied mechanisms underlie the free sialic acid storage disorders

Salla disease and infantile sialic acid storage disorder are autosomal recessive neurodegenerative diseases characterized by loss of a lysosomal sialic acid transport activity and the resultant accumulation of free sialic acid in lysosomes. Genetic analysis of these diseases has identified several unique mutations in a single gene encoding a protein designated sialin (Verheijen, F. W., Verbeek, E., Aula, N., Beerens, C. E., Havelaar, A. C., Joosse, M., Peltonen, L., Aula, P., Galjaard, H., van der Spek, P. J., and Mancini, G. M. (1999) Nat. Genet. 23, 462-465; Aula, N., Salomaki, P., Timonen, R., Verheijen, F., Mancini, G., Mansson, J. E., Aula, P., and Peltonen, L. (2000) Am. J. Hum. Genet. 67, 832-840). From the biochemical phenotype of the diseases and the predicted polytopic structure of the protein, it has been suggested that sialin functions as a lysosomal sialic acid transporter. Here we directly demonstrate that this activity is mediated by sialin and that the recombinant protein has functional characteristics similar to the native lysosomal sialic acid transport system. Furthermore, we describe the effect of disease-causing mutations on the protein. We find that the majority of the mutations are associated with a complete loss of activity, while the mutations associated with the milder forms of the disease lead to reduced, but residual, function. Thus, there is a direct correlation between sialin function and the disease state. In addition, we find with one mutation that the protein is retained in the endoplasmic reticulum, indicating that altered trafficking of sialin is also associated with disease. This analysis of the molecular mechanism of sialic acid storage disorders is a further step in identifying therapeutic approaches to these diseases.     

Molecular pathogenesis of sialic acid storage diseases: insight gained from four missense mutations and a putative polymorphism of human sialin

Background information. Free sialic acid storage diseases are caused by mutations of a lysosomal sialic acid transporter called sialin. We showed recently that the milder clinical form, Salla disease, and a related non‐Finish case, are characterized by residual transport, whereas sialin mutants found in lethal infantile cases are inactive. In the present study, we have characterized the molecular effects of a putative polymorphism (M316I) and of four pathogenic mutations associated with either infantile (G127E and R57C) or Salla‐like (G409E) phenotypes, or both (G328E). The transport activity of human sialin was analysed using a novel assay that was based on a construct without the functional lysosomal sorting motif, which is expressed at the plasma membrane. Results. The lysosomal localization of human sialin was not (M316I and G328E) or only partially (R57C, G127E and G409E) affected by the missense mutations. In contrast, all pathogenic mutations abolished transport, whereas the putative M316I polymorphism induced an approx. 5‐fold decrease of sialic acid transport. Conclusions. The molecular effects of the R57C and G127E mutations strengthen the conclusion that the infantile phenotype is caused by loss‐of‐function mutations. On the other hand, the milder severity of the heterozygous G409E patient may reflect an incomplete expression of the splicing mutation present on the second allele. In the case of the G328E mutation, found in the homozygous state in a clinically heterogeneous family, the apparent severity of the transport phenotype suggests that the genetic or environmental factors underlying this clinical heterogeneity might be protective.     

Embryonic expression patterns of Drosophila ACS family genes related to the human sialin gene

The anion/cation symporter (ACS) family is a large subfamily of the major facilitator superfamily (MFS) of transporters. ACS family permeases are widely distributed in nature and transport either organic or inorganic anions in response to chemiosmotic cation gradients. The only protein in the ACS family to which a human disease has been linked, is sialin, the proton-driven lysosomal carrier for sialic acid. Genetic defects in sialin cause a lysosomal storage disease in humans. Here we have identified a group of conserved Drosophila ACS family genes related to sialin and studied their expression patterns throughout embryogenesis. Drosophila sialin-related genes are expressed in a wide variety of tissues. Expression is frequently observed throughout various parts of the intestinal tract, including Malpighian tubules and salivary glands. Additionally, some genes are expressed in vitellophages (yolk nuclei), nervous system, respiratory tract and a number of other embryonic tissues. These data will aid the establishment of a fruitfly model of human lysosomal storage disorders, the most common cause of neurodegeneration in childhood.     

