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C alpha-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O-2) and consumes 1 mol O-2 per mol FGly formed. For maximal activity FGE requires an O-2 concentration of 9% (105 mu M). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O-2-dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O-2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O-2 without involvement of any activating cofactor.

Leukocyte adhesion deficiency II has been described in only 2 patients; herein we report extensive investigation of another patient. The physical stigmata were detected during prenatal ultrasonographic investigation. Sialyl-Lewis X (sLe x ) was absent from the surface of polymorphonuclear neutrophils, and cell binding to E- and P-selectin was severely impaired, causing an immunodeficiency. The elevation of peripheral neutrophil counts occurred within several days after birth. A severe hypofucosylation of glycoconjugates bearing fucose in different glycosidic links was present in all cell types investigated, demonstrating that leukocyte adhesion deficiency II is not only a disorder of leukocytes but a generalized inherited metabolic disease affecting the metabolism of fucose. (J Pediatr 1999;134:681-8)

The extracellular sulfatases Sulf1 and Sulf2 remove specific 6-O-sulfate groups from heparan sulfate, thereby modulating numerous signalling pathways underlying development and homeostasis. In vitro data have suggested that the two enzymes show functional redundancy. To elucidate their in vivo functions and to further address the question of a putative redundancy, we have generated Sulf1- and Sulf2-deficient mice. Phenotypic analysis of these animals revealed higher embryonic lethality of Sulf2 knockout mice, which can be associated with neuroanatomical malformations during embryogenesis. Sulf1 seems not to be essential for developmental or postnatal viability, as mice deficient in this sulfatase show no overt phenotype. However, neurite outgrowth deficits were observed in hippocampal and cerebellar neurons of both mutant mouse lines, suggesting that not only Sulf2 but also Sulf1 function plays a role in the developing nervous system. Behavioural analysis revealed differential deficits with regard to cage activity and spatial learning for Sulf1- and Sulf2-deficient mouse lines. In addition, Sulf1-specific deficits were shown for synaptic plasticity in the CA1 region of the hippocampus, associated with a reduced spine density. These results reveal that Sulf1 and Sulf2 fulfil non-redundant functions in vivo in the development and maintenance of the murine nervous system.

Multiple sulfatase deficiency (MSD), mucolipidosis (MIL) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease. and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been addressed. Further. the discovery of FGE as an essential sulfatase activating enzyme has considerable impact on enzyme replacement or gene therapy of lysosomal storage disorders caused by single sulfatase deficiencies. (C) 2008 Elsevier B.V. All rights reserved.

As assay for the detection in leukocytes of homozygous and heterozygous carriers of Sanfilippo syndrome type C is described. In one family with two patients suffering from Sanfilippo C syndrome, the affected individuals had no residual activity to acetyl-CoA: alpha-glucosaminide N-acetyltransferase. The determination of the acetyl-CoA: alpha-glucosaminide N-acetyltransferase/ beta-glucuronidase ratio allows the discrimination between obligate heterozygotes and normal individuals and may be used for carrier detection.

Formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the oxidation of a specific cysteine residue to formylglycine, which is the catalytic residue required for sulfate ester hydrolysis. This post-translational modification occurs in the endoplasmic reticulum (ER), and is an essential step in the biogenesis of this enzyme family. A paralog of FGE (pFGE) also localizes to the ER. It shares many properties with FGE, but lacks formylglycine-generating activity. There is evidence that FGE and pFGE act in concert, possibly by forming complexes with sulfatases and one another. Here we show that human pFGE, but not FGE, is retained in the ER through its C-terminal tetrapeptide PGEL, a noncanonical variant of the classic KDEL ER-retention signal. Surprisingly, PGEL, although having two nonconsensus residues (PG), confers efficient ER retention when fused to a secretory protein. Inducible coexpression of pFGE at different levels in FGE-expressing cells did not significantly influence the kinetics of FGE secretion, suggesting that pFGE is not a retention factor for FGE in vivo. PGEL is accessible at the surface of the pFGE structure. It is found in 21 mammalian species with available pFGE sequences. Other species carry either canonical signals (eight mammals and 26 nonmammals) or different noncanonical variants (six mammals and six nonmammals). Among the latter, SGEL was tested and found to also confer ER retention. Although evolutionarily conserved for mammalian pFGE, the PGEL signal is found only in one further human protein entering the ER. Its consequences for KDEL receptor-mediated ER retrieval and benefit for pFGE functionality remain to be fully resolved.

Contains fulltext : 49358.pdf (Publisher’s version ) (Closed access) HS (heparan sulfate) is essential for normal embryonic development. This requirement is due to the obligatory role for HS in the signalling pathways of many growth factors and morphogens that bind to sulfated domains in the HS polymer chain. The sulfation patterning of HS is determined by a complex interplay of Golgi-located N- and O-sulfotransferases which sulfate the heparan precursor and cell surface endosulfatases that selectively remove 6-O-sulfates from mature HS chains. In the present study we generated single or double knock-out mice for the two murine endosulfatases mSulf1 and mSulf2. Detailed structural analysis of HS from mSulf1-/- fibroblasts showed a striking increase in 6-O-sulfation, which was not seen in mSulf2-/- HS. Intriguingly, the level of 6-O-sulfation in the double mSulf1-/-/2-/- HS was significantly higher than that observed in the mSulf1-/- counterpart. These data imply that mSulf1 and mSulf2 are functionally co-operative. Unlike their avian orthologues, mammalian Sulf activities are not restricted to the highly sulfated S-domains of HS. Mitogenesis assays with FGF2 (fibroblast growth factor 2) revealed that Sulf activity decreases the activating potential of newly-synthesized HS, suggesting an important role for these enzymes in cell growth regulation in embryonic and adult tissues.

