Several cooperating binding sites mediate the interaction of a lysosomal enzyme with phosphotransferase.

Lysosomal targeting of soluble lysosomal hydrolases is mediated by mannose 6-phosphate receptors, which recognize and bind mannose 6-phosphate residues in the oligosaccharide chains of proteins destined for delivery to lysosomes. This recognition marker is generated by the sequential action of two enzymes, the first of which, UDP-N-acetylglucosamine phosphotransferase, recognizes lysosomal enzymes on the basis of a structural determinant in their polypeptide chains. This recognition event is a key step in lysosomal targeting of soluble proteins, but the exact nature of the recognition determinant is not well understood. In this study we have characterized the phosphotransferase recognition signals of human lysosomal aspartylglucosaminidase (AGA) using transient expression of polypeptides carrying targeted amino acid substitutions. We found that three lysine residues and a tyrosine residing in three spatially distinct regions of the AGA polypeptide are necessary for phosphorylation of the oligosaccharides. Two of the lysines are especially important for the lysosomal targeting efficiency of AGA, which seems to be mostly dictated by the degree of phosphorylation of the alpha subunit oligosaccharide. On the basis of the results of this and previous studies we suggest a general model for recognition of lysosomal enzymes by the phosphotransferase.     

In vitro mutagenesis of potential N-glycosylation sites of arylsulfatase A. Effects on glycosylation, phosphorylation, and intracellular sorting.

The correct intracellular sorting of lysosomal enzymes such as arylsulfatase A depends on the presence of mannose 6-phosphate residues on high mannose type oligosaccharides. The arylsulfatase A cDNA contains three potential N-glycosylation sites, two of which are utilized. We have mutated one or two of the N-glycosylation sites and analyzed the glycosylation, phosphorylation, and intracellular sorting of the mutant arylsulfatase A polypeptides. The results show that each of the three glycosylation sites (I, II, and III) can be glycosylated, but glycosylation at sites I and II is mutually exclusive. In mutants with one oligosaccharide side chain at positions I, II, or III all side chains can acquire mannose 6-phosphate residues irrespective of their location. This demonstrates spatial flexibility of the phosphotransferase, which specifically recognizes lysosomal enzymes and initiates the addition of mannose 6-phosphate residues on oligosaccharide side chains. However, these mutants have different intracellular sorting efficiencies and seem to use different (mannose 6-phosphate receptor-dependent and -independent) sorting pathways.     

Identification of the minimal lysosomal enzyme recognition domain in cathepsin D

Specific recognition of lysosomal hydrolases by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues, is governed by a common protein determinant. Previously, we generated a lysosomal enzyme recognition domain in the secretory protein glycopepsinogen by substituting in two regions (lysine 203 and amino acids 265-293 of the beta loop) from cathepsin D, a highly related lysosomal protease. Here we show that substitution of just two lysines (Lys-203 and Lys-267) stimulates mannose phosphorylation 116-fold. Substitution of additional residues in the beta loop, particularly lysines, increased phosphorylation 4-fold further, approaching the level obtained with intact cathepsin D. All the phosphorylation occurred at the carboxyl lobe glycan, indicating that additional elements are required for phosphorylation of the amino lobe glycan. These data support the proposal that as few as two lysines in the correct orientation to each other and to the glycan can serve as the minimal elements of the lysosomal enzyme recognition domain. However, our findings show that the spacing between lysines is flexible and other residues contribute to the recognition marker.     

Is the bovine lysosomal phospholipase B‐like protein an amidase?

