Maria Francisca Coutinho

Mannose-6-phosphate pathway: A review on its role in lysosomal function and dysfunction.

Lysosomal hydrolases are synthesized in the rough endoplasmic reticulum and specifically transported through the Golgi apparatus to the trans-Golgi network, from which transport vesicles bud to deliver them to the endosomal/lysosomal compartment. The explanation of how are the lysosomal enzymes accurately recognized and selected over many other proteins in the trans-Golgi network relies on being tagged with a unique marker: the mannose-6-phosphate (M6P) group, which is added exclusively to the N-linked oligosaccharides of lysosomal soluble hydrolases, as they pass through the cis-Golgi network. Generation of the M6P recognition marker depends on a reaction involving two different enzymes: UDP-N-acetylglucosamine 1-phosphotransferase and α-N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase. The M6P groups are then recognized by two independent transmembrane M6P receptors, present in the trans-Golgi network: the cation-independent M6P receptor and/or the cation-dependent M6P receptor. These proteins bind to lysosomal hydrolases on the lumenal side of the membrane and to adaptins in assembling clathrin coats on the cytosolic side. In this way, the M6P receptors help package the hydrolases into vesicles that bud from the trans-Golgi network to deliver their contents to endosomes that ultimately will develop into mature lysosomes, where recently-delivered hydrolases may start digesting the endocyted material. The above described process is known as the M6P-dependent pathway and is responsible for transporting most lysosomal enzymes. This review synthesizes the current knowledge on each of the major proteins involved in the M6P-dependent pathway. Impairments in this pathway will also be addressed, highlighting the lysosomal storage disorders associated to GlcNAc-1-phosphotransferase loss of function: mucolipidosis type II and III.

Glycosaminoglycan Storage Disorders: A Review

Impaired degradation of glycosaminoglycans (GAGs) with consequent intralysosomal accumulation of undegraded products causes a group of lysosomal storage disorders known as mucopolysaccharidoses (MPSs). Characteristically, MPSs are recognized by increased excretion in urine of partially degraded GAGs which ultimately result in progressive cell, tissue, and organ dysfunction. There are eleven different enzymes involved in the stepwise degradation of GAGs. Deficiencies in each of those enzymes result in seven different MPSs, all sharing a series of clinical features, though in variable degrees. Usually MPS are characterized by a chronic and progressive course, with different degrees of severity. Typical symptoms include organomegaly, dysostosis multiplex, and coarse facies. Central nervous system, hearing, vision, and cardiovascular function may also be affected. Here, we provide an overview of the molecular basis, enzymatic defects, clinical manifestations, and diagnosis of each MPS, focusing also on the available animal models and describing potential perspectives of therapy for each one.

A shortcut to the lysosome: The mannose-6-phosphate-independent pathway

Lysosomal hydrolases have long been known to be responsible for the degradation of different substrates in the cell. These acid hydrolases are synthesized in the rough endoplasmic reticulum and transported through the Golgi apparatus to the trans-Golgi network (TGN). From there, they are delivered to endosomal/lysosomal compartments, where they finally become active due to the acidic pH characteristic of the lysosomal compartment. The majority of the enzymes leave the TGN after modification with mannose-6-phosphate (M6P) residues, which are specifically recognized by M6P receptors (MPRs), ensuring their transport to the endosomal/lysosomal system. Although M6P receptors play a major role in the intracellular transport of newly synthesized lysosomal enzymes in mammalian cells, several lines of evidence suggest the existence of alternative processes of lysosomal targeting. Among them, the two that are mediated by the M6P alternative receptors, lysosomal integral membrane protein (LIMP-2) and sortilin, have gained unequivocal support. LIMP-2 was shown to be implicated in the delivery of beta-glucocerebrosidase (GCase) to the lysosomes, whereas sortilin has been suggested to be a multifunctional receptor capable of binding several different ligands, including neurotensin and receptor-associated protein (RAP), and of targeting several proteins to the lysosome, including sphingolipid activator proteins (prosaposin and GM2 activator protein), acid sphingomyelinase and cathepsins D and H. Here, we review the current knowledge on these two proteins: their discovery, study, structural features and cellular function, with special attention to their role as alternative receptors to lysosomal trafficking. Recent studies associating both LIMP2 and sortilin to disease are also extensively reviewed.

