Enzyme replacement therapy in a mouse model of aspartylglycosaminuria

Dunder, Ulla; Kaartinen, Vesa; Valtonen, Pirjo; Väänänen, Eira; Kosma, Veli Matti; Heisterkamp, Nora; Groffen, John; Mononen, Ilkka

doi:10.1096/fasebj.14.2.361

Cited by 65 publications

(56 citation statements)

References 20 publications

(30 reference statements)

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“…The above mentioned results, along with our earlier results that enzyme replacement therapy with glycosylasparaginase effectively removes the lysosomal storage of GlcNAc-Asn from glycosylasparaginase-deficient cell lines 4 and mice, 5 led us to consider GA as a potential cytotoxic enzyme against L-asparagine-dependent leukemia cells. We supplemented the culture medium of two different leukemic cell lines, human B-cell precursor leukemia SUP-B15 cells and human T-lineage leukemia CCRF-CEM cells, either with GA, dephosphorylated (c) The L-asparaginase activity in the EBV-transformed lymphoblasts after addition of GA 40 U/l ('), ErAII 40 U/l (J), ErAII 150 U/l (&), ErAII 1500 U/l (K) into the culture medium, or in the absence of enzymes (n).…”

Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 80%

“…Interestingly, enzyme replacement therapy with glycosylasparaginase effectively clears non-neuronal tissues from lysosomal storage of glycoasparagines typical of glycosylasparaginase-deficient mice. 5 From the therapeutic point of view, the administration of glycosylasparaginaseFan endogenous human protein with no L-glutaminase activity 3 Fmight cause fewer side effects to the human body than that of bacterial L-asparaginases. Thus, the combined evidence shown above and the serious side effects associated with L-asparaginase therapy using bacterial L-asparaginases suggest that the applicability of human glycosylasparaginase as a potential anticancer agent to induce apoptosis in L-asparagine-dependent cancer cells should be studied further.…”

Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 99%

“…3 Glycosylasparaginase is transported in active form into various non-neuronal cell types, including Epstein-Barr virus-transformed (EBV) glycosylasparaginase-deficient lymphoblasts, and it effectively corrects the metabolic defects in glycosylasparaginase-deficient cell lines 4 and mice. 5 The finding that human glycosylasparaginase also hydrolyzes L-asparagine to L-aspartic acid and ammonia-like bacterial L-asparaginases 6 without any L-glutaminase activity 3 led us to investigate its potential cytotoxic activity toward leukemia cells that are dependent on their external supply of L-asparagine. We investigated the ability of human recombinant GA to deplete the intra-and extracellular Asn reservoirs in vitro using EBVtransformed glycosylasparaginase-deficient lymphoblasts from an Letters to the Editor 1167 Leukemia aspartylglycosaminuria patient.…”

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confidence: 99%

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Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