The formylglycine (FGly)-generating enzyme (FGE) uses molecular oxygen to oxidize a conserved cysteine residue in all eukaryotic sulfatases to the catalytically active FGly. Sulfatases degrade and remodel sulfate esters, and inactivity of FGE results in multiple sulfatase deficiency, a fatal disease. The previously determined FGE crystal structure revealed two crucial cysteine residues in the active site, one of which was thought to be implicated in substrate binding. The other cysteine residue partakes in a novel oxygenase mechanism that does not rely on any cofactors. Here, we present crystal structures of the individual FGE cysteine mutants and employ chemical probing of wild-type FGE, which defined the cysteines to differ strongly in their reactivity. This striking difference in reactivity is explained by the distinct roles of these cysteine residues in the catalytic mechanism. Hitherto, an enzyme–substrate complex as an essential cornerstone for the structural evaluation of the FGly formation mechanism has remained elusive. We also present two FGE–substrate complexes with pentamer and heptamer peptides that mimic sulfatases. The peptides isolate a small cavity that is a likely binding site for molecular oxygen and could host reactive oxygen intermediates during cysteine oxidation. Importantly, these FGE–peptide complexes directly unveil the molecular bases of FGE substrate binding and specificity. Because of the conserved nature of FGE sequences in other organisms, this binding mechanism is of general validity. Furthermore, several disease-causing mutations in both FGE and sulfatases are explained by this binding mechanism.

Mannose-6-phosphate receptors (MPRs) have been identified in a wide range of species from humans to invertebrates such as molluscs. A characteristic of all MPRs is their common property to recognize mannose-6-phosphate residues that are labelling lysosomal enzymes and to mediate their targeting to lysosomes in mammalian cells by the corresponding receptor proteins. We present here the analysis of full-length sequences for MPR 46 from zebrafish (Danio rerio) and its functional analysis. This is the first non-mammalian MPR 46 to be characterised. The amino acid sequences of the zebrafish MPR 46 displays 70% similarity to the human MPR 46 protein. In particular, all essential cysteine residues, the transmembrane domain as well as the cytoplasmic tail residues harbouring the signals for endocytosis and Golgi-localizing, gamma-ear-containing, ARF-binding protein (GGA)-mediated sorting at the trans-Golgi network, are highly conserved. The zebrafish MPR 46 has the arginine residue known to be essential for mannose-6-phosphate binding and other additional characteristic residues of the mannose-6-phosphate ligand-binding pocket. Like the mammalian MPR 46, zebrafish MPR 46 binds to the multimeric mannose-6-phosphate ligand phosphomannan and can rescue the missorting of lysosomal enzymes in mammalian MPR-deficient cells. The conserved C-terminal acidic dileucine motif (DxxLL) in the cytoplasmic domain of zebrafish MPR 46 essential for the interaction of the GGAs with the receptor domains interacts with the human GGA1-VHS domain. Interestingly, the serine residue suggested to regulate the interaction between the tail and the GGAs in a phosphorylation-dependent manner is substituted by a proline residue in fish.

Niemann-Pick disease type C (NPC), caused by mutations in the NPC1 gene or the NPC2 gene, is characterized by the accumulation of unesterified cholesterol and other lipids in endo/lysosomal compartments. NPC2 is a small, soluble, lysosomal protein that is targeted to this compartment via a mannose 6-phosphate-inhibitable pathway. To obtain insight into the roles of mannose 6-phosphate receptors (MPRs) in NPC2 targeting, we here examine the trafficking and function of NPC2 in fibroblast lines deficient in one or both of the two MPRs, MPR46 and MPR300. We demonstrate that either MPR alone is sufficient to transport NPC2 to the endo/lysosomal compartment, although MPR300 seems to be more efficient than MPR46. In the absence of both MPRs, NPC2 is secreted into the culture medium, and only a small amount of intracellular NPC2 can be detected, mainly in the endoplasmic reticulum. This leads to massive accumulation of unesterified cholesterol in the endo/lysosomal compartment of the MPR46/300-deficient fibroblasts, a phenotype similar to that of the NPC patient fibroblasts. In addition, we observed an upregulation of NPC1 protein and mRNA in the MPR-double-deficient cells. Taken together, our results suggest that the lysosomal targeting of NPC2 is strictly dependent on MPRs in fibroblasts.

Although of clinical importance, little is known about the mechanism of seizure in neuronal ceroid lipofuscinosis (NCL). In the present study, we have attempted to elucidate the mechanism underlying the seizure of cathepsin D-deficient (CD-/-) mice that show a novel type of lysosomal storage disease with a phenotype resembling late infantile NCL. In hippocampal slices prepared from CD-/- mice at post-natal day (P)24, spontaneous burst discharges were recorded from CA3 pyramidal cells. At P24, the mean amplitude of IPSPs after stimulation of the mossy fibres was significantly smaller than that of wild-type mice, which was substantiated by the decreased level of gamma-aminobutyric acid (GABA) contents in the hippocampus measured by high-performance liquid chromatography (HPLC). At this stage, activated microglia were found to accumulate in the pyramidal cell layer of the hippocampal CA3 subfield of CD-/- mice. However, there was no significant change in the numerical density of GABAergic interneurons in the CA3 subfield of CD-/- mice at P24, estimated by counting the number of glutamate decarboxylase (GAD) 67-immunoreactive somata. In the hippocampus and the cortex of CD-/- mice at P24, some GABAergic interneurons displayed extremely high somatic granular immunoreactivites for GAD67, suggesting the lysosomal accumulation of GAD67. GAD67 levels in axon terminals abutting on to perisomatic regions of hippocampal CA3 pyramidal cells was not significantly changed in CD-/- mice even at P24, whereas the total protein levels of GAD67 in both the hippocampus and the cortex of CD-/- mice after P24 were significantly decreased as a result of degradation. Furthermore, the recombinant human GAD65/67 was rapidly digested by the lysosomal fraction prepared from the whole brain of wild-type and CD-/- mice. These observations strongly suggest that the reduction of GABA contents, presumably because of lysosomal degradation of GAD67 and lysosomal accumulation of its degraded forms, are responsible for the dysfunction of GABAergic interneurons in the hippocampal CA3 subfield of CD-/- mice.

Among the autophagic vacuolar myopathies (AVMs), a subgroup is characterized pathologically by unusual autophagic vacuoles with sarcolemmal features (AVSF) and includes Danon disease and X-linked myopathy with excessive autophagy. The diagnostic importance and detailed morphologic features of AVSF in different AVMs have not been well established, and the mechanism of AVSF formation is not known. To address these issues, we have performed detailed histologic studies of myopathies with AVSF and other AVMs. In Danon disease and related AVMs, at the light microscopic level, autophagic vacuoles appeared to be accumulations of lysosomes, which, by electron microscopy consisted of clusters of autophagic vacuoles, indicative of autolysosomes. Some autolysosomes were surrounded by membranes with sarcolemmal proteins, acetylcholinesterase activity, and basal lamina. In Danon disease, the number of fibers with AVSF increased linearly with age while the number with autolysosomal accumulations decreased slightly, suggesting that AVSF are produced secondarily in response to autolysosomes. Most of the AVSF form enclosed spaces, indicating that the vacuolar membranes may be formed in situ rather than through sarcolemmal indentation. This unique intracytoplasmic membrane structure was not found in other AVMs. In conclusion, AVSF with acetylcholinesterase activity are autolysosomes surrounded by secondarily generated intracytoplasmic sarcolemma-like structure and delineates a subgroup of AVMs.

