Keratan sulphate levels in mucopolysaccharidoses and mucolipidoses.

The mucopolysaccharidoses (MPS) is characterized by accumulation of glycosaminoglycans (GAGs), and mucolipidosis (ML) by accumulation of GAGs and sphingolipids. Each type of MPS accumulates specific GAGs. The lysosomal enzymes N-acetylgalactosamine-6-sulphate sulphatase and beta-galactosidase involve the stepwise degradation of keratan sulphate (KS). Deficiency of these enzymes results in elevation of KS levels in the body fluids and in tissues, leading to MPS IV disease. In this study, we evaluated blood and urine KS levels in types of MPS and ML other than MPS IV. Eighty-five plasma samples came from MPS I (n = 18), MPS II (n = 28), MPS III (n = 20), MPS VI (n = 3), MPS VII (n = 5) and ML (n = 11) patients while 127 urine samples came from MPS I (n = 34), MPS II (n = 34), MPS III (n = 32), MPS VI (n = 7), MPS VII (n = 9) and ML (n = 11) patients. KS levels were determined using the ELISA method. Plasma KS levels varied with age in both control and patient populations. In all age groups, the mean values of plasma KS in MPS and ML patients were significantly higher than those in the age-matched controls. Plasma KS values in four newborn patients were above the mean + 2SD of the age-matched controls (mean, 41 ng/ml). Overall, 85.9% of individual values in non-type IV MPS and ML patients were above the mean + 2SD of the age-matched controls. For urine KS levels, 24.4% of individual values in patients were above the mean + 2SD of the age-matched controls. In conclusion, KS in blood is elevated in each type of non-type IV MPS examined, in contrast to the conventional understanding. This finding suggests that measurement of KS level provides a new diagnostic biomarker in a wide variety of mucopolysaccharidoses and mucolipidoses in addition to MPS IV.

Biodistribution, kinetics, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase in the murine model of mucopolysaccharidosis VII.

Enzyme replacement therapy (ERT) has been shown to be effective at reducing the accumulation of undegraded substrates in lysosomal storage diseases. Most ERT studies have been performed with recombinant proteins that are mixtures of phosphorylated and non-phosphorylated enzyme. Because different cell types use different receptors to take up phosphorylated or non-phosphorylated enzyme, it is difficult to determine which form of enzyme contributed to the clinical response. Here we compare the uptake, distribution, and efficacy of highly phosphorylated and non-phosphorylated beta-glucuronidase (GUSB) in the MPS VII mouse. Highly phosphorylated murine GUSB was efficiently taken up by a wide range of tissues. In contrast, non-phosphorylated murine GUSB was taken up primarily by tissues of the reticuloendothelial (RE) system. Although the tissue distribution was different, the half-lives of both enzymes in any particular tissue were similar. Both preparations of enzyme were capable of preventing the accumulation of lysosomal storage in cell types they targeted. An important difference in clinical efficacy emerged in that phosphorylated GUSB was more efficient than non-phosphorylated enzyme at preventing the hearing loss associated with this disease. These data suggest that both forms of enzyme contribute to the clinical responses of ERT in MPS VII mice but that enzyme preparations containing phosphorylated GUSB are more broadly effective than non-phosphorylated enzyme.

Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells.

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC ) XII is observed in certain human cancers. This bitopic membrane protein contains an N-terminal extracellular catalytic domain, a membrane-spanning alpha-helix, and a small intracellular C-terminal domain. We have determined the three-dimensional structure of the extracellular catalytic domain of human CA XII by x-ray crystallographic methods at 1.55-A resolution. The structure reveals a prototypical CA fold; however, two CA XII domains associate to form an isologous dimer, an observation that is confirmed by studies of the enzyme in solution. The identification of signature GXXXG and GXXXS motifs in the transmembrane sequence that facilitate helix-helix association is additionally consistent with dimeric architecture. The dimer interface is situated so that the active site clefts of each monomer are clearly exposed on one face of the dimer, and the C termini are located together on the opposite face of the dimer to facilitate membrane interaction. The amino acid composition of the active-site cleft closely resembles that of the other CA isozymes in the immediate vicinity of the catalytic zinc ion, but differs in the region of the nearby alpha-helical "130's segment." The structure of the CA XII-acetazolamide complex is also reported at 1.50-A resolution, and prospects for the design of CA XII-specific inhibitors of possible chemotherapeutic value are discussed.

