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.     

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.     

At the acidic edge: emerging functions for lysosomal membrane proteins

It has recently become clear that lysosomes have more complex functions than simply being the end-point on a degradative pathway. Similarly, it is now emerging that there are interesting functions for the limiting membranes around these organelles and their associated proteins. Although it has been known for several decades that the lysosomal membrane contains several highly N-glycosylated proteins, including the lysosome-associated membrane proteins LAMP-1 and LAMP-2 and lysosomal integral membrane protein-2/lysosomal membrane glycoprotein-85 (LIMP-2/LGP85), specific functions of these proteins have only recently begun to be recognized. Although the normal functions of LAMP-1 can be substituted by the structurally related LAMP-2, LAMP-2 itself has more specific tasks. Knockout of LAMP-2 in mice has revealed roles for LAMP-2 in lysosomal enzyme targeting, autophagy and lysosomal biogenesis. LAMP-2 deficiency in humans leads to Danon disease, a fatal cardiomyopathy and myopathy. Furthermore, there is evidence that LAMP-2 functions in chaperone-mediated autophagy. LIMP-2/LGP85 also seems to have specific functions in maintaining endosomal transport and lysosomal biogenesis. The pivotal function of lysosomal membrane proteins is also highlighted by the recent identification of disease-causing mutations in cystine and sialic acid transporter proteins, leading to nephropathic cystinosis and Salla disease.     

Regulation of lamp2a levels in the lysosomal membrane

The selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy depends, at least in part, on the levels of a substrate receptor at the lysosomal membrane. We have previously identified this receptor as the lysosome-associated membrane protein type 2a (lamp2a) and showed that levels of lamp2a at the lysosomal membrane directly correlate with the activity of the proteolytic pathway. Here we show that levels of lamp2a at the lysosomal membrane are mainly controlled by changes in its half-life and its distribution between the lysosomal membrane and the matrix. The lysosomal degradation of lamp2a requires the combined action of at least two different proteolytic activities at the lysosomal membrane. Lamp2a is released from the membrane by the action of these proteases, and then the truncated lamp2a is rapidly degraded within the lysosomal matrix. Membrane degradation of lamp2a is a regulated process that is inhibited in the presence of substrates for chaperone-mediated autophagy and under conditions that activate that type of autophagy. Uptake of substrate proteins also results in transport of some intact lamp2a from the lysosomal membrane into the matrix. This fraction of lamp2a can be reinserted back into the lysosomal membrane. The traffic of lamp2a through the lysosomal matrix is not mediated by vesicles, and lamp2a reinsertion requires the lysosomal membrane potential and protein components of the lysosomal membrane. The distribution of lamp2a between the lysosomal membrane and matrix is a dynamic process that contributes to the regulation of lysosomal membrane levels of lamp2a and consequently to the activity of the chaperone-mediated autophagic pathway.     

Loss of normal allele of the APC gene in an adrenocortical carcinoma from a patient with familial adenomatous polyposis

Summary Salla disease is a lysosomal storage disorder due to impaired transport of free sialic acid across the lysosomal membrane. The clinical presentation of this autosomal recessive trait is severe psychomotor retardation from early infancy on. In order to determine the gene locus for the disease we have initiated a genetic linkage study using polymorphic gene markers in rep-resentative family material comprising about 60% of all families known to be affected with Salla disease. Here we present an exclusion map based on combined linkage data from 64 informative loci on 19 autosomes. Theoretically, at least 55% of the genome has been excluded as a locus for the disease gene, while some chromosome areas, particularly the long arm of chromosome 2, are highlighted as possible sites for the gene locus.     

Mechanisms of chaperone-mediated autophagy

Chaperone-mediated autophagy is one of several lysosomal pathways of proteolysis. This pathway is activated by physiological stresses such as prolonged starvation. Cytosolic proteins with particular peptide sequence motifs are recognized by a complex of molecular chaperones and delivered to lysosomes. No vesicular traffic is required for this protein degradation pathway, so it differs from microautophagy and macroautophagy. Protein substrates bind to a receptor in the lysosomal membrane, the lysosome-associated membrane protein (lamp) type 2a. Levels of lamp2a in the lysosomal membrane are controlled by alterations in the lamp2a half-life as well as by the dynamic distribution of the protein between the lysosomal membrane and the lumen. Substrate proteins are unfolded before transport into the lysosome lumen, and the transport of substrate proteins requires a molecular chaperone within the lysosomal lumen. The exact roles of this lysosomal chaperone remain to be defined. The mechanisms of chaperone-mediated autophagy are similar to mechanisms of protein import into mitochondria, chloroplasts, and the endoplasmic reticulum.     