Cytotoxic T lymphocytes (CTL) play an important role in the rejection of allogeneic cells and organs. CTL secrete granzymes and perforin as cytotoxic effector molecules. The mannose 6-phosphate receptor (Mpr)300 has been reported to function as receptor for granzyme B on target cells and to be essential for the rejection of allogeneic cells in vivo. Using mouse embryonal fibroblasts from Mpr300 and Mpr46 knockout mice, we show that both Mpr 300 and Mpr46 are dispensable on target cells for lysis and apoptosis mediated by alloreactive CTL in vitro and for allorejection in vivo. In agreement with a postulated function of Mpr300 as a tumor suppressor gene, deficiency of Mpr300 appears to promote cellular proliferation and tumorigenicity but not resistance to allorejection.

Cα-formylglycine (FGly) is the catalytic residue in the active site of eukaryotic sulfatases. It is posttranslationally generated from a cysteine in the endoplasmic reticulum. The genetic defect of FGly formation causes multiple sulfatase deficiency (MSD), a lysosomal storage disorder. We purified the FGly generating enzyme (FGE) and identified its gene and nine mutations in seven Mp. patients. In patient fibroblasts, the activity of sulfatases is partially restored by transduction of FGE encoding cDNA, but not by cDNA carrying an MSD mutation. The gene encoding FGE is highly conserved among pro- and eukaryotes and has a paralog of unknown function in vertebrates. FGE is localized in the endoplasmic reticulum and is predicted to have a tri-partite domain structure. peerReviewed

Calpha-formylglycine is the catalytic residue of sulfatases. Formylglycine is generated by posttranslational modification of a cysteine (pro- and eukaryotes) or serine (prokaryotes) located in a conserved (C/S)XPXR motif. The modifying enzymes are unknown. AtsB, an iron-sulfur protein, is strictly required for modification of Ser(72) in the periplasmic sulfatase AtsA of Klebsiella pneumoniae. Here we show (i) that AtsB is a cytosolic protein acting on newly synthesized serine-type sulfatases, (ii) that AtsB-mediated FGly formation is dependent on AtsA's signal peptide, and (iii) that the cytosolic cysteine-type sulfatase of Pseudomonas aeruginosa can be converted into a substrate of AtsB if the cysteine is substituted by serine and a signal peptide is added. Thus, formylglycine formation in serine-type sulfatases depends both on AtsB and on the presence of a signal peptide, and AtsB can act on sulfatases of other species. AtsB physically interacts with AtsA in a Ser(72)-dependent manner, as shown in yeast two-hybrid and GST pull-down experiments. This strongly suggests that AtsB is the serine-modifying enzyme and that AtsB relies on a cytosolic function of the sulfatase's signal peptide.

C-alpha-Formylglycine, the catalytic amino acid residue in the active site of sulfatases, is generated by post-translational modification of a cysteine or serine residue. We describe a highly sensitive procedure for the detection of C-alpha-formylglycine-containing peptides in tryptic digests of sulfatase proteins, The protocol is based on the formation of hydrazone derivatives of C-alpha-formylglycine-containing peptides when using dinitrophenylhydrazine as a matrix for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives desorb and ionize with high efficiency and can be detected in the sub-femtomole range. The presence of C-alpha-formylglycine is indicated by a mass increment of 180.13 u, corresponding to the hydrazone moiety, and also by a unique C-terminal fragment ion, characteristic of sulfatases, that becomes prominent in MALDI post-source decay mass spectra of the hydrazone derivatives. Copyright (C) 2003 John Wiley Sons, Ltd.

Improved data quality now makes it feasible to exploit the weak anomalous signal derived only from the sulfurs inherent to the protein or in particular from halide ions incorporated by soaking. The latter technique requires the location of a high number of partially occupied halide sites. This number appears to be roughly proportional to the exposed protein surface. This paper explores the application of dual-space ab initio methods as implemented in the program SHELXD to the location of substructures of sulfur in SAD experiments, bromide in SAD and MAD experiments and iodide using SAD and SIRAS to determine the anomalous-atom substructure. Sets of atoms consistent with the Patterson function were generated as a starting point for the dual-space recycling procedure in SHELXD. The substructure is then expanded to the full structure by maximum-likelihood phasing with SHARP and density modification with the program DM. Success in the location of the substructures and subsequent phasing depends critically on the quality of the data and on the extent of the anomalous signal. This varies with each crystal and soak, but for the same crystal the significance of the anomalous signal was found to be highly sensitive to the redundancy of the intensity measurements, which in some cases made all the difference. This is illustrated by the determination of the previously unknown structure of repeat 11 of the human mannose-6-phosphate/insulin-like growth factor II receptor (Man6P/IGFII-receptor), with 310 amino acids in the asymmetric unit, which was phased by soaking the crystals in a cryoprotectant solution containing halide anions.

Deficiency of the endoplasmic reticulum enzyme dolichyl-phosphate mannose (Dol-P-Man):Man(7)GlcNAc(2)-PP-dolichyl mannosyltransferase leads to a new type of congenital disorder of glycosylation, designated type Ig. The patient 1 presented with a multisystemic disorder with microcephaly, developmental retardation, convulsions and dysmorphic signs. The isoelectric focusing pattern of the patient's serum transferrin showed the partial loss of complete N-glycan side chains. In skin fibroblasts from the patient, the activity of Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase was severely reduced leading to the accumulation of Man(7)GlcNAc(2)-PP-Dol, which was transferred to newly synthesized glycoproteins. Sequencing of the Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase cDNA revealed a compound heterozygosity for two point mutations, leading to the exchange of leucine(158) for a proline residue and a premature translation stop with loss of the C-terminal 74 amino acids. The parents were heterozygous for one of the two mutations. Retroviral expression of the wild-type Dol-P-Man:Man(7)GlcNAc(2)-PP-Dol mannosyltransferase cDNA in patient's fibroblasts normalized the mannosyltransferase activity.