Active site mutant transgene confers tolerance to human beta-glucuronidase without affecting the phenotype of MPS VII mice.

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is an autosomal recessive lysosomal storage disorder due to an inherited deficiency of beta-glucuronidase. A naturally occurring mouse model for this disease was discovered at The Jackson Laboratory and shown to be due to homozygosity for a 1-bp deletion in exon 10 of the gus gene. The murine model MPS VII (gus(mps/mps)) has been very well characterized and used extensively to evaluate experimental strategies for lysosomal storage diseases, including bone marrow transplantation, enzyme replacement therapy, and gene therapy. To enhance the value of this model for enzyme and gene therapy, we produced a transgenic mouse expressing the human beta-glucuronidase cDNA with an amino acid substitution at the active site nucleophile (E540A) and bred it onto the MPS VII (gus(mps/mps)) background. We demonstrate here that the mutant mice bearing the active site mutant human transgene retain the clinical, morphological, biochemical, and histopathological characteristics of the original MPS VII (gus(mps/mps)) mouse. However, they are now tolerant to immune challenge with human beta-glucuronidase. This "tolerant MPS VII mouse model" should be useful for preclinical trials evaluating the effectiveness of enzyme and/or gene therapy with the human gene products likely to be administered to human patients with MPS VII.

Expression of membrane-associated carbonic anhydrase XIV on neurons and axons in mouse and human brain.

Although long suspected from histochemical evidence for carbonic anhydrase (CA) activity on neurons and observations that CA inhibitors enhance the extracellular alkaline shifts associated with synaptic transmission, an extracellular CA in brain had not been identified. A candidate for this CA was suggested by the recent discovery of membrane CA (CA XIV) whose mRNA is expressed in mouse and human brain and in several other tissues. For immunolocalization of CA XIV in mouse and human brain, we developed two antibodies, one against a secretory form of enzymatically active recombinant mouse CA XIV, and one against a synthetic peptide corresponding to the 24 C-terminal amino acids in the human enzyme. Immunostaining for CA XIV was found on neuronal membranes and axons in both mouse and human brain. The highest expression was seen on large neuronal bodies and axons in the anterolateral part of pons and medulla oblongata. Other CA XIV-positive sites included the hippocampus, corpus callosum, cerebellar white matter and peduncles, pyramidal tract, and choroid plexus. Mouse brain also showed a positive reaction in the molecular layer of the cerebral cortex and granular cellular layer of the cerebellum. These observations make CA XIV a likely candidate for the extracellular CA postulated to have an important role in modulating excitatory synaptic transmission in brain.

Purification and kinetic analysis of recombinant CA XII, a membrane carbonic anhydrase overexpressed in certain cancers.

Carbonic anhydrase XII (CA XII) is a transmembrane glycoprotein with an active extracellular CA domain that is overexpressed on cell surfaces of certain cancers. Its expression has been linked to tumor invasiveness. To characterize its catalytic properties, we purified recombinant secretory forms of wild-type and mutant CA XIIs. The catalytic properties of these enzymes in the hydration of CO(2) were measured at steady state by stopped-flow spectrophotometry and at chemical equilibrium by the exchange of (18)O between CO(2) and water determined by mass spectrometry. The catalysis of CO(2) hydration by soluble CA XII has a maximal value of k(cat)/K(m) at 34 microM(-1) small middle dots(-1), which is similar to those of the membrane-associated CA IV and to soluble CA I. The pH profiles of this catalysis and the catalyzed hydrolysis of 4-nitrophenylacetate indicate that the pK(a) of the zinc-bound water in CA XII is 7.1. His64 in CA XII acts as a proton shuttle residue, as evidenced by the reduced rate constant for proton transfer in the mutants containing the replacements His64 --> Ala and His64 --> Arg, as well as by the selective inhibition of the proton transfer step by cupric ions in wild-type CA XII. The catalytic rate of CO(2) hydration by the soluble form of CA XII is identical with that of the membrane-bound enzyme. These observations suggest a role for CA XII in CO(2)/HCO(3)(-) homeostasis in cells in which it is normally expressed. They are also compatible with a role for CA XII in acidifying the microenvironment of cancer cells in which CA XII is overexpressed, providing a mechanism for CA XII to augment tumor invasiveness and suggesting CA XII as a potential target for chemotherapeutic agents.