Studies of lysosomal sialic acid metabolism: retention of sialic acid by Salla disease lysosomes

Purified rat liver lysosomes were incubated in 0.2 M sialic acid resulting in an increase in lysosomal free sialic acid of 3.8 +/- 1.5 nmol/unit beta hexosaminidase. Sialic acid loss by these lysosomes was stimulated 2-3 fold by 25 mM sodium phosphate. Loss of sialic acid by lysosomes from cultured human diploid fibroblasts was similar to that observed in rat liver lysosomes while loss of sialic acid by lysosomes from cultured fibroblasts from a patient with infantile Salla disease occurred much more slowly. Salla disease appears to be the consequence of defective lysosomal transport of sialic acid and is analogous to cystinosis, a disorder of lysosomal amino acid transport.     

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.     

The chaperone-mediated autophagy receptor organizes in dynamic protein complexes at the lysosomal membrane

Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane.     

Synthesis of rat-liver lactate dehydrogenase and characterization of its mRNA

Salla disease is a lysosomal storage disorder of unknown etiology, characterized biochemically by increased urinary excretion of N-acetylneuraminic acid. This compound has now been shown to occur in abnormally large amounts in liver and cultured skin fibroblasts from these patients. Quantification of N-acetylneuraminic acid was performed using a new gas-chromatography/mass spectrometric single-ion method which is sensitive and specific. No abnormalities in the activity of several enzymes involved in sialic acid metabolism (N-acetylneuraminate:pyruvate lyase, neuraminidase, CMP-N-acetylneuraminate N-acylneuraminohydrolase and CTP:N-acyl-neuraminate cytidylyltransferase) were demonstrable. A possible explanation for the defect is a malfunctioning active transport of N-acetylneuraminic acid across the lysosomal membrane.     

Expression analysis and chromosomal assignment of PRA1 and RILP genes

PRA1 (prenylated Rab acceptor) is a general regulator of Rab proteins, while RILP (Rab interacting lysosomal protein) is a specific effector for Rab7. It has been shown that PRA1 interacts with Rab proteins and with VAMP2. Therefore PRA1 is probably an important factor for membrane traffic, linking together the function of Rab proteins and SNAREs. RILP has a key role in the control of transport to degradative compartments together with Rab7 and probably links Rab7 function to the cytoskeleton. Here we have studied by Northern blot the expression of the two genes in several different human tissues. The 0.8-kb mRNA for human PRA1 is ubiquitously expressed, while the two mRNAs for RILP are differentially expressed. In addition, we have assigned the human PRA1 gene to chromosome 19q13.13-q13.2 and the human RILP gene to chromosome 17p13.3.     

Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor

Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of unknown physiological function. We now demonstrate that this protease activity triggers the degradation of the lysosome-associated membrane protein type 2a (lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of lamp2a is important because its level in the lysosomal membrane is a rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a degradation, higher levels of lamp2a and higher rates of CMA. Restoration of PPCA protease activity increases rates of lamp2a degradation, reduces levels of lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the lysosomal membrane and cleaves lamp2a near the boundary between the luminal and transmembrane domains. In addition to the well-studied role of PPCA in targeting and protecting two lysosomal glycosidases, we have defined a role for the proteolytic activity of this multifunctional protein.     

The genetic locus for free sialic acid storage disease maps to the long arm of chromosome 6.

Salla disease (SD), or adult-type free sialic acid storage disease, is an autosomal recessive lysosomal storage disorder characterized by impaired transport of free sialic acid across the lysosomal membrane and severe psychomotor retardation. Random linkage analysis of a sample of 27 Finnish families allowed us to localize the SD locus to the long arm of chromosome 6. The highest lod score of 8.95 was obtained with a microsatellite marker of locus D6S286 at theta = .00. Evidence for linkage disequilibrium was observed between the SD locus and the alleles of three closely linked markers, suggesting that the length of the critical region for the SD locus is in the order of 190 kb.     