The main function of lysosomal proteins is to degrade cellular macromolecules. We purified a novel lysosomal protein to homogeneity from bovine kidneys. By gene annotation, this protein is defined as a bovine phospholipase B‐like protein 1 (bPLBD1) and, to better understand its biological function, we solved its structure at 1.9 Å resolution. We showed that bPLBD1 has uniform noncomplex‐type N‐glycosylation and that it localized to the lysosome. The first step in lysosomal protein transport, the initiation of mannose‐6‐phosphorylation by a N‐acetylglucosamine‐1‐phosphotransferase, requires recognition of at least two distinct lysines on the protein surface. We identified candidate lysines by analyzing the structural and sequentially conserved N‐glycosylation sites and lysines in bPLBD1 and in the homologous mouse PLBD2. Our model suggests that N408 is the primarily phosphorylated glycan, and K358 a key residue for N‐acetylglucosamine‐1‐phosphotransferase recognition. Two other lysines, K334 and K342, provide the required second site for N‐acetylglucosamine‐1‐phosphotransferase recognition. bPLBD1 is an N‐terminal nucleophile (Ntn) hydrolase. By comparison with other Ntn‐hydrolases, we conclude that the acyl moiety of PLBD1 substrate must be small to fit the putative binding pocket, whereas the space for the rest of the substrate is a large open cleft. Finally, as all the known substrates of Ntn‐hydrolases have amide bonds, we suggest that bPLBD1 may be an amidase or peptidase instead of lipase, explaining the difficulty in finding a good substrate for any members of the PLBD family. Proteins 2014; 82:300–311. © 2013 Wiley Periodicals, Inc.     

Arylsulfatase G, a novel lysosomal sulfatase

The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site C(alpha)-formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder.     

Arylsulfatase K,a Novel Lysosomal Sulfatase

The human sulfatase family has 17 members, 13 of which have been characterized biochemically. These enzymes specifically hydrolyze sulfate esters in glycosaminoglycans, sulfolipids, or steroid sulfates, thereby playing key roles in cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked to severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase K (ARSK), was identified bioinformatically through its conserved sulfatase signature sequence directing posttranslational generation of the catalytic formylglycine residue in sulfatases. However, overall sequence identity of ARSK with other human sulfatases is low (18–22%). Here we demonstrate that ARSK indeed shows desulfation activity toward arylsulfate pseudosubstrates. When expressed in human cells, ARSK was detected as a 68-kDa glycoprotein carrying at least four N-glycans of both the complex and high-mannose type. Purified ARSK turned over p-nitrocatechol and p-nitrophenyl sulfate. This activity was dependent on cysteine 80, which was verified to undergo conversion to formylglycine. Kinetic parameters were similar to those of several lysosomal sulfatases involved in degradation of sulfated glycosaminoglycans. An acidic pH optimum (∼4.6) and colocalization with LAMP1 verified lysosomal functioning of ARSK. Further, it carries mannose 6-phosphate, indicating lysosomal sorting via mannose 6-phosphate receptors. ARSK mRNA expression was found in all tissues tested, suggesting a ubiquitous physiological substrate and a so far non-classified lysosomal storage disorder in the case of ARSK deficiency, as shown before for all other lysosomal sulfatases.     

Developmental profiles of arylsulfatases A and B in rat cerebral cortex and spinal cord.

Arylsulfatases A (EC 3.1.6.1) and B (EC 3.1.6.12) are lysosomal enzymes that can remove sulfate groups from sulfatides and sulfo-glycosaminoglycans, respectively. The activities of these enzymes in cerebral cortex and in spinal cord of developing rat pups were measured. The tissues were homogenized and the arylsulfatases A and B in the soluble fraction were separated from each other by anion exchange chromatography on DE-52 cellulose. Subsequently, the enzyme activities were assayed with p-nitrocatechol sulfate as substrate at 37 degrees C and pH 5.6. We observed a developmental profile of arylsulfatase A, similar to that previously reported for cerebroside sulfatase (EC 3.1.6.8; (Van der Pal et al. (1990) Biochim. Biophys. Acta 1043, 91-96]. The activity of arylsulfatase A increased gradually during development, whereas arylsulfatase B rose more steeply, peaked around day 15 and declined thereafter. As a consequence the ratio between B and A forms of arylsulfatase dropped from about 4 in 1-week-old pups to 2.2 (cortex) and 0.7 (cord) in 7-week-old rat pups.     