Mitochondrial carbonic anhydrase CA VB: differences in tissue distribution and pattern of evolution from those of CA VA suggest distinct physiological roles.

A cDNA for a second mouse mitochondrial carbonic anhydrase (CA) called CA VB was identified by homology to the previously characterized murine CA V, now called CA VA. The full-length cDNA encodes a 317-aa precursor that contains a 33-aa classical mitochondrial leader sequence. Comparison of products expressed from cDNAs for murine CA VB and CA VA in COS cells revealed that both expressed active CAs that localized in mitochondria, and showed comparable activities in crude extracts and in mitochondria isolated from transfected COS cells. Northern blot analyses of total RNAs from mouse tissues and Western blot analyses of mouse tissue homogenates showed differences in tissue-specific expression between CA VB and CA VA. CA VB was readily detected in most tissues, while CA VA expression was limited to liver, skeletal muscle, and kidney. The human orthologue of murine CA VB was recently reported also. Comparison of the CA domain sequence of human CA VB with that reported here shows that the CA domains of CA VB are much more highly conserved between mouse and human (95% identity) than the CA domains of mouse and human CA VAs (78% identity). Analysis of phylogenetic relationships between these and other available human and mouse CA isozyme sequences revealed that mammalian CA VB evolved much more slowly than CA VA, accepting amino acid substitutions at least 4.5 times more slowly since each evolved from its respective human-mouse ancestral gene around 90 million years ago. Both the differences in tissue distribution and the much greater evolutionary constraints on CA VB sequences suggest that CA VB and CA VA have evolved to assume different physiological roles.

Expression of a novel transmembrane carbonic anhydrase isozyme XII in normal human gut and colorectal tumors.

Carbonic anhydrase isozyme XII is a recently discovered member of the alpha-carbonic anhydrase gene family with a suggested role in von Hippel-Lindau gene-mediated carcinogenesis. Increased expression of its mRNA has been observed in renal and lung carcinomas. This paper presents the localization of CA XII in the normal human gut and in colorectal tumors. Immunohistochemistry performed using a polyclonal antibody raised against truncated CA XII revealed prominent polarized staining for CA XII in the basolateral plasma membrane of the enterocytes of the normal large intestine, the reaction being most intense in the surface epithelial cuff region. Most colorectal tumors displayed abnormal expression of CA XII; the most dramatic change was observed in the deep parts of the adenomatous mucosa, where the positive immunoreaction clearly increased along with the grade of dysplasia. Adenomas with severe dysplasia and carcinomas showed an equal, diffuse staining pattern. The results indicate region-specific regulation of CA XII expression along the cranial-caudal axis of the human gut, whereas its diffuse expression in the most malignant tumors seems to correlate with their biological behavior.

Identification of carbonic anhydrase XII as the membrane isozyme expressed in the normal human endometrial epithelium.

Although previous studies demonstrated carbonic anhydrase (CA) activity in the human endometrium, the CA isozyme(s) responsible for this activity has not been established. In this report, we provide the first evidence that the CA isozyme XII, a recently identified transmembrane isozyme that is expressed in normal kidney and greatly overexpressed in some renal cancers, is present in endometrium. We show by immunohistochemistry that CA XII is expressed in the basolateral plasma membrane of epithelial cells of normal human endometrium. Expression of CA XII in uterus was confirmed by Northern blotting. Detergent-solubilized CA XII was isolated from human endometrium by inhibitor affinity chromatography and characterized by isoelectric focusing and Western blot as a polypeptide with a pI of 6.3. The high expression of CA XII in the endometrial epithelium suggests that it may be functionally linked to the pH-dependent events in spermatozoa that precede fertilization. Its basolateral location and extracellular active site could also allow it to influence the morphological changes in endometrium that occur during the menstrual cycle.