溶质转运蛋白超家族的功能及结构研究进展

溶质转运蛋白(solute carriers,SLC)超家族是人类细胞膜(含胞内膜)上最重要的膜转运蛋白家族之一,它参与了细胞间的物质运输、能量传递、营养代谢、信号传导等重要生理活动。SLC转运蛋白超家族包含52个亚家族,共有400多名成员。研究表明,人类基因突变所致SLC蛋白表达异常或功能缺陷与糖尿病、高血压、抑郁症等多种重大疾病密切相关,使得该家族蛋白的功能研究近年来备受关注。SLC转运家族蛋白三维结构的解析有助于阐述其底物选择性结合与转运的精确分子机制,为研究该家族功能相关疾病的分子机理以及针对理性药物研发奠定了精细的三维结构基础。本文对近年来溶质转运蛋白超家族的结构及功能研究进展进行了总结,试图对该家族的共性规律进行阐述。     

Molecular basis of essential amino acid transport from studies of insect nutrient amino acid transporters of the SLC6 family (NAT-SLC6)

Two protein families that represent major components of essential amino acid transport in insects have been identified. They are annotated as the SLC6 and SLC7 families of transporters according to phylogenetic proximity to characterized amino acid transporters (HUGO nomenclature). Members of these families have been identified as important apical and basolateral parts of transepithelial essential amino acid absorption in the metazoan alimentary canal. Synergistically, they play critical physiological roles as essential substrate providers to diverse metabolic processes, including generic protein synthesis. This review briefly clarifies the requirements for amino acid transport and a variety of amino acid transport mechanisms, including the aforementioned families. Further it focuses on the large group of Nutrient Amino acid Transporters (NATs), which comprise a recently identified subfamily of the Neurotransmitter Sodium Symporter family (NSS or SLC6). The first insect NAT, cloned from the caterpillar gut, has a broad substrate spectrum similar to mammalian B(0) transporters. Several new NAT-SLC6 members have been characterized in an effort to explore mechanisms for the essential amino acid absorption in model dipteran insects. The identification and functional characterization of new B(0)-like and narrow specificity transporters of essential amino acids in fruit fly and mosquitoes leads to a fundamentally important insight: that NATs evolved and act together as the integrated active core of a transport network that mediates active alimentary absorption and systemic distribution of essential amino acids. This role of NATs is projected from the most primitive prokaryotes to the most complex metazoan organisms, and represents an interesting platform for unraveling the molecular evolution of amino acid transport and modeling amino acid transport disorders. The comparative study of NATs elucidates important adaptive differences between essential amino acid transportomes of invertebrate and vertebrate organisms, outlining a new possibility for selective targeting of essential amino acid absorption mechanisms to control medically and economically important arthropods and other invertebrate organisms.     

Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase

Paramyxoviruses are the main cause of respiratory disease in children. One of two viral surface glycoproteins, the hemagglutinin-neuraminidase (HN), has several functions in addition to being the major surface antigen that induces neutralizing antibodies. Here we present the crystal structures of Newcastle disease virus HN alone and in complex with either an inhibitor or with the beta-anomer of sialic acid. The inhibitor complex reveals a typical neuraminidase active site within a beta-propeller fold. Comparison of the structures of the two complexes reveal differences in the active site, suggesting that the catalytic site is activated by a conformational switch. This site may provide both sialic acid binding and hydrolysis functions since there is no evidence for a second sialic acid binding site in HN. Evidence for a single site with dual functions is examined and supported by mutagenesis studies. The structure provides the basis for the structure-based design of inhibitors for a range of paramyxovirus-induced diseases.     

The spectrum of SLC17A5-gene mutations resulting in free sialic acid-storage diseases indicates some genotype-phenotype correlation