Mannose 6-phosphate-independent targeting of lysosomal enzymes in I- cell disease B lymphoblasts

B lymphocytes from patients with I-cell disease (ICD) maintain normal cellular levels of lysosomal enzymes despite a deficiency of the enzyme UDP-N-acetylglucosamine: lysosomal enzyme N-acetylglucosamine-1- phosphotransferase. We find that an ICD B lymphoblastoid cell line targets about 45% of the lysosomal protease cathepsin D to dense lysosomes. This targeting occurs in the absence of detectable mannose 6- phosphate residues on the cathepsin D and is not observed in ICD fibroblasts. The secretory protein pepsinogen, which is closely related to cathepsin D in both amino acid sequence and three-dimensional structure, is mostly excluded from dense lysosomes, indicating that the lymphoblast targeting pathway is specific. Carbohydrate residues are not required for lysosomal targeting, since a non-glycosylated mutant cathepsin D is sorted with comparable efficiency to the wild type protein. Analysis of a number of cathepsin D/pepsinogen chimeric proteins indicates that an extensive polypeptide determinant in the cathepsin D carboxyl lobe can confer efficient lysosomal sorting when introduced into the pepsinogen sequence. This determinant overlaps but is not identical to the recognition marker for phosphotransferase. These results indicate that a specific protein recognition event underlies Man-6-P-independent lysosomal sorting in ICD lymphoblasts.     

Phosphorylation of Asn-linked oligosaccharides located at novel sites on the lysosomal enzyme cathepsin D.

We have examined the phosphorylation of Asn-linked oligosaccharides introduced at seven novel sites on human cathepsin D to determine whether the location of an oligosaccharide on a lysosomal enzyme affects its ability to serve as a substrate for UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (phosphotransferase), the enzyme that catalyzes the initial step in the biosynthesis of mannose 6-phosphate residues. The glycosylation sites were introduced into the cathepsin D cDNA by site-directed mutagenesis and were selected to be widely distributed over the surface of the molecule. When the constructs were expressed in Xenopus oocytes, the oligosaccharides at each glycosylation site were phosphorylated at levels considerably above background (19-70% phosphorylation versus < 0.4% for the secretory protein glycopepsinogen). However, oligosaccharides located closer to the essential components of the phosphotransferase recognition domain (lysine 203 and amino acids 265-292) were phosphorylated better than oligosaccharides located further away. Similar results were obtained for oligosaccharides at homologous sites on a pepsinogen/cathepsin D chimera containing only lysine 203 and residues 265-319 of cathepsin D, although the absolute levels of phosphorylation were lower. These results demonstrate that there is considerable flexibility in the placement of glycosylation sites on cathepsin D in terms of the ability of the oligosaccharides to serve as substrates for phosphotransferase, although oligosaccharides located closer to the phosphotransferase recognition determinant are preferentially phosphorylated.     

Lysosomal enzyme phosphorylation. I. Protein recognition determinants in both lobes of procathepsin D mediate its interaction with UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase.

We have investigated the nature of a protein domain that is shared among lysosomal hydrolases and is recognized by UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the initial enzyme in the biosynthesis of mannose 6-phosphate residues. Previously, elements of this recognition domain were identified using a chimeric protein approach. The combined substitution of two regions (amino acids 188-230, particularly lysine 203, and 265-292) from the carboxyl lobe of the lysosomal hydrolase cathepsin D into the homologous positions of the related secretory protein glycopepsinogen was sufficient to confer recognition by phosphotransferase and subsequent phosphorylation of the oligosaccharides when this chimeric protein was expressed in Xenopus oocytes. (Baranski, T. J., Faust, P. L., and Kornfeld, S. (1990) Cell 63, 281-291). The current study demonstrates that when these two regions are replaced in cathepsin D by the homologous glycopepsinogen amino acids, the resultant chimeric molecule is poorly phosphorylated. However, when either of these regions is substituted individually, the chimeric molecules are well phosphorylated. The phosphorylation of these latter chimeric proteins is dependent on the presence of procathepsin D amino lobe elements. By analyzing a series of chimeric proteins that contain all eight combinations of three consecutive segments of the entire amino lobe of procathepsin D, it was found that multiple regions of the amino lobe of cathepsin D enhance phosphorylation of the chimeric proteins. These elements may be part of an extended carboxyl lobe recognition domain or comprise a second independent recognition domain.     

