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Heteropolymeric beta-glucuronidases are detected in somatic cell hybrids between mouse cells and human fibroblasts deficient in beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) by electrophoresis and column chromatography. Specific antisera against human beta-glucuronidase prepared in mice recognize these heteropolymeric beta-glucuronidases. Our results demonstrate that synthesis of mutant subunits of variant beta-glucuronidase continues in deficient human fibroblasts; these mutant subunits of human beta-glucuronidase retain the ability to associate with normal subunits of mouse beta-glucuronidase to form the enzymatically and immunologically active tetramer. These studies demonstrate the usefulness of interspecific cell hybrids for study of structural gene mutations of polymeric enzymes, such as those of beta-glucuronidase in mucopolysaccharidosis type VII.
Deficiency of beta-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced beta-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low beta-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the beta-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and 35SO4-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low beta-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low beta-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low beta-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801deltaT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold.
A significant portion of murine hepatocyte beta-glucuronidase is maintained within the endoplasmic reticulum (ER) by complex formation with the esterase active site of the protein egasyn. The carboxyl-terminal propeptide of the precursor form of glucuronidase appears important in localization of glucuronidase to the ER since a naturally occurring mutation in it is associated with decreased levels of ER glucuronidase. A sequence similarity was noted between the carboxyl-terminal propeptide and portions of the conserved sequences of the reactive site region of members of the serpin (serine proteinase inhibitor) superfamily. Also, previous studies had shown that a synthetic peptide, corresponding to the propeptide region, was a specific and potent inhibitor of the esterase activity of purified egasyn. Taken together, these results suggest that (a) the egasyn-glucuronidase system may use a novel mechanism related to that of serine proteinases and their inhibitors in complex formation and in subsequent localization of glucuronidase within the ER and that (b) a possible function of ER glucuronidase is to modulate the esterase activity of egasyn.
The severity of human mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, depends on the relative activity of the enzyme beta-glucuronidase. Loss of beta-glucuronidase activity can cause hydrops fetalis, with in utero or postnatal death of the patient. In this report, we show that beta-glucuronidase activity is not detectable by a standard fluorometric assay in C3H/HeOuJ (C3H) mice homozygous for a new mutation, gusmps2J. These gusmps2J/gusmps2J mice are born and survive much longer than the previously characterized beta-glucuronidase-null B6.C-H-2(bm1)/ByBir-gusmps (gusmps/gusmps) mice. Northern blot analysis of liver from gusmps2J/gusmps2J mice demonstrates a 750-bp reduction in size of beta-glucuronidase mRNA. A 5.4-kb insertion in the Gus-sh nucleotide sequence from these mice was localized by Southern blot analysis to intron 8. The ends of the inserted sequences were cloned by inverse PCR and revealed an intracisternal A-particle (IAP) element inserted near the 3' end of the intron. The sequence of the long terminal repeat (LTR) regions of the IAP most closely matches that of a composite LTR found in transposed IAPs previously identified in the C3H strain. The inserted IAP may contribute to diminished beta-glucuronidase activity either by interfering with transcription or by destabilizing the message. The resulting phenotype is much less severe than that previously described in the gusmps/gusmps mouse and provides an opportunity to study MPS VII on a genetic background that clearly modulates disease severity.
We have detected a plant beta-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric beta-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45 degrees C. It is relatively resistant to proteases and insensitive to -SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by the Escherichia coli beta-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay the E. coli beta-glucuronidase in transgenic plants without interference from the endogenous plant activity.
Lysosomal beta-glucuronidase shows a dual localization in mouse liver, where a significant fraction is retained in the endoplasmic reticulum (ER) by interaction with an ER-resident carboxyl esterase called egasyn. This interaction of mouse egasyn (mEg) with murine beta-glucuronidase (mGUSB) involves binding of the C-terminal 8 residues of the mGUSB to the carboxylesterase active site of the mEg. We isolated the recombinant human homologue of the mouse egasyn cDNA and found that it too binds human beta-glucuronidase (hGUSB). However, the binding appears not to involve the active site of the human egasyn (hEg) and does not involve the C-terminal 18 amino acids of hGUSB. The full-length cDNA encoding hEg was isolated from a human liver cDNA library using full-length mEg cDNA as a probe. The 1941-bp cDNA differs by only a few bases from two previously reported cDNAs for human liver carboxylesterase, allowing the anti-human carboxylesterase antiserum to be used for immunoprecipitation of human egasyn. The cDNA expressed bis-p-nitrophenyl phosphate (BPNP)-inhibitable esterase activity in COS cells. When expressed in COS cells, it is localized to the ER. The intracellular hEg coimmunoprecipitated with full-length hGUSB and with a truncated hGUSB missing the C-terminal 18-amino-acid residue when extracts of COS cells expressing both proteins were treated with anti-hGUSB antibody. It did not coimmunoprecipitate with mGUSB from extracts of coexpressing COS cells. Unlike mEg, hEg was not released from the hEg-GUSB complex with BPNP. Thus, hEg resembles mEg in that it binds hGUSB. However, it differs from mEg in that (i) it does not appear to use the esterase active site for binding since treatment with BPNP did not release hEg from hGUSB and (ii) it does not use the C terminus of GUSB for binding, since a C-terminal truncated hGUSB (the C-terminal 18 amino acids are removed) bound as well as nontruncated hGUSB. Evidence is presented that an internal segment of 51 amino acids between 228 and 279 residues contributes to binding of hGUSB by hEg.