In LAMP-2–deficient mice autophagic vacuoles accumulate in many tissues, including liver, pancreas, muscle, and heart. Here we extend the phenotype analysis using cultured hepatocytes. In LAMP-2–deficient hepatocytes the half-life of both early and late autophagic vacuoles was prolonged as evaluated by quantitative electron microscopy. However, an endocytic tracer reached the autophagic vacuoles, indicating delivery of endo/lysosomal constituents to autophagic vacuoles. Enzyme activity measurements showed that the trafficking of some lysosomal enzymes to lysosomes was impaired. Immunoprecipitation of metabolically labeled cathepsin D indicated reduced intracellular retention and processing in the knockout cells. The steady-state level of 300-kDa mannose 6-phosphate receptor was slightly lower in LAMP-2–deficient hepatocytes, whereas that of 46-kDa mannose 6-phosphate receptor was decreased to 30% of controls due to a shorter half-life. Less receptor was found in the Golgi region and in vesicles and tubules surrounding multivesicular endosomes, suggesting impaired recycling from endosomes to the Golgi. More receptor was found in autophagic vacuoles, which may explain its shorter half-life. Our data indicate that in hepatocytes LAMP-2 deficiency either directly or indirectly leads to impaired recycling of 46-kDa mannose 6-phosphate receptors and partial mistargeting of a subset of lysosomal enzymes. Autophagic vacuoles may accumulate due to impaired capacity for lysosomal degradation.

The heterotetrameric AP-1A adaptor complex of clathrin-coated vesicles is ubiquitously expressed. The mu 1-adaptin subunit of the complex exists as the ubiquitous mu 1A and the polarized epithelia-specific mu 1B, which are 80% identical. In polarized epithelia, mu 1B is incorporated into the AP-1B complex, which is required for basolateral plasma membrane sorting of the low-density lipoprotein receptor. Binding of AP-1B to subdomains of the trans-Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed mu 1B in mu 1A-deficient fibroblasts to test for mu 1B function in non-polarized cells. AP-1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP-1B restored the AP-1A-dependent sorting of mannose 6-phosphate receptors between endosomes and the TGN. This demonstrates that mu 1A and mu 1B do have overlapping sorting functions and indicates that AP-1A and AP-1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.

In one of the most common mutations causing metachromatic leukodystrophy, the P426L-allele of arylsulfatase A (ASA), the deficiency of ASA results from its instability in lysosomes. Inhibition of lysosomal cysteine proteinases protects the P426L-ASA and restores the sulfatide catabolism in fibroblasts of the patients. P426L-ASA, but not wild type ASA, was cleaved by purified cathepsin L at threonine 421 yielding 54- and 9-kDa fragments. X-ray crystallography at 2.5-A resolution showed that cleavage is not due to a difference in the protein fold that would expose the peptide bond following threonine 421 to proteases. Octamerization, which depends on protonation of Glu-424, was impaired for P426L-ASA. The mutation lowers the pH for the octamer/dimer equilibrium by 0.6 pH units from pH 5.8 to 5.2. A second oligomerization mutant (ASA-A464R) was generated that failed to octamerize even at pH 4.8. A464R-ASA was degraded in lysosomes to catalytically active 54-kDa intermediate. In cathepsin L-deficient fibroblasts, degradation of P426L-ASA and A464R-ASA to the 54-kDa fragment was reduced, while further degradation was blocked. This indicates that defective oligomerization of ASA allows degradation of ASA to a catalytically active 54-kDa intermediate by lysosomal cysteine proteinases, including cathepsin L. Further degradation of the 54-kDa intermediate critically depends on cathepsin L and is modified by the structure of the 9-kDa cleavage product.

C(alpha)-formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay that allowed us to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome association helped to maintain the modification competence of nascent chains but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mol, and the pH optimum is approximately 10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca(2+). Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.

Cathepsin D (CD) deficiency has been shown to induce ceroid-lipofuscin storage in lysosomes of mouse CNS neuron (Koike et al., 2000). To understand the behavior of microglial cells corresponding to these neuronal changes, CD-deficient (CD-/-) mice, which die at approximately postnatal day (P) 25 by intestinal necrosis, were examined using morphological as well as biochemical approaches. Light and electron microscopic observations revealed that microglia showing large round cell bodies with few processes appeared in the cerebral cortex and thalamus after P16. At P24, microglia often encircled neurons that were occupied with autolysosomes, indicating increased phagocytic activity. These morphologically transformed microglia markedly expressed inducible nitric oxide synthase (iNOS), which was also detected in the intestine of the mice. To assess the role of microglial nitric oxide (NO) in neuropathological changes in CD-/- mice, l-N(G)-nitro-arginine methylester (l-NAME), a competitive NOS inhibitor, or S-methylisothiourea hemisulfate (SMT), an iNOS inhibitor, was administered intraperitoneally for 13 consecutive days. The total number of terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling-positive cells counted in the thalamus was found to be significantly decreased by chronic treatment of l-NAME or SMT, whereas neither the neuronal accumulation of ceroid-lipofuscin nor the microglial phagocytic activity was affected by these treatments. Moreover, the chronic treatment of l-NAME or SMT completely suppressed hemorrhage-necrotic changes in the small intestine of CD-/- mice, resulting in normal growth of the body weight of the mice. These results suggest that NO production via iNOS activity in microglia and peripheral macrophages contributes to secondary tissue damages such as neuronal apoptosis and intestinal necrosis, respectively.

Background: Sulfatases constitute a family of enzymes with a highly conserved active site region including a Cα−formylglycine that is posttranslationally generated by the oxidation of a conserved cysteine or serine residue. The crystal structures of two human arylsulfatases, ASA and ASB, along with ASA mutants and their complexes led to different proposals for the catalytic mechanism in the hydrolysis of sulfate esters. Results: The crystal structure of a bacterial sulfatase from Pseudomonas aeruginosa (PAS) has been determined at 1.3 A. Fold and active site region are strikingly similar to those of the known human sulfatases. The structure allows a precise determination of the active site region, unequivocally showing the presence of a Cα−formylglycine hydrate as the key catalytic residue. Furthermore, the cation located in the active site is unambiguously characterized as calcium by both its B value and the geometry of its coordination sphere. The active site contains a noncovalently bonded sulfate that occupies the same position as the one in para -nitrocatecholsulfate in previously studied ASA complexes. Conclusions: The structure of PAS shows that the resting state of the key catalytic residue in sulfatases is a formylglycine hydrate. These structural data establish a mechanism for sulfate ester cleavage involving an aldehyde hydrate as the functional group that initiates the reaction through a nucleophilic attack on the sulfur atom in the substrate. The alcohol is eliminated from a reaction intermediate containing pentacoordinated sulfur. Subsequent elimination of the sulfate regenerates the aldehyde, which is again hydrated. The metal cation involved in stabilizing the charge and anchoring the substrate during catalysis is established as calcium.