A specific ultrastructural stain for arylsulfatase A activity in human cultured fibroblasts

A staining reaction was developed to specifically detect arylsulfatase A activity in the presence of arylsulfatases B and C. Nitrocatechol, generated by all arylsulfatases from the substrate p-nitrocatechol sulfate, can be coupled to produce Hatchett 's brown which reacts with 3,3'-diaminobenzidine to yield an osmiophilic polymer visible under the electron microscope. The reaction was made specific for arylsulfatase A by inhibiting arylsulfatase C activity with low pH and arylsulfatase B activity with pyrophosphate. The specificity was confirmed both by electrophoretic analysis and by patient fibroblasts deficient only in arylsulfatase A activity. Under optimal conditions for preserving structural integrity and enzyme activity, enzyme reaction deposits were found mainly around vesicles. Some of these vesicles were large and heterogeneous (48-330 nm in diameter), distributed randomly within the cytoplasm, but most of the positive-reacting vesicles were uniform in size (86 +/- 18 nm in diameter) and distributed in a peripheral zone about 0.1-0.5 micron wide. These periplasmic vesicles might be partly fused with each other or with the plasma membrane. In conclusion, a specific stain for arylsulfatase A activity suitable for light and electron microscopy and the optimal conditions for structural and enzymatic preservations were developed. Although this enzyme has been considered to be lysosomal in origin, most of the activity was detected in periplasmic vesicles near the cell surface.     

Compensatory expression of human N-Acetylglucosaminyl-1-phosphotransferase subunits in mucolipidosis type III gamma

The N-Acetylglucosaminyl-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate (M6P) recognition markers essential for efficient transport of lysosomal hydrolases to lysosomes. The phosphotransferase is composed of six subunits (α₂, β₂, γ₂). The α- and β-subunits are catalytically active and encoded by a single gene, GNPTAB, whereas the γ-subunit encoded by GNPTG is proposed to recognize conformational structures common to lysosomal enzymes. Defects in GNPTG cause mucolipidosis type III gamma, which is characterized by missorting and cellular loss of lysosomal enzymes leading to lysosomal accumulation of storage material. Using plasmon resonance spectrometry, we showed that recombinant γ-subunit failed to bind the lysosomal enzyme arylsulfatase A. Additionally, the overexpression of the γ-subunit in COS7 cells did not result in hypersecretion of newly synthesized lysosomal enzymes expected for competition for binding sites of the endogenous phosphotransferase complex. Analysis of fibroblasts exhibiting a novel mutation in GNPTG (c.619insT, p.K207IfsX7) revealed that the expression of GNPTAB was increased whereas in γ-subunit overexpressing cells the GNPTAB mRNA was reduced. The data suggest that the γ-subunit is important for the balance of phosphotransferase subunits rather for general binding of lysosomal enzymes.     

Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii.

The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase.     

Investigation of the conserved lysines of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase

Sequence analysis has revealed two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Crystal structures of ternary complexes of the Class II enzyme from Pseudomonas mevalonii revealed lysine 267 critically positioned at the active site. This observation suggested a revised catalytic mechanism in which lysine 267 facilitates hydride transfer from reduced coenzyme by polarizing the carbonyl group of HMG-CoA and subsequently of bound mevaldehyde, an inference supported by mutagenesis of lysine 267 to aminoethylcysteine. For this mechanism to be general, Class I HMG-CoA reductases ought also to possess an active site lysine. Three lysines are conserved among all Class I HMG-CoA reductases. The three conserved lysines of Syrian hamster HMG-CoA reductase were mutated to alanine. All three mutant enzymes had reduced but detectable activity. Of the three conserved lysines, sequence alignments implicate lysine 734 of the hamster enzyme as the most likely cognate of P. mevalonii lysine 267. Low activity of enzyme K734A did not reflect an altered structure. Substrate recognition was essentially normal, and both circular dichroism spectroscopy and analytical ultracentrifugation implied a native structure. Enzyme K734A also formed an active heterodimer when coexpressed with inactive mutant enzyme D766N. We infer that a lysine is indeed essential for catalysis by the Class I HMG-CoA reductases and that the revised mechanism for catalysis is general for all HMG-CoA reductases.     