Lysosomal free sialic acid-storage diseases include the allelic disorders Salla disease (SD) and infantile sialic acid-storage disease (ISSD). The defective gene, SLC17A5, coding for the lysosomal free sialic acid transporter was recently isolated by positional cloning. In the present study, we have identified a large number of mutations in SLC17A5 in patients presenting with either Salla disease or the ISSD phenotype. We also report for the first time the exon-intron boundaries of SLC17A5. All Finnish patients with SD (n=80) had a missense mutation changing a highly conserved arginine to cysteine (R39C); 91% of them were homozygotes for this old founder mutation. The compound-heterozygote patients, with the founder mutation in only one allele, presented with a more severe phenotype than did the homozygote patients. The same R39C mutation was also found both in most of the Swedish patients with SD and in a heterozygous form in five patients from central Europe who presented with an unusually severe (intermediate) SD phenotype. Ten different mutations, including deletions, insertions, and missense and nonsense mutations, were identified in patients with the most severe ISSD phenotype, most of whom were compound heterozygotes. Our results indicate some genotype-phenotype correlation in free sialic acid-storage diseases, suggesting that the phenotype associated with the homozygote R39C mutation is milder than that associated with other mutations.     

Sialic acid storage disease and related disorders

This paper gives an overview of the two sialic acid storage disorders, Salla disease and infantile sialic acid storage disease, and the related disorders cystinosis, sialuria, sialidosis, and galactosialidosis. Sialic acid storage disease and cystinosis are models for a deficient lysosomal transport of monosaccharides and amino acids, respectively. Several gene mutations leading to the production of the faulty membrane proteins sialin and cystinosin have been identified in recent years. Knowledge of the underlying pathophysiology is a prerequisite for future research projects, which will focus on the expression of the disease genes in living systems and the physical characterization of these proteins by X-ray crystallography and nuclear magnetic resonance spectroscopy.     

Diversity of microbial sialic acid metabolism.

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.     

Genome Wide Identification,Phylogeny and Expression of Zinc Transporter Genes in Common Carp

Background

Zinc is an essential trace element in organisms, which serves as a cofactor for hundreds of enzymes that are involved in many pivotal biological processes including growth, development, reproduction and immunity. Therefore, the homeostasis of zinc in the cell is fundamental. The zinc transporter gene family is a large gene family that encodes proteins which regulate the movement of zinc across cellular and intracellular membranes. However, studies on teleost zinc transporters are mainly limited to model species.

Methodology/Principal Findings

We identified a set of 37 zinc transporters in common carp genome, including 17 from SLC30 family (ZnT), and 20 from SLC39 family (ZIP). Phylogenetic and syntenic analysis revealed that most of the zinc transporters are highly conserved, though recent gene duplication and gene losses do exist. Through examining the copy number of zinc transporter genes across several vertebrate genomes, thirteen zinc transporters in common carp are found to have undergone the gene duplications, including SLC30A1, SLC30A2, SLC30A5, SLC30A7, SLC30A9, SLC30A10, SLC39A1, SLC39A3, SLC39A4, SLC39A5, SLC39A6, SLC39A7 and SLC39A9. The expression patterns of all zinc transporters were established in various tissues, including blood, brain, gill, heart, intestine, liver, muscle, skin, spleen and kidney, and showed that most of the zinc transporters were ubiquitously expressed, indicating the critical role of zinc transporters in common carp.

Conclusions

To some extent, examination of gene families with detailed phylogenetic or orthology analysis could verify the authenticity and accuracy of assembly and annotation of the recently published common carp whole genome sequences. The gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp zinc transporters provides an important genomic resource for future biochemical, toxicological and physiological studies of zinc in teleost.     

Bound to be different: neurotransmitter transporters meet their bacterial cousins

The neurotransmitter transporters belonging to the solute carrier 6 (SLC6) family, including the gamma-aminobutyric acid (GAT), norepinephrine (NET), serotonin (SERT) and dopamine (DAT) transporters are extremely important drug targets of great clinical relevance. These Na+, Cl(-)-dependent transporters primarily function following neurotransmission to reset neuronal signaling by transporting neurotransmitter out of the synapse and back into the pre-synaptic neuron. Recent studies have tracked down an elusive binding site for Cl(-) that facilitates neurotransmitter transport using structural differences evident with bacterial family members (e.g., the Aquifex aeolicus leucine transporter LeuT Aa) that lack Cl(-) dependence. Additionally, the crystal structures of antidepressant-bound LeuT Aa reveals a surprising mode of drug interaction that may have relevance for medication development. The study of sequence and structural divergence between LeuT Aa and human SLC6 family transporters can thus inform us as to how and why neurotransmitter transporters evolved a reliance on extracellular Cl(-) to propel the transport cycle; what residue changes and helical rearrangements give rise to recognition of different substrates; and how drugs such as antidepressants, cocaine, and amphetamines halt (or reverse) the transport process.     