Molecular analysis of the GNPTAB and GNPTG genes in 13 patients with mucolipidosis type II or type III – identification of eight novel mutations

Mucolipidosis II (ML II) and mucolipidosis III (ML III) are diseases in which the activity of the uridine diphosphate (UDP)‐N‐acetylglucosamine:lysosomal enzyme N‐acetylglucosamine‐1‐phosphotransferase (GlcNAc‐phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage‐disease phenotype. GlcNAc‐phosphotransferase is a multimeric transmembrane enzyme composed of three subunits (α, β and γ) encoded by two genes –GNPTAB and GNPTG. Defects in GNPTAB result in ML II and III whereas mutations in GNPTG were only found in ML III patients. We have performed a molecular analysis of the GNPTAB and GNPTG genes in 13 mucolipidosis II and III patients (10 Portuguese, one Finnish, one Spanish of Arab origin and one Indian). Mutations were identified by the study of both cDNA and gDNA. The GNPTAB and GNPTG mRNA expressions were determined by quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR). The study led to the identification of 11 different mutations. Eight of these mutations are novel, six in the GNPTAB gene [c.121delG (V41FfsX42), c.440delC (A147AfsX5), c.2249_50insA (N750KfsX8), c.242G>T (W81L), c.1208T>C (I403T) and c.1999G>T (p.E667X)] and two in the GNPTG gene [c.610‐1G>T and c.639delT (F213LfsX7)]. With regard to the mRNA expression studies, the values obtained by qRT‐PCR indicate the possible existence of feedback regulation mechanisms between α/β and the γ subunits.

Mucolipidosis II-Related Mutations Inhibit the Exit from the Endoplasmic Reticulum and Proteolytic Cleavage of GlcNAc-1-Phosphotransferase Precursor Protein (GNPTAB)

Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β‐subunit precursor protein of GlcNAc‐1‐phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α‐subunit‐specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild‐type and mutant α/β‐subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β‐subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc‐1‐phosphotransferase that may help to explain the clinical phenotype of MLII patients.

Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders.

Lysosomal storage diseases (LSDs) are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT), with no impact on neuropathology. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why additional therapeutic approaches are being (and have to be) investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate reduction therapy (SRT), whose aim is to prevent storage not by correcting the original enzymatic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s). Here we review the concept of substrate reduction, highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, especially, as complementary approach for LSDs.

From bedside to cell biology: A century of history on lysosomal dysfunction

Lysosomal storage disorders (LSDs) are a group of rare genetic diseases, generally caused by a deficiency of specific lysosomal enzymes, which results in abnormal accumulation of undegraded substrates. The first clinical reports describing what were later shown to be LSDs were published more than a hundred years ago. In general, the history and pathophysiology of LSDs has impacted on our current knowledge of lysosomal biology. Classically, depending on the nature of the substrates, LSDs can be divided into different subgroups. The mucopolysaccharidoses (MPSs) are those caused by impaired degradation of glycosaminoglycans (GAGs). Amongst LSDs, the MPSs are a major group of pathologies with crucial historical relevance, since their study has revealed important biological pathways and highlighted interconnecting pathological cascades which are still being unveiled nowadays. Here we review the major historical discoveries in the field of LSDs and their impact on basic cellular knowledge and practical applications. Attention will be focused on the MPSs, with occasional references to other LSDs. We will show as studies on the metabolic basis of this group of diseases have increased our knowledge of the complex degradative pathways associated with the lysosome and established the basis to the development of specific therapeutic approaches aiming at correcting or, at least ameliorating their associated phenotypes.

Origin and spread of a common deletion causing mucolipidosis type II: insights from patterns of haplotypic diversity

Coutinho MF, Encarnação M, Gomes R, da Silva Santos L, Martins S, Sirois‐Gagnon D, Bargal R, Filocamo M, Raas‐Rothschild A, Tappino B, Laprise C, Cury GK, Schwartz IV, Artigalás O, Prata MJ, Alves S. Origin and spread of a common deletion causing mucolipidosis type II: insights from patterns of haplotypic diversity.