The two clathrin-associated adaptor complexes AP1 and AP2 are known to participate in the formation of clathrin-coated vesicles at the trans-Golgi network and at the plasma membrane. During this process adaptors are involved in the sequestration of vesicle cargo by binding to the sorting signals within the cytoplasmic domains of the cargo proteins and in the recruitment of the clathrin coat. After budding of the clathrin-coated vesicles, the clathrin and adaptors dissociate from the vesicles. Here we show that in vitro binding of AP2 to sorting signals, which is one of the initial steps in receptor-mediated endocytosis, is modulated by adaptor phosphorylation. AP2 was phosphorylated by incubating purified AP2 in the presence of ATP and dephosphorylated by incubation with alkaline phosphatase. Affinity for tyrosine-, leucine-based and noncanonical sorting motifs was 15-33 times higher for phosphorylated than for dephosphorylated AP2. Also the binding of AP2 to membranes was regulated by adaptor phosphorylation/dephosphorylation and was about 8-fold higher for phosphorylated than for dephosphorylated AP2. Moreover, AP2 isolated from cytosol is higher phosphorylated than membrane-extracted and exhibits a 5-fold higher binding affinity than AP2 extracted from membranes. Taken together these data point to a cycle of phosphorylation/dephosphorylation as a mechanism for regulating the reversible association of AP2 with membranes and sorting signals during the process of receptor-mediated endocytosis.

Lysosomal cysteine proteinases of the papain family are involved in lysosomal bulk proteolysis, major histocompatibility complex class II mediated antigen presentation, prohormone processing, and extracellular matrix remodeling. Cathepsin L (CTSL) is a ubiquitously expressed major representative of the papain-like family of cysteine proteinases. To investigate CTSL in vivo functions, the gene was inactivated by gene targeting in embryonic stem cells. CTSL-deficient mice develop periodic hair loss and epidermal hyperplasia, acanthosis, and hyperkeratosis. The hair loss is due to alterations of hair follicle morphogenesis and cycling, dilatation of hair follicle canals, and disturbed club hair formation. Hyperproliferation of hair follicle epithelial cells and basal epidermal keratinocytes-both of ectodermal origin-are the primary characteristics underlying the mutant phenotype. Pathological inflammatory responses have been excluded as a putative cause of the skin and hair disorder. The phenotype of CTSL-deficient mice is reminiscent of the spontaneous mouse mutant furless (fs). Analyses of the ctsl gene of fs mice revealed a G149R mutation inactivating the proteinase activity. CTSL is the first lysosomal proteinase shown to be essential for epidermal homeostasis and regular hair follicle morphogenesis and cycling.

The heterotetrameric AP-1 complex is involved in the formation of clathrin-coated vesicles at the trans-Golgi network (TGN) and interacts with sorting signals in the cytoplasmic tails of cargo molecules. Targeted disruption of the mouse mu1A-adaptin gene causes embryonic lethality at day 13.5. In cells deficient in micro1A-adaptin the remaining AP-1 adaptins do not bind to the TGN. Polarized epithelial cells are the only cells of micro1A-adaptin-deficient embryos that show gamma-adaptin binding to membranes, indicating the formation of an epithelial specific AP-1B complex and demonstrating the absence of additional mu1A homologs. Mannose 6-phosphate receptors are cargo molecules that exit the TGN via AP-1-clathrin-coated vesicles. The steady-state distribution of the mannose 6-phosphate receptors MPR46 and MPR300 in mu1A-deficient cells is shifted to endosomes at the expense of the TGN. MPR46 fails to recycle back from the endosome to the TGN, indicating that AP-1 is required for retrograde endosome to TGN transport of the receptor.

The two known mannose 6-phosphate receptors (MPR 46 and MPR 300) mediate the transport of mannose 6-phosphate-containing lysosomal proteins to lysosomes. Endocytosis of extracellular mannose 6-phosphate ligands can only be mediated by MPR 300. Neither type of MPR appears to be sufficient for targetting the full complement of lysosomal enzymes to lysosomes. The complements of lysosomal enzymes transported by either of the two receptors are distinct but largely overlapping. Chimeric receptors were constructed in which the transmembrane and cytoplasmic domains of the two receptors were systematically exchanged. After expression of the chimeric receptors in cells lacking endogenous MPRs the binding of ligands, the subcellular distribution and the sorting efficiency for lysosomal enzymes were analyzed. All chimeras were functional, and their subcellular distribution was similar to that of wild type MPRs. The ability to endocytose lysosomal enzymes was restricted to receptors with the lumenal domain of MPR 300. The efficiency to sort lysosomal enzymes correlated with the lumenal and cytoplasmic domains of MPR 300. In contrast to the wild type receptors, a significant fraction of most of the chimeric receptors was misrouted to lysosomes, indicating that the signals determining the routing of MPRs have been fitted for the parent receptor polypeptides.

Type IV of the carbohydrate deficient glycoprotein syndromes (CDGS) is characterized by microcephaly, severe epilepsy, minimal psychomotor development and partial deficiency of sialic acids in serum glycoproteins. Here we show that the molecular defect in the index patient is a missense mutation in the gene encoding the mannosyltransferase that transfers mannose from dolichyl-phosphate mannose on to the lipid-linked oligosaccharide (LLO) intermediate Man(5)GlcNAc(2)-PP-dolichol. The defect results in the accumulation of the LLO intermediate and, due to its leaky nature, a residual formation of full-length LLOs. N-glycosylation is abnormal because of the transfer of truncated oligosaccharides in addition to that of full-length oligosaccharides and because of the incomplete utilization of N-glycosylation sites. The mannosyltransferase is the structural and functional orthologue of the Saccharomyces cerevisiae ALG3 gene.