Chromosomal mapping of lysosomal enzyme structural genes in the domestic cat

A panel of 42 rodent x cat somatic cell hybrids has been used to assign seven structural genes for lysosomal enzymes to specific chromosomes in the domestic cat. The assignments include alpha-glucosidase (GANAB) to chromosome D1, alpha-galactosidase (GLA) to the X chromosome, beta-galactosidase 1 (GLB1) to chromosome B3, beta-glucuronidase (GUSB) to chromosome E3, alpha-mannosidase A (MANA) to chromosome B3, alpha-L-fucosidase (FUCA) to chromosome C1, and hexosaminidase A (HEXA) to chromosome B3. In all cases, the feline lysosomal enzyme genes were located in linkage groups which were syntenic with their homologous positions in the human gene map. These assignments expand the genetic map of the cat and reaffirm the extensive syntenic homology between the chromosome maps of man and cat.     

IκB Is a Substrate for a Selective Pathway of Lysosomal Proteolysis

In lysosomes isolated from rat liver and spleen, a percentage of the intracellular inhibitor of the nuclear factor κ B (IκB) can be detected in the lysosomal matrix where it is rapidly degraded. Levels of IκB are significantly higher in a lysosomal subpopulation that is active in the direct uptake of specific cytosolic proteins. IκB is directly transported into isolated lysosomes in a process that requires binding of IκB to the heat shock protein of 73 kDa (hsc73), the cytosolic molecular chaperone involved in this pathway, and to the lysosomal glycoprotein of 96 kDa (lgp96), the receptor protein in the lysosomal membrane. Other substrates for this degradation pathway competitively inhibit IκB uptake by lysosomes. Ubiquitination and phosphorylation of IκB are not required for its targeting to lysosomes. The lysosomal degradation of IκB is activated under conditions of nutrient deprivation. Thus, the half-life of a long-lived pool of IκB is 4.4 d in serum-supplemented Chinese hamster ovary cells but only 0.9 d in serum-deprived Chinese hamster ovary cells. This increase in IκB degradation can be completely blocked by lysosomal inhibitors. In Chinese hamster ovary cells exhibiting an increased activity of the hsc73-mediated lysosomal degradation pathway due to overexpression of lamp2, the human form of lgp96, the degradation of IκB is increased. There are both short- and long-lived pools of IκB, and it is the long-lived pool that is subjected to the selective lysosomal degradation pathway. In the presence of antioxidants, the half-life of the long-lived pool of IκB is significantly increased. Thus, the production of intracellular reactive oxygen species during serum starvation may be one of the mechanisms mediating IκB degradation in lysosomes. This selective pathway of lysosomal degradation of IκB is physiologically important since prolonged serum deprivation results in an increase in the nuclear activity of nuclear factor κ B. In addition, the response of nuclear factor κ B to several stimuli increases when this lysosomal pathway of proteolysis is activated.     

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.     

Defective lysosomal egress of free sialic acid (N-acetylneuraminic acid) in fibroblasts of patients with infantile free sialic acid storage disease

Egress of free NeuAc from normal lysosome-rich granular fractions was assessed at NeuAc concentrations of up to 221 pmol/hexosaminidase unit, achieved by exposure of growing fibroblasts to 40-125 nM N-acetylmannosamine for up to 7 days. The normal velocity of NeuAc egress increased with NeuAc loading and with temperature, exhibiting a Q10 of 2.4, characteristic of carrier-mediated transport. Fibroblasts cultured from five patients with infantile free sialic acid storage disease (ISSD) contained approximately 139 nmol of free NeuAc/mg of whole cell protein, or 100 times the normal level. Differential centrifugation, as well as density gradient analysis using 25% Percoll, showed that the stored NeuAc cosedimented with the lysosomal enzyme beta-hexosaminidase. The velocity of appearance of free NeuAc outside ISSD granular fractions was negligible, even at initial loading levels of up to 3500 pmol/hexosaminidase unit. The lack of egress from ISSD granular fractions was found for both endogenous and N-acetylmannosamine-derived NeuAc. Fibroblasts from ISSD parents did not accumulate excess free NeuAc and did not display a velocity of NeuAc egress significantly different from normal. The defect in ISSD, like that in Salla disease, appears to be an impairment of carrier-mediated transport of free NeuAc across the lysosomal membrane. Clinical and biochemical differences between Salla disease and ISSD may reflect differences in the amount of residual NeuAc transport capacity.     

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.