Active site residues of human beta-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue.

Human beta-glucuronidase (hGUSB) is a member of family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. Amino acid sequence and structural homology of hGUSB and Escherichia coli beta-galactosidase active sites led us to propose that residues Glu(451), Glu(540), and Tyr(504) in hGUSB are involved in catalysis, Glu(451) being the acid-base residue and Glu(540) the nucleophile. To test this hypothesis, we introduced mutations in these residues and determined their effects on enzymes expressed in COS cells and GUSB-deficient fibroblasts. The extremely low activity in cells expressing Glu(451), Glu(540), and Tyr(504) hGUSBs supported their roles in catalysis. For kinetic analysis, wild type and mutant enzymes were produced in baculovirus and purified to homogeneity by affinity chromatography. The k(cat)/K(m) values (mM(-1).s(-1)) of the E540A, E451A, and Y504A enzymes were 34,000-, 9100-, and 830-fold lower than that of wild type hGUSB, respectively. High concentrations of azide stimulated the activity of the E451A mutant enzyme, supporting the role of Glu(451) as the acid-base catalyst. We conclude that, like their homologues in E. coli beta-galactosidase, Glu(540) is the nucleophilic residue, Glu(451) the acid-base catalyst, and Tyr(504) is also important for catalysis, although its role is unclear. All three residues are located in the active site cavity previously determined by structural analysis of hGUSB.

The mannose 6-phosphate/insulin-like growth factor-II receptor is a substrate of type V transforming growth factor-beta receptor.

The type V transforming growth factor beta (TGF-beta) receptor (TbetaR-V) is a ligand-stimulated acidotropic Ser-specific protein kinase that recognizes a motif of SXE/S(P)/D. This motif is present in the cytoplasmic domain of the mannose 6-phosphate/insulin-like growth factor-II (Man-6-P/IGF-II) receptor. We have explored the possibility that the Man-6-P/IGF-II receptor is a substrate of TbetaR-V. Purified bovine Man-6-P/IGF-II receptor was phosphorylated by purified bovine TbetaR-V in the presence of [gamma-32P]ATP and MnCl2 with an apparent Km of 130 nM. TGF-beta stimulated the phosphorylation of the Man-6-P/IGF-II receptor at 0 degrees C in mouse L cells overexpressing the Man-6-P/IGF-II receptor and in wild-type mink lung epithelial (Mv1Lu cells) metabolically labeled with [32P]orthophosphate. The in vitro and in vivo phosphorylation of the Man-6-P/IGF-II receptor occurred at the putative phosphorylation sites as revealed by phosphopeptide mapping and amino acid sequence analysis. TGF-beta stimulated Man-6-P/IGF-II receptor-mediated uptake (approximately 2-fold after 12 h treatment) of exogenous beta-glucuronidase in Mv1Lu cells and type II TGF-beta receptor (TbetaR-II)-defective mutant cells (DR26 cells) but not in type I TGF-beta receptor (TbetaR-I)-defective mutant cells (R-1B cells) and human colorectal carcinoma cells (RII-37 cells) expressing TbetaR-I and TbetaR-II but lacking TbetaR-V. These results suggest the Man-6-P/IGF-II receptor serves as an in vitro and in vivo substrate of TbetaR-V and that both TbetaR-V and TbetaR-I may play a role in mediating the TGF-beta-stimulated uptake of exogenous beta-glucuronidase.

Enzyme replacement therapy improves reproductive performance in mucopolysaccharidosis type VII mice but does not prevent postnatal losses.