Lysosomal enzyme phosphorylation. II. Protein recognition determinants in either lobe of procathepsin D are sufficient for phosphorylation of both the amino and carboxyl lobe oligosaccharides.

Cathepsin D is a bilobed lysosomal aspartyl protease that contains one Asn-linked oligosaccharide/lobe. Each lobe also contains protein determinants that serve as recognition domains for binding of UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase, the first enzyme in the biosynthesis of the mannose 6-phosphate residues on lysosomal enzymes. In this study we examined whether the location of the protein recognition domain influences the relative phosphorylation of the amino and carboxyl lobe oligosaccharides. To do this, chimeric proteins containing either amino or carboxyl lobe sequences of cathepsin D substituted into a glycosylated form of the homologous secretory protein pepsinogen were expressed in Xenopus oocytes. The amino and carboxyl lobe oligosaccharides were then isolated from the various chimeric proteins and independently analyzed for their mannose 6-phosphate content. This analysis has shown that a phosphotransferase recognition domain located on either lobe of a cathepsin D/glycopepsinogen chimeric molecule is sufficient to allow phosphorylation of oligosaccharides on both lobes. However, phosphorylation of the oligosaccharide on the lobe containing the recognition domain is favored. We also found that the majority of the carboxyl lobe oligosaccharides of cathepsin D acquire two phosphates, whereas the amino lobe oligosaccharides only acquire one phosphate.     

Renin, a secretory glycoprotein, acquires phosphomannosyl residues

Renin is an aspartyl protease which is highly homologous to the lysosomal aspartyl protease cathepsin D. During its biosynthesis, cathepsin D acquires phosphomannosyl residues that enable it to bind to the mannose 6-phosphate (Man-6-P) receptor and to be targeted to lysosomes. The phosphorylation of lysosomal enzymes by UDP- GlcNAc:lysosomal enzyme N-acetylglucosaminylphosphotransferase (phosphotransferase) occurs by recognition of a protein domain that is thought to be present only on lysosomal enzymes. In order to determine whether renin, being structurally similar to cathepsin D, also acquires phosphomannosyl residues, human renin was expressed from cloned DNA in Xenopus oocytes and a mouse L cell line and its biosynthesis and posttranslational modifications were characterized. In Xenopus oocytes, the majority of the renin remained intracellular and underwent a proteolytic cleavage which removed the propiece. Most of the renin synthesized by oocytes was able to bind to a Man-6-P receptor affinity column (53%, 57%, and 90%, in different experiments), indicating the presence of phosphomannosyl residues. In the L cells, the majority of the renin was secreted but 5-6% of the renin molecules contained phosphomannosyl residues as demonstrated by binding of [35S]methionine- labeled renin to the Man-6-P receptor as well as direct analysis of [2- 3H]mannose-labeled oligosaccharides. Although the level of renin phosphorylation differed greatly between the two cell types examined, these results demonstrate that renin is recognized by the phosphotransferase and suggest that renin contains at least part of the lysosomal protein recognition domain.     

Affinity labelling of enzymes with triazine dyes. Isolation of a peptide in the catalytic domain of horse-liver alcohol dehydrogenase using Procion blue MX-R as a structural probe

The incorporation of [3H]leucine and [32P]phosphate into three lysosomal enzymes, cathepsin D, beta-hexosaminidase and arylsulfatase A by fibroblasts from six patients affected with mucolipidosis III was determined. In the mutant cells the incorporation of 32P in the enzymes was reduced by 70-97% as compared to controls. The residual phosphorylation of lysosomal enzymes is definitely higher than in fibroblasts from patients with mucolipidosis II, where apparently non-phosphorylated enzymes are formed. In mucolipidosis III the major part of the newly formed enzymes accumulated extracellularly and the cellular enzymes were recovered mainly in their processed forms. In mucolipidosis III arylsulfatase A and the processed forms of cathepsin D exhibited a heterogeneity that was not observed in controls. beta-Hexosaminidase and cathepsin D secreted by mucolipidosis III fibroblasts contained only a small amount of phosphorylated oligosaccharides with either one or two phosphate groups per oligosaccharide. As in controls the major fraction of phosphate was present as acid-labile phosphodiester resistant to alkaline phosphatase. The residual phosphorylation of lysosomal enzymes may be related to the partial intracellular retention and processing of these enzymes in fibroblasts from patients with mucolipidosis III.     