In mammals, Mannose 6-phosphate receptor proteins (MPR 300 and MPR 46) mediate transport of lysosomal enzymes to lysosomes. Both receptors have been found in non-mammalian vertebrates including fish. To investigate the presence of MPRs in invertebrates, MPR 300 protein was isolated from the mollusc unio by affinity chromatography. It was shown to exhibit biochemical and immunological properties similar to mammalian MPR 300.

Arylsulfatase A belongs to the sulfatase family whose members carry a Cα-formylglycine that is post-translationally generated by oxidation of a conserved cysteine or serine residue. The formylglycine acts as an aldehyde hydrate with two geminal hydroxyls being involved in catalysis of sulfate ester cleavage. In arylsulfatase A and N-acetylgalactosamine 4-sulfatase this formylglycine was found to form the active site together with a divalent cation and a number of polar residues, tightly interconnected by a net of hydrogen bonds. Most of these putative active site residues are highly conserved among the eukaryotic and prokaryotic members of the sulfatase family. To analyze their function in binding and cleaving sulfate esters, we substituted a total of nine putative active site residues of human ASA by alanine (Asp29, Asp30, Asp281, Asn282, His125, His229, Lys123, Lys302, and Ser150). In addition the Mg2+-complexing residues (Asp29, Asp30, Asp281, and Asn282) were substituted conservatively by either asparagine or aspartate. In all mutants V max was decreased to 1–26% of wild type activity. The K m was more than 10-fold increased in K123A and K302A and up to 5-fold in the other mutants. In all mutants the pH optimum was increased from 4.5 by 0.2–0.8 units. These results indicate that each of the nine residues examined is critical for catalytic activity, Lys123 and Lys302 by binding the substrate and the others by direct (His125 and Asp281) or indirect participation in catalysis. The shift in the pH optimum is explained by two deprotonation steps that have been proposed for sulfate ester cleavage.

Lysosomal membranes contain two highly glycosylated proteins, designated LAMP-1 and LAMP-2, as major components. LAMP-1 and LAMP-2 are structurally related. To investigate the physiological role of LAMP-1, we have generated mice deficient for this protein. LAMP-1-deficient mice are viable and fertile. In LAMP-1-deficient brain, a mild regional astrogliosis and altered immunoreactivity against cathepsin-D was observed. Histological and ultrastructural analyses of all other tissues did not reveal abnormalities. Lysosomal properties, such as enzyme activities, lysosomal pH, osmotic stability, density, shape, and subcellular distribution were not changed in comparison with controls. Western blot analyses of LAMP-1-deficient and heterozygote tissues revealed an up-regulation of the LAMP-2 protein pointing to a compensatory effect of LAMP-2 in response to the LAMP-1 deficiency. The increase of LAMP-2 was neither correlated with an increase in the level of lamp-2 mRNAs nor with increased half-life time of LAMP-2. This findings suggest a translational regulation of LAMP-2 expression.

Intracellular protein transport and sorting by vesicles in the secretory and endocytic pathways requires the formation of a protein coat on the membrane. The heterotetrameric adaptor protein complex 1 (AP-1) promotes the formation of clathrin-coated vesicles at the trans-Golgi network. AP-1 interacts with various sorting signals in the cytoplasmic tails of cargo molecules, thus indicating a function in protein sorting. We generated mutants of the gamma-adaptin subunit of AP-1 in mice to investigate its role in post-Golgi vesicle transport and sorting processes. gamma-Adaptin-deficient embryos develop until day 3.5 post coitus and die during the prenidation period, revealing that AP-1 is essential for viability. In heterozygous mice the amount of AP-1 complexes is reduced to half of controls. Free beta1- or micro1 chains were not detectable, indicating that they are unstable unless they are part of AP-1 complexes. Heterozygous mice weigh less then their wild-type littermates and show impaired T cell development.

Cathepsin K is a recently identified lysosomal cysteine proteinase. It is abundant in osteoclasts, where it is believed to play a vital role in the resorption and remodeling of bone. Pycnodysostosis is a rare inherited osteochondrodysplasia that is caused by mutations of the cathepsin-K gene, characterized by osteosclerosis, short stature, and acroosteolysis of the distal phalanges. With a view to delineating the role of cathepsin K in bone resorption, we generated mice with a targeted disruption of this proteinase. Cathepsin-K-deficient mice survive and are fertile, but display an osteopetrotic phenotype with excessive trabeculation of the bone-marrow space. Cathepsin-K-deficient osteoclasts manifested a modified ultrastructural appearance: their resorptive surface was poorly defined with a broad demineralized matrix fringe containing undigested fine collagen fibrils; their ruffled borders lacked crystal-like inclusions, and they were devoid of collagen-fibril-containing cytoplasmic vacuoles. Assaying the resorptive activity of cathepsin-K-deficient osteoclasts in vitro revealed this function to be severely impaired, which supports the contention that cathepsin K is of major importance in bone remodeling.

Eukaryotic sulfatases carry an alpha-formylglycine residue that is essential for activity and is located within the catalytic site. This formylglycine is generated by posttranslational modification of a conserved cysteine residue. The arylsulfatase gene of Pseudomonas aeruginosa also encodes a cysteine at the critical position. This protein could be expressed in active form in a sulfatase-deficient strain of P. aeruginosa, thereby restoring growth on aromatic sulfates as sole sulfur source, and in Escherichia coli, Analysis of the mature protein expressed in E. coli revealed the presence of formylglycine at the expected position, showing that the cysteine is also converted to formylglycine in a prokaryotic sulfatase. Substituting the relevant cysteine by a serine codon in the P. aeruginosa gene led to expression of inactive sulfatase protein, lacking the formylglycine. The machinery catalyzing the modification of the Pseudomonas sulfatase in E. coli therefore resembles the eukaryotic machinery, accepting cysteine but not serine as a modification substrate, By contrast, in the arylsulfatase of Klebsiella pneumoniae a formylglycine is found generated by modification of a serine residue. The expression of both the Klebsiella and the Pseudomonas sulfatases as active enzymes in E. coli suggests that two modification systems are present, or that a common modification system is modulated by a cofactor.