Mice with mucopolysaccharidosis type VII (MPS VII) are devoid of beta-glucuronidase and accumulate glycosaminoglycans in lysosomes resulting in bone dysplasia, learning disabilities, and decreased mobility. MPS VII males do not breed and, while MPS VII females occasionally mate with heterozygous males, they do not maintain their young postnatally. Heterozygous matings produce less than 25% MPS VII offspring, but until now it was unclear whether this results from prenatal or postnatal losses. The administration of recombinant beta-glucuronidase from birth significantly reduces glycosaminoglycan storage in most tissues, increases life span, and improves the animal's cognitive ability and mobility. To determine whether reproductive failure is corrected by such therapy, male and female MPS VII mice were injected with enzyme at weekly intervals from birth to 5 wk of age (6xinj). Enzyme-replaced MPS VII mice bred when mated together. The 6xinj MPS VII males mated repeatedly until they were killed 135 d postinjection. All mated 6xinj MPS VII females gave birth to two litters, but maintained few of their young. Selective loss of MPS VII offspring was observed in matings between heterozygotes. Analysis of 379 preterm fetuses from heterozygous matings showed a frequency of 24.6% MPS VII pups, indicating that the decreased number of MPS VII pups produced by mating heterozygotes results from postnatal losses. The ovaries of young adult MPS VII mice have follicles and corpora lutea, and the testes generate sperm. Results suggest that the reproductive failure in MPS VII mice is related to impaired mobility and/or impaired cognitive function, and enzyme replacement restores mating capacity.

Identification of Glu-540 as the catalytic nucleophile of human beta-glucuronidase using electrospray mass spectrometry.

Human beta-glucuronidase is a member of the Family 2 glycosylhydrolases that cleaves beta-D-glucuronic acid residues from the nonreducing termini of glycosaminoglycans. The enzyme is shown to catalyze glycoside bond hydrolysis with net retention of anomeric configuration, presumably via a mechanism involving a covalent glucuronyl-enzyme intermediate. Incubation of human beta-glucuronidase with 2-deoxy-2-fluoro-beta-D-glucuronyl fluoride resulted in time-dependent inactivation of the enzyme through the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme, as observed by electrospray mass spectrometry. Regeneration of the free enzyme by hydrolysis or transglycosylation and removal of excess inactivator demonstrated that the covalent intermediate was kinetically competent. Peptic digestion of the 2-deoxy-2-fluoro-alpha-D-glucuronyl-enzyme intermediate and subsequent analysis by liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry indicated the presence of a 2-deoxy-2-fluoro-alpha-D-glucuronyl peptide. Sequence determination of the labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Glu-540 as the catalytic nucleophile within the sequence SEYGAET.

Human carbonic anhydrase XII: cDNA cloning, expression, and chromosomal localization of a carbonic anhydrase gene that is overexpressed in some renal cell cancers.

We report the cloning and characterization of a tumor-associated carbonic anhydrase (CA) that was identified in a human renal cell carcinoma (RCC) by serological expression screening with autologous antibodies. The cDNA sequence predicts a 354-amino acid polypeptide with a molecular mass of 39,448 Da that has features of a type I membrane protein. The predicted sequence includes a 29-amino acid signal sequence, a 261-amino acid CA domain, an additional short extracellular segment, a 26-amino acid hydrophobic transmembrane domain, and a hydrophilic C-terminal cytoplasmic tail of 29 amino acids that contains two potential phosphorylation sites. The extracellular CA domain shows 30-42% homology with known human CAs, contains all three Zn-binding histidine residues found in active CAs, and contains two potential sites for asparagine glycosylation. When expressed in COS cells, the cDNA produced a 43- to 44-kDa protein in membranes that had around one-sixth the CA activity of membranes from COS cells transfected with the same vector expressing bovine CA IV. We have designated this human protein CA XII. Northern blot analysis of normal tissues demonstrated a 4.5-kb transcript only in kidney and intestine. However, in 10% of patients with RCC, the CA XII transcript was expressed at much higher levels in the RCC than in surrounding normal kidney tissue. The CA XII gene was mapped by using fluorescence in situ hybridization to 15q22. CA XII is the second catalytically active membrane CA reported to be overexpressed in certain cancers. Its relationship to oncogenesis and its potential as a clinically useful tumor marker clearly merit further investigation.

Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs.

The X-ray structure of the homotetrameric lysosomal acid hydrolase, human beta-glucuronidase (332,000 Mr), has been determined at 2.6 A resolution. The tetramer has approximate dihedral symmetry and each promoter consists of three structural domains with topologies similar to a jelly roll barrel, an immunoglobulin constant domain and a TIM barrel respectively. Residues 179-204 form a beta-hairpin motif similar to the putative lysosomal targeting motif of cathepsin D, supporting the view that lysosomal targeting has a structural basis. The active site of the enzyme is formed from a large cleft at the interface of two monomers. Residues Glu 451 and Glu 540 are proposed to be important for catalysis. The structure establishes a framework for understanding mutations that lead to the human genetic disease mucopolysaccharidosis VII, and for using the enzyme in anti-cancer therapy.

Enzyme replacement therapy for murine mucopolysaccharidosis type VII.

Recombinant mouse beta-glucuronidase administered intravenously to newborn mice with mucopolysaccharidosis type VII (MPS VII) is rapidly cleared from the circulation and localized in many tissues. Here we determine the tissue distribution of injected enzyme and describe its effects on the histopathology in 6-wk-old MPS VII mice that received either one injection of 28,000 U recombinant beta-glucuronidase at 5 wk of age or received six injections of 28,000 U given at weekly intervals beginning at birth. These mice were compared with untreated 6-wk-old MPS VII mice. The single injection decreased lysosomal distention in the fixed tissue macrophage system. MPS VII mice that received multiple injections had 27.8, 3.5, and 3.3% of normal levels of beta-glucuronidase in liver, spleen, and kidney, respectively. Brain had detectable beta-glucuronidase, ranging from 2.0-12.1% of normal. Secondary elevations of alpha-galactosidase and beta-hexosaminidase in brain, spleen, liver, and kidney were decreased compared with untreated MPS VII mice. Although no improvement was observed in chondrocytes, glia, and some neurons, the skeleton had less clinical and pathological evidence of disease and the brain had reduced lysosomal storage in meninges and selected neuronal groups. These data show that recombinant beta-glucuronidase treatment begun in newborn MPS VII mice provides enzyme to most tissues and significantly reduces or prevents the accumulation of lysosomal storage during the first 6 wk of life. Whether therapy begun later in life can achieve this level of correction remains to be established.

C-terminal processing of human beta-glucuronidase. The propeptide is required for full expression of catalytic activity, intracellular retention, and proper phosphorylation.

beta-Glucuronidase undergoes proteolytic C-terminal processing during or after its transport to lysosomes or endosomes. We determined the C-terminal processing site for human placental beta-glucuronidase to be the peptide bond between Thr633-Arg634. To evaluate the role of the 18-amino acid peptide removed in C-terminal processing, we changed the codon for Arg634 to a stop codon by site-directed mutagenesis and studied expression of the truncated mutant enzyme in COS-7 cells. An increased fraction of newly synthesized enzyme from R634Stop cDNA was secreted. Pulse-chase experiments provided no evidence for increased degradation of the intracellular R634Stop enzyme. The total amount of catalytic activity expressed from the R634Stop mutant cDNA was only half that seen with the wild type cDNA, and the Kcat of the mutant enzyme was 52% that of wild type enzyme. These results indicate that the C-terminal propeptide in the precursor is important for beta-glucuronidase to achieve maximal activity. The truncated enzyme formed hybrid tetramers in cotransfection experiments with the cDNA for rat beta-glucuronidase. There appeared to be no decrease in stability of the R634Stop enzyme, since chaotropic agents, heat treatment, and pH had similar effects on the mutant and the wild type enzymes. The uptake rate of the truncated mutant (R634Stop) enzyme by beta-glucuronidase-deficient human fibroblast cells was only 55-60% that of the wild type enzyme. Binding to the immobilized cation-independent mannose-6-phosphate receptor and measurement of the 32P-labeled phosphorylated oligosaccharides revealed that the truncated mutant enzyme was 32-34% less phosphorylated and appeared to contain proportionately more covered phosphate groups than the wild type enzyme. These results suggest that the propeptide influences the accessibility to both processing enzymes that produce the mannose-6-phosphate recognition marker on beta-glucuronidase.