Characterization of the mannose 6-phosphate receptor (Mr 300 kDa) protein dependent pathway of lysosomal enzyme targeting in Biomphalaria glabrata mollusc cells

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) are involved in the targeting of newly synthesized lysosomal enzymes and only MPR 300 also participates in the endocytosis of various exogenous ligands. The present study describes for the first time the MPR 300 dependent pathway of lysosomal enzyme sorting in the Biomphalaria glabrata embryonic (Bge) cells. Lysosomal enzymes (arylsulfatase A, β-hexosaminidase and α-fucosidase) were identified by their enzymatic activities and by immunoprecipitation with specific antisera. Exposure of Bge cells to unio MPR 300 antiserum resulted in a dramatic loss of MPR 300 protein with a shortened half life of ∼20 min as compared to control cells exposed to preimmune serum in which the half life of MPR 300 was of ∼13 h. Loss of receptor proteins resulted in a significant misrouting of newly synthesized lysosomal enzymes and their secretion in cell culture medium as demonstrated by immunoprecipitation. The ability of Bge cells to uptake and internalize labeled arylsulfatase A, β-hexosaminidase and α-fucosidase enzymes contained in cell secretion products also indicated the role of B. glabrata MPR 300 (CIMPR) protein in internalization and targeting of lysosomal enzymes. M6P dependent binding of lysosomal enzymes to MPR 300 was shown by confocal microscopy and coimmunoprecipitation experiments.     

Pathochemistry, pathogenesis and enzyme replacement in multiple-sulfatase deficiency

Multiple-sulfatase deficiency (MSD) is now considered to be heterogeneous and could be classified into three or four clinical phenotypes according to the onset of the disease: neonatal, late infantile, juvenile and possibly adult type. Neonatal-type MSD shows severe clinical involvement and practically no arylsulfatase A, B and C activities in cultured skin fibroblasts. Furthermore, arylsulfatase A activity in neonatal-type MSD was not enhanced by the addition of thiosulfate. Therefore, it is distinct from late infantile-type MSD. The degradation of 14C-sulfatide can occur in MSD-cultured skin fibroblasts and was much higher than in late infantile-type MLD. The addition of thiol protease such as leupeptin to cultured MSD skin fibroblasts enhanced arylsulfatase A activity as well as the degradation of 14C-sulfatide. This suggests that the decreased activities of arylsulfatase A is due to an acceleration of the enzyme degradation. Enzyme replacement by the addition of arylsulfatases of different sources (human liver, brain, fungus) was carried out in cultured MSD skin fibroblasts. Human enzymes of arylsulfatase A and B were mostly taken up by MSD cells rather than those of fungus origin. By the exposure to leukocytes to cultured skin fibroblasts, MSD cells corrected arylsulfatase A and B activities.     

Synthesis and stability of arylsulfatase A and B in fibroblasts from multiple sulfatase deficiency

Fibroblasts from patients with multiple sulfatase deficiency were analyzed for activities of arylsulfatase A and B, iduronate 2-sulfatase and sulfamatase. A group of patients (group I) severely deficient in all sulfatases (residual activities less than or equal to 10% of control) were differentiated from patients (group II) with residual sulfatase activities of up to 90% of control. The synthesis and stability of arylsulfatase A and B were determined in pulse-chase labelling experiments. The apparent rate of synthesis of arylsulfatase A and B varied from 30% to normal in both fibroblasts from group I and II multiple sulfatase deficiency. In group I the molecular activity of the arylsulfatase A and B was more than 10-fold lower than in control fibroblasts. In group II the molecular activity of the arylsulfatase A was twofold to threefold lower and that of arylsulfatase B half of normal. In fibroblasts of both groups the stability of arylsulfatase A polypeptides was significantly diminished. For arylsulfatase B the instability was restricted to the mature 47000-Mr polypeptide and was variable within both groups. These results demonstrate that multiple sulfatase deficiency is a heterogeneous disorder, in which the primary defects can impair both the catalytic properties and the stability of sulfatases.