In fibroblasts from five patients with carbohydrate-deficient glycoprotein syndrome type 1, the incorporation of [2-3H] mannose into mannose phosphates, GDP-mannose, GDP-fucose, dolichol-P-mannose, lipid-linked oligosaccharides, and glycoprotein fraction was determined. We observed a 3- to 5-fold reduction of incorporation of radioactivity into mannose 1-phosphate, GDP-mannose, GDP-fucose, dolichol-P-mannose, and nascent glycoproteins. The incorporation of radioactivity into mannose 6-phosphate was normal. The formation of lipid linked oligosaccharides was only slightly affected (

Mannose 6-phosphate receptor deficient mice were generated by crossing mice carrying null alleles for Igf2 and the 300 kDa and 46 kDa mannose 6-phosphate receptors, Mpr300 and Mpr46. Pre- and perinatal lethality of mice nullizygous for Igf2, Mpr300 and Mpr46 was increased. Triple deficient mice surviving the first postnatal day had normal viability and developed a phenotype resembling human I-cell disease. The triple deficient mice were characterized by dwarfism, facial dysplasia, waddling gait, dysostosis multiplex, elevated lysosomal enzymes in serum and histological signs of lysosomal storage predominantly in fibroblasts, but also in parenchymal cells of brain and liver. A paternally inherited Mpr300 wild type allele that is normally inactive in mice due to imprinting was reactivated in some tissues of mice lacking IGF II and MPR 46 and carrying a maternal Mpr300 null allele. Inspite of the partial reactivation the phenotype of these mice was similar to that of triple deficient mice.

Point mutations in the presenilin-1 gene (PS1) are a major cause of familial Alzheimer's disease. They result in a selective increase in the production of the amyloidogenic peptide amyloid-beta(1-42) by proteolytic processing of the amyloid precursor protein (APP). Here we investigate whether PS1 is also involved in normal APP processing in neuronal cultures derived from PS1-deficient mouse embryos. Cleavage by alpha- and beta-secretase of the extracellular domain of APP was not affected by the absence of PS1, whereas cleavage by gamma-secretase of the transmembrane domain of APP was prevented, causing carboxyl-terminal fragments of APP to accumulate and a fivefold drop in the production of amyloid peptide. Pulse-chase experiments indicated that PS1 deficiency specifically decreased the turnover of the membrane-associated fragments of APP. As in the regulation of cholesterol metabolism by proteolysis of a membrane-bound transcription factor, PS1 appears to facilitate a proteolytic activity that cleaves the integral membrane domain of APP. Our results indicate that mutations in PS1 that manifest clinically cause a gain of function and that inhibition of PS1 activity is a potential target for anti-amyloidogenic therapy in Alzheimer's disease.

Two mannose 6-phosphate receptors (MPR 300 and MPR 46) are involved in transport of lysosomal enzymes. Both receptors are expressed in all mammalian species studied so far and in chicken. Here we present the first report on affinity purification of both MPRs from the liver tissues of reptiles and amphibians using Sepharose divinyl sulfone phosphomannan at pH 7.0. MPR 300 from both species show similar electrophoretic mobility as mammalian MPR 300 and cross-react with an antibody directed against MPR 300 from goat liver. Furthermore, MPR 46 from reptilian liver and amphibian oocytes cross-react with peptide-specific antibodies against the cytoplasmic domain of human MPR 46 (anti-MSC1).

The cytoplasmic tail of the human 46 kDa mannose 6-phosphate receptor (MPR 46) is necessary for rapid internalization of the receptor and sufficient to mediate internalization of a resident plasma membrane protein. To localize the internalization sequences within the 67 amino acids of the cytoplasmic tail, the tail was progressively shortened from its C-terminus, internal deletions of between four and eight amino acids were introduced into the tail, and individual residues were substituted by alanine, glycine or serine. Three sequences were identified that contribute to the internalization of MPR 46. The first is located within the 23 juxtamembrane cytoplasmic residues of the tail. It contains four essential residues within a heptapeptide and does not resemble known internalization signals. The second sequence contains as a critical residue Tyr-45. The third region is located within the C-terminal seven residues and contains a di-leucine pair as essential residues. The first and third sequences were shown to function as autonomous internalization sequences. Substitution of critically important residues within a single internalization sequence was tolerated, with no or only a moderate decrease in the internalization rate. When essential residues from two or all three internalization sequences were substituted, however, the internalization rate was decreased by more than 60% and 90% respectively. This indicates that the autonomous internalization signals in the cytoplasmic tail of MPR 46 function in an additive manner, but are partly redundant.

Lysosomal acid phosphatase (LAP) is a tartrate-sensitive enzyme with ubiquitous expression. Neither the physiological substrates nor the functional significance is known. Mice with a deficiency of LAP generated by targeted disruption of the LAP gene are fertile and develop normally. Microscopic examination of various peripheral organs revealed progredient lysosomal storage in podocytes and tubular epithelial cells of the kidney, with regionally different ultrastructural appearance of the stored material. Within the central nervous system, lysosomal storage was detected to a regionally different extent in microglia, ependymal cells, and astroglia concomitant with the development of a progressive astrogliosis and microglial activation. Whereas behavioral and neuromotor analyses were unable to distinguish between control and deficient mice, approximately 7% of the deficient animals developed generalized seizures. From the age of 6 months onward, conspicuous alterations of bone structure became apparent, resulting in a kyphoscoliotic malformation of the lower thoracic vertebral column. We conclude from these findings that LAP has a unique function in only a subset of cells, where its deficiency causes the storage of a heterogeneously appearing material in lysosomes. The causal relationship of the enzyme defect to the clinical manifestations remains to be determined.

betaA4-Amyloid peptide, the main component of the amyloid plaques in the brain of Alzheimer's disease patients is produced from amyloid precursor protein (APP) by proteolytical processing. Several lines of evidence suggest a direct role for cathepsin D, the major endosomal/lysosomal aspartic endopeptidase, in betaA4-amyloid peptide generation. Here we tested this hypothesis using primary cultures of hippocampal neurons derived from cathepsin D-deficient (knock out) mice and expressing wild-type human APP and two clinical APP variants via recombinant Semliki Forest virus. We demonstrate APP secretory processing, production of carboxyl-terminal amyloid fragments, and secretion of the betaA4-amyloid peptide in the complete absence of cathepsin D. The results rule out cathepsin D as a critical component of alpha-, beta-, or gamma-secretase and therefore as a primary target for drugs aimed at decreasing the betaA4-amyloid peptide burden in Alzheimer's disease.