Antiserum to the inhibitor of beta=glucuronidase isolated from porcine sublingual gland was prepared in rabbits. Double immunodiffusion with the inhibitor produced a single precipitin line. However, neutralization of the inhibitor was produced by the antiserum and also by normal serum. Anti-beta-glucuronidase inhibitor isolated from human serum, by fractionation with (NH4)2 SO4 followed by DEAE-cellulose, Sephadex G-200 and Sepharose 4B chromatography, was identified as alpha2-macroglobulin by using ultracentrifuge analysis and immunoelectrophoresis. The mechanism of interaction of beta-glucuronidase inhibitor with alpha2-macroglobulin was also studied.
The use of erythrocyte entrapment as a strategy to deliver and protect exogenously administered enzymes for replacement therapy in selected genetic diseases has been evaluated in a mammalian model system. The uptake, tissue distribution, intracellular localization, and in vivo lifetime of erythrocyte-entrapped bovine beta-glucuronidase were determined by a selective thermal inactivation assay after intravenous administration into beta-glucuronidase-deficient mice. The exogenous activity was cleared from the circulation with a half-life of about 20 min and was no longer detectable at 2 hr. A concomitant uptake of the injected enzyme was observed in murine tissues, primarily the liver; approximately 30% of the bovine activity was recovered at 30 min and maximal hepatic uptake, 71% of dose, was detected at 2 hr. Hepatic recovery of the bovine activity was observed to decrease in a biphasic pattern to nondetectable levels by 5 days. The recovery of the entrapped activity was characterized by a latency of detection in hepatic tissue up to 13 hr postinjection. At each time point more than 80% (84-100%) of the recovered bovine activity was detected in the lysosomally enriched hepatic subcellular fraction. Maximal recoveries of 10% and 15% of administered dose were observed in splenic and renal tissues, respectively, soon after enzyme administration. In comparison to results obtained after intravenous administration of unentrapped bovine beta-glucuronidase, erythrocyte-entrapped activity was retained fourfold longer in the circulation, fivefold longer in hepatic tissue, and was more efficiently delivered to a variety of tissues.
Human placental beta-glucuronidase could be identified in rat organs by heating organ extracts to 65 degrees which selectively inactivated endogenous rat enzyme. Infused enzyme was rapidly cleared from rat plasma (t0.5 of 3.5 min). From 50-60% of the infused dose was accounted for in rat liver and spleen 24 hr after infusion. Removal of abdominal viscera, including the spleen, and interruption of the portal circulation before infusion slowed the plasma disappearance (t0.5 of 60 min) and allowed significant uptake by bone and other organs. Subcellular fractionation of liver 18 hr postinfusion localized the human enzyme in the mitochondrial-lysosomal fraction. The half-disappearance times of infused human enzyme were 2.6 days in rat liver and 5.8 days in rat spleen. Periodate treatment of human placental beta-glucuronidase destroyed 90% of its binding to concanavalin A-Sephadex rose, reduced its heat stability, and abolished its rapid clearance from rat plasma after infusion.
Signal sequences and endoplasmic reticulum (ER) retention signals are known to play central roles in targeting and translocation in the secretory pathway, but molecular aspects about their involvement are poorly understood. We tested the effectiveness of deduced signal sequences from various genes (hydroxyproline-rich glycoprotein [HRGP] from Phaseolus vulgaris; Serpin from Manduca sexta) to direct a modified beta-glucuronidase (GUS) protein into the secretory pathway in transgenic tobacco (Nicotiana tabacum L.). The reporter protein was not secreted to the cell wall/extracellular space as monitored using extracellular fluid analysis (low- or high-ionic-strength conditions) but occurred in membranes with a density of 1.16 to 1.20 g/mL. Membrane-bound GUS equilibrated with the plasma membrane (PM) and the ER on linear sucrose gradients with or without ethylenediaminetetraacetic acid, suggesting that GUS associates with the ER and the PM. Confocal microscopy of fixed cultured cells prepared from GUS control and HRGP signal peptide (SP)-GUS-expressing plants suggested only cytosolic localization in GUS-expressing plants but substantial peripheral localization in HRGP SP-GUS plants, which is consistent with GUS being associated with the PM. Aqueous two-phase partitioning of microsomal membranes from HRGP SP-GUS and Serpin SP-GUS transgenic leaves also indicated that GUS activity was enriched in the ER and the PM. These observations, together with hydrophobic moment plot analysis, suggest that properties of the SP-GUS protein result in its retention in the secretory pathway and PM.
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