Crystallization and preliminary crystallographic studies of human beta-glucuronidase.

Crystals of human beta-glucuronidase have been obtained by the vapor diffusion method, using 2-methyl-2,4-pentanediol as a precipitant. The crystals belong to the orthorhombic space group P222(1), with cell dimensions a = 134.5 A, b = 95.1 A, c = 124.4 A. The unit cell contains two copies of the tetrameric enzyme. Complete native data have been collected to a resolution of 2.6 A.

The role of glycosylation and phosphorylation in the expression of active human beta-glucuronidase.

Phosphorylation of mannose residues on N-linked oligosaccharide side chains of lysosomal enzymes targets them to lysosomes. We used site-directed mutagenesis to observe the effect of eliminating selective glycosylation sites from human beta-glucuronidase on enzyme sorting. Expression studies allowed us to determine which of four potential sites were glycosylated, preferentially phosphorylated, and required for catalytic activity. All four sites of the human enzyme were glycosylated, whereas in the mouse and rat enzymes, only three of four sites are used. Sites 2 and 3 were preferentially phosphorylated. Elimination of sites 2 and 3 in combination markedly decreased sorting to lysosomes and increased enzyme secretion. Each of the four glycosylation sites could be eliminated individually without drastic reduction in catalytic activity. Activity was progressively lost as combinations of two, three, and four sites were eliminated. Wild-type enzyme produced in the presence of tunicamycin was also inactive, indicating that glycosylation is required for realization of enzyme activity. However, active enzyme could be deglycosylated with only minimal loss of activity. Mutant enzyme completely lacking glycosylation did not form tetramers. Partial restoration of tetramerization was achieved by the co-expression of normal rat enzyme, provided that the normal rat enzyme supplied at least two subunits to the tetramer.

Mutational analysis of a patient with mucopolysaccharidosis type VII, and identification of pseudogenes.

PCR of cDNA produced from patient fibroblasts allowed us to determine the paternal mutation in the first patient reported with beta-glucuronidase-deficiency mucopolysaccharidosis type VII (MPS VII). The G-->T transversion 1,881 bp downstream of the ATG translation initiation codon destroys an MboII restriction site and converts Trp627 to Cys (W627C). Digestion of genomic DNA PCR fragments with MboII indicated that the patient and the father were heterozygous for this missense mutation in exon 12. Failure to find cDNAs from patient RNA which did not contain this mutation suggested that the maternal mutation leads to greatly reduced synthesis or reduced stability of mRNA from the mutant allele. In order to identify the maternal mutation, it was necessary to analyze genomic sequences. This approach was complicated by the finding of multiple unprocessed pseudogenes and/or closely related genes. Using PCR with a panel of human/rodent hybrid cell lines, we found that these pseudogenes were present over chromosomes 5-7, 20, and 22 and the Y chromosome. Conditions were defined which allowed us to amplify and characterize genomic sequences for the true beta-glucuronidase gene despite this background of related sequences. The patient proved to be heterozygous for a second mutation, in which a C-->T transition introduces a termination codon (R356STOP) in exon 7. The mother was also heterozygous for this mutation. Expression of a cDNA containing the maternal mutation produced no enzyme activity, as expected. Expression of the paternal mutation in COS-7 cells produced a surprisingly high (65% of control) level of activity. However, activity was 13% of control in transiently transfected murine MPS VII cells. The level of activity of this mutant allele appears to correlate with the level of overexpression, suggesting that high concentrations of mutant monomers can drive the folding and tetramerization of mutant enzyme to produce an active and stable enzyme.