In mammals, the sorting of newly synthesized lysosomal enzymes is accomplished by two mannose 6-phosphate receptors (MPR) designated MPR46 and MPR300. MPR300 has an additional function in clearing the nonglycosylated insulin-like growth factor II (IGFII). The distinct expression pattern of the two MPR has been ascribed to the control of MPR300 expression by IGFII. In lower vertebrates, such as chickens or frogs, only MPR300 homologues have been described. These MPR300 homologues do not bind IGFII. In the present study, we examined whether lower vertebrates such as chickens also express two types of MPR and, if so, whether the expression pattern is distinct or similar. We were able to clone chicken cDNA fragments homologous to mammalian MPR46 and MPR300 and to show the synthesis of respective MPR polypeptides, thus establishing the existence of two types of MPR also in a nonmammalian species. Further, we analyzed the expression of the two MPR in chicken by Northern blotting and in situ hybridization. High levels of MPR46 and MPR300 RNA were detectable in epithelia, ganglia, and uropoietic system of chicken embryos. In a number of embryonic and adult tissues, varying ratios of MPR46 and MPR300 RNA were observed. The expression pattern for both MPR46 and MPR300 was distinct, although less pronounced than in mice. We conclude that functional differences unrelated to the additional function of the mammalian MPR300 as a receptor clearing IGFII are responsible for the distinct expression of the two MPR in nonmammalian, and probably also in mammalian, species.

Mammalian cells express two different mannose 6-phosphate receptors (MPR 46 and MPR 300), which both mediate targeting of Man-6-P-containing lysosomal proteins to lysosomes. To assess the contribution of either and both MPRs to the transport of lysosomal proteins, fibroblasts were established from mouse embryos that were homozygous for disrupted alleles of either MPR 46 or MPR 300 or both MPRs. Fibroblasts missing both MPRs secreted most of the newly synthesized lysosomal proteins and were unable to maintain the catabolic function of lysosomes. The intracellular levels of lysosomal proteins decreased to < 20%, and undigested material accumulated in the lysosomal compartment. Fibroblasts lacking either MPR exhibited only a partial missorting and maintained, in general, half-normal to normal levels of lysosomal proteins. The same species of lysosomal proteins were found in secretions of double MPR-deficient fibroblasts as in secretions of single MPR-deficient fibroblasts, but at different ratios. This clearly indicates that neither MPR has an exclusive affinity for one or several lysosomal proteins. Furthermore, neither MPR can substitute in vivo for the loss of the other. It is proposed that the heterogeneity of the Man-6-P recognition marker within a lysosomal protein and among different lysosomal proteins has necessitated the evolution of two MPRs with complementary binding properties to ensure an efficient targeting of lysosomal proteins.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) binds insulin-like growth factor II (IGF-II) with high affinity. To localize the IGF-II binding site within the 15 repeating units that form the extracytoplasmic domain of the receptor, purified human M6P/IGF-II receptor was digested with thermolysin, and the fragments were analyzed for their ability to bind 125I-IGF-II in a cross-linking assay. Two IGF-II-binding receptor fragments of 23 and 37 kDa were purified. Sequence analysis revealed that the fragments consist of disulfide connected peptides comprising amino acids 1331-1566 and 1331-1697 of the receptor repeats 9-12. In a second approach we expressed truncated forms of the M6P/IGF-II receptor fused to the C terminus of the extracytoplasmic domain of the 46-kDa mannose 6-phosphate receptor. Fusion proteins containing M6P/IGF-II receptor repeats 10-15, 10-11, or 11-15 bound IGF-II, whereas a fusion protein containing the single repeat 10 failed to bind. This result indicates that repeat 11 (amino acids 1508-1650) is sufficient for binding of IGF-II. Residues 1508-1566, which are shared by the 23-kDa IGF-II-binding fragment and repeat 11, are proposed to form the IGF-II binding site of the M6P/IGF-II receptor.

Lysosomal membrane glycoproteins are highly glycosylated proteins decorating the luminal surface of lysosomal membranes. Their biosynthetic route from the rough endoplasmic reticulum to the lysosomal compartment has been elucidated during recent years. Signals for intracellular sorting have been identified and characterized. The function of these proteins remains to be determined. Besides resident proteins the lysosomal membrane harbours at least one transient passenger, lysosomal acid phosphatase, which is sorted as a membrane-bound precursor like resident lysosomal membrane proteins and liberated from the membrane by limited proteolysis upon arrival in dense lysosomes.

A cloned mouse genomic DNA fragment containing the gene encoding cathepsin D (Catd) encompasses 11 kb of genomic DNA and is composed of 9 exons. Using recombinant inbred strains, we localized the Catd gene on chromosome 4, tightly linked to the loci Mtv-13, Cyp4a, Ms15-1, and Pmv-19. The exon-intron organization of the Catd gene was shown to be very similar to that of its human counterpart. Presence of a CpG island, absence of a TATA box, and initiation of transcription at more than one site indicate that the Catd gene is a "housekeeping" gene. A 1.2-kb fragment containing the 5'-flanking region of the gene displayed promoter activity in BHK-21 cells. Comparison of the nucleotide sequences of mouse and human cathepsin D promoter regions revealed conservation of three potential regulatory elements: an E box, a GC box and a potential cAMP-responsive element. In contrast to the 5' region of human cathepsin D, the murine gene contains three CCAAT boxes but lacks any of the four AP2 binding sites found in the human gene.

Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI, MPS VI) is an autosomally inherited lysosomal storage disorder caused by a deficiency of N-acetylgalactosamine-4-sulfatase (EC 3.1.6.1; 4-sulfatase). In order to determine the gene defect in a clinically severe MPS VI patient, polymerase chain reaction (PCR) products were generated from the patient's fibroblast mRNA and also from a 4-sulfatase cDNA clone and subjected to the chemical cleavage technique to detect mismatched bases, which were then identified by direct DNA sequencing of the PCR products. The patient was homozygous for an early frameshift mutation caused by the deletion of a G at position 238 (ΔG238), which produces a truncated 4-sulfatase with an altered amino acid sequence from amino acid 80 to a premature stop codon at codon 113 relative to the normal 4-sulfatase reading frame of 533 amino acids. Since the mutation occurs only 40 amino acids past the signal peptidase cleavage site, it is most likely that this will result in a protein with no 4-sulfatase activity. This is consistent with the severe clinical presentation and the absence of 4-sulfatase enzyme activity or mutant 4-sulfatase protein in the patient. The patient was also found to be homozygous for two polymorphisms, i.e., a G to A transition at nucleotide 1072 resulting in a valine358 to methionine substitution (V358M) and a silent A to G transition in the third base of the proline397 codon at nucleotide 1191. © 1992 Wiley-Liss, Inc.