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.     

The SLC6 orphans are forming a family of amino acid transporters

Transporters in the human genome are grouped in solute carrier families (SLC). The SLC6 family is one of the biggest transporter families in the human genome comprising 20 members. It is usually referred to as the neurotransmitter transporter family because its founding members encode transporters for the neurotransmitters GABA, noradrenaline, serotonin and dopamine. The family also includes a number of 'orphan' transporters, the function of which has remained elusive until recently. Identification of the broadly specific neutral amino acid transporter SLC6A19 (also called B(0)AT1) suggested that all orphan transporters may in fact be amino acid transporters. This was subsequently confirmed by the identification of SLC6A20 as the long-sought IMINO system, a proline transporter found in kidney, intestine and brain. Very recently, SLC6A15 was identified as the neutral amino acid transporter B(0)AT2. All amino acid transporters appear to cotransport only 1Na(+) together with the amino acid substrate. Both, B(0)AT1 and B(0)AT2 are chloride independent, whereas IMINO is chloride dependent. The amino acid transporters of the SLC6 family are functionally and sequence related to the recently crystallized leucine transporter from Aquifex aeolicus. The structure elegantly explains many of the mechanistic features of the SLC6 amino acid transporters.     

LPAR5, GNAT3 and partial amino acid transporters messenger RNA expression patterns in digestive tracts,metabolic organs and muscle tissues of growing goats

Sufficient amino acid (AA) transport is essential to ensure the normal physiological function and growth of growing animals. The processes of AA sensing and transport in humans and murine animals, but rarely in goats, have been arousing great interest recently. This study was conducted to investigate the messenger RNA expression patterns of lysophosphatidic acid receptor 5 (LPAR5), guanine nucleotide-binding protein α-transducing 3 (GNAT3) and important partial AA transporters in digestive tracts, metabolic organs and muscles of growing goats. The results showed that these genes were widely expressed in goats, and had different expression patterns. LPAR5, GNAT3, solute carrier (SLC38A2), SLC7A7, SLC7A1 and SLC3A1 were rarely expressed in the rumen, but were highly expressed in the abomasum and intestine which are the main sites of AA absorption. GNAT3, SLC38A1, SLC38A2, SLC6A19, SLC7A7 and SLC7A1 showed comparatively high expression in the pancreas and the vital digestive glands, and the relatively high expression of these nine genes were noted in the tibialis posterior, the active muscle in energy metabolism. The correlation analysis showed that there were certain positive correlation among most genes. The current results indicate that the AA sensing and transport occur extensively in the abomasum and small intestine, metabolic organs and muscle tissues of ruminants, and that related genes have tissue specificity.     

Diversity of Microbial Sialic Acid Metabolism

Sialic acids are structurally unique nine-carbon keto sugars occupying the interface between the host and commensal or pathogenic microorganisms. An important function of host sialic acid is to regulate innate immunity, and microbes have evolved various strategies for subverting this process by decorating their surfaces with sialylated oligosaccharides that mimic those of the host. These subversive strategies include a de novo synthetic pathway and at least two truncated pathways that depend on scavenging host-derived intermediates. A fourth strategy involves modification of sialidases so that instead of transferring sialic acid to water (hydrolysis), a second active site is created for binding alternative acceptors. Sialic acids also are excellent sources of carbon, nitrogen, energy, and precursors of cell wall biosynthesis. The catabolic strategies for exploiting host sialic acids as nutritional sources are as diverse as the biosynthetic mechanisms, including examples of horizontal gene transfer and multiple transport systems. Finally, as compounds coating the surfaces of virtually every vertebrate cell, sialic acids provide information about the host environment that, at least in Escherichia coli, is interpreted by the global regulator encoded by nanR. In addition to regulating the catabolism of sialic acids through the nan operon, NanR controls at least two other operons of unknown function and appears to participate in the regulation of type 1 fimbrial phase variation. Sialic acid is, therefore, a host molecule to be copied (molecular mimicry), eaten (nutrition), and interpreted (cell signaling) by diverse metabolic machinery in all major groups of mammalian pathogens and commensals.