Lysosomal multienzymatic complex-related diseases: a genetic study among Portuguese patients

Coutinho MF, Lacerda L, Macedo‐Ribeiro S, Baptista E, Ribeiro H, Prata MJ, Alves S. Lysosomal multienzymatic complex‐related diseases: a genetic study among Portuguese patients.

Molecular characterization of Portuguese patients with mucopolysaccharidosis IIIC: two novel mutations in the HGSNAT gene.

To the Editor: Mucopolysaccharidosis IIIC (MPS IIIC, Sanfilippo syndrome C) belongs to a class of lysosomal storage disorders known as mucopolysaccharidosis characterized by a deficiency in one of a group of enzymes responsible for the catabolism of glycosaminoglycans (1). MPS IIIC is caused by the inherited deficiency of the lysosomal membrane enzyme acetyl-coenzyme A: a-glucosaminide Nacetyltransferase (N-acetyltransferase), which leads to impaired degradation of heparan sulfate (1). Hallmark symptoms of MPS IIIC include mental retardation and hearing loss as well as relatively minor visceral manifestations (1, 2). It is known that the gene encoding N-acetyltransferase – HGSNAT – is located on chromosome 8p11.1andcontains18exons (3, 4).ThecomplementaryDNA(cDNA)codes foraproductof 635amino acids (previously named transmembrane protein 76 – TMEM 76), of which the N-terminal 30 amino acids are predicted to form a cleavable signal peptide, while along the remainder of the protein, there are11 transmembranedomainsandupto5N-linked glycosylation sites (3, 4). However, because these findings only came to light in 2006 when the HGSNAT gene was identified by two independent groups (3, 4), the molecular defects underlying MPS IIIC still remain largely uncharacterized. We have developed a new molecular method to characterize patients with MPS IIIC for the HGSNAT gene through cDNA analysis and identified two novel mutations: an insertion (c.525dupT) and a splice-site mutation (c.3722A/G), both of which are very likely deleterious to the function of HGSNAT. Our sample included three unrelated Portuguese patients with MPS IIIC whose clinical diagnosis was biochemically confirmed by demonstrating abnormal excretion of heparan sulfate in urine and HGSNAT deficiency in fibroblasts. Total cellular RNA was isolated from cultured fibroblasts using the High Pure RNA Isolation Kit’ (Roche, Basel, Switzerland) and reverse transcribed using the First-Strand cDNA Synthesis Kit’ (Amersham Biosciences, Munich, Germany). We designed specific primers to amplify the HGSNAT cDNA in seven overlapping fragments. Genomic DNA was also isolated from cultured fibroblasts, and polymerase chain reaction (PCR) amplification of HGSNAT exons including adjacent intronic regions was performed with specific primers. Primers and PCR conditions are provided as online supplementary material. The three patients only harbored two different mutations c.525dupT and c.372-2A/G (Table 1), both of which were previously unreported. Their presence was confirmed in patient’s cDNA and genomic DNA, and none was detected in 100 chromosomes from healthy Portuguese. Both mutations are very likely deleterious to the function of HGSNAT because they predictably result in a total loss of HGSNAT protein function. The insertion mutation c.525dupT causes the introduction of a premature STOP codon downstream that results in the translation of a product with 445 amino acids less than the normal protein. The splice-site mutation c.3722A/G leads to the skipping of exon 4 that also causes the introduction of a premature STOP codon downstream. Both transcripts will probably be degraded through the cellular mechanism of nonsensemediated messenger RNA (mRNA) decay, which is well known to be responsible for the elimination of mRNAs that contain premature termination codons (5). In full agreement with the finding of Hřebı́ček et al., when HGSNAT was sequenced, all studied individuals revealed the presence of an alternative transcript unlikely to be functional because it presented exons 9 and 10 spliced out (4). However, it can be excluded as causative of the MPS IIIC phenotype because of being a minoritary transcript that also appeared in an unaffected individual.