2009

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ReferencesA lysosomal enzyme glycosylasparaginase (GA; aspartylglucosaminidase; EC 3.5.1.26) hydrolyzes the N-glycosidic carbohydrate-to-protein linkage region, aspartylglucosamine, to L-aspartic acid and l-amino-N-acetylglucosamine through a reaction mechanism similar to L-asparaginase. 3 Glycosylasparaginase is transported in active form into various non-neuronal cell types, including Epstein-Barr virus-transformed (EBV) glycosylasparaginase-deficient lymphoblasts, and it effectively corrects the metabolic defects in glycosylasparaginase-deficient cell lines 4 and mice. 5 The finding that human glycosylasparaginase also hydrolyzes L-asparagine to L-aspartic acid and ammonia-like bacterial L-asparaginases 6 without any L-glutaminase activity 3 led us to investigate its potential cytotoxic activity toward leukemia cells that are dependent on their external supply of L-asparagine. We investigated the ability of human recombinant GA to deplete the intra-and extracellular Asn reservoirs in vitro using EBVtransformed glycosylasparaginase-deficient lymphoblasts from an Letters to the Editor 1167 Leukemia aspartylglycosaminuria patient. 4 Lymphoblasts were continuously grown in an RPMI-1640 (HyClone, Logan, Utah, USA) medium supplemented with 15% of fetal calf serum (BioClear, Wiltshire, UK), 2 mM L-glutamine (HyClone) and 1.5-2.0 mM L-asparagine (Fluka Chemie AG, Buchs, Switzerland) and various concentrations of either GA or Erwinia L-asparaginase (Erwinase, ErAII) for 24 h. The kinetic parameters of the enzymes for the hydrolysis of L-asparagine were determined by a spectrophotometric method as described. 7,8 At pH 7.5, the K m of glycosylasparaginase, Erwinia L-asparaginase and Escherichia coli L-asparaginase was 538, 90 and 130 mM and V max 1.49, 16 and 23 mM/min, respectively. One unit of enzyme causes hydrolysis of 1 mmol of L-asparagine per minute under standard conditions.The depletion of the extracellular Asn concentration in the culture medium from 2.3 to 0.2 mmol/l in the presence of 10 or 40 U/l of glycosylasparaginase or 10 U/l of ErAII took 18, 4 and 4 h, respectively. In the presence of 1500 U/l of ErAII, similar Asn concentration decrease occurred within 5 min. The Asn level in the cell culture medium remained unchanged at approximately 2 mmol/l in the absence of either glycosylasparaginase or ErAII (Figure 1a).To determine whether the enzymes can deplete the intracellular Asn reservoirs of EBV-transformed lymphoblasts, we analyzed both the Asn concentration and the enzyme activity inside the cells during the incubation. In the presence of 10 or 40 U/l of glycosylasparaginase or 10 U/l of ErAII in the cell culture medium, the Asn concentration inside the GA-deficient cells first increased within the first 2 h of incubation from the initial approximately 80 nmol/mg protein and then decreased below 6 nmol/mg protein during the next 16-18, 6 or 10-12 h, respectively (Figure 1b). In the presence of 1500 U/l of ErAII in the culture medium, the intracellular Asn concentration decreased from 89 to 14 nmol/m...

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Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 80%

Section: Depletion Of L-asparagine Supply and Apoptosis Of Leukemia Cmentioning

confidence: 99%

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confidence: 99%

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Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

2009

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“…Using a high enough dose of recombinant enzyme might be a nontoxic way to saturate the MR clearance system and enhance MPR-mediated enzyme delivery. Favorable effects in overcoming the blood-brain barrier to ERT in certain parts of the CNS with high doses of infused GUS may have been partially attributable to saturation of the MR clearance system (18)(19)(20)(21).…”

Section: Comparison Of Effectiveness Of P-gus In Reducing Lysosomal Smentioning

confidence: 99%

Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors

Sly¹,

Vogler²,

Grubb³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Enzyme replacement therapy (ERT) is available for several lysosomal storage diseases. Except for Gaucher disease, for which an enzyme with exposed mannosyl residues targets mannose receptors (MR) on macrophages, ERT targets primarily the mannose 6-phosphate receptor (MPR). Most recombinant lysosomal enzymes contain oligosaccharides with both terminal mannosyl and mannose 6-phosphate residues. Effective MPR-mediated delivery may be compromised by rapid clearance of infused enzyme by the MR on fixed tissue macrophages, especially Kupffer cells. To evaluate the impact of this obstacle to ERT, we introduced the MR-null mutation onto the mucopolysaccharidosis type VII (MPS VII) background and produced doubly deficient MR ؊/؊ MPS VII mice. The availability of both MR ؉/؉ and MR ؊/؊ mice allowed us to study the effects of eliminating the MR on MR-and MPR-mediated plasma clearance and tissue distribution of infused phosphorylated (P) and nonphosphorylated (NP) forms of human ␤-glucuronidase (GUS). In MR ؉/؉ MPS VII mice, the MR clearance system predominated at doses up to 6.4 mg͞kg P-GUS. Genetically eliminating the MR slowed plasma clearance of both P-and NP-GUS and enhanced the effectiveness of P-GUS in clearing storage in kidney, bone, and retina. Saturating the MR clearance system by high doses of enzyme also improved targeting to MPR-containing tissues such as muscle, kidney, heart, and hepatocytes. Although ablating the MR clearance system genetically is not practical clinically, blocking the MR-mediated clearance system with high doses of enzyme is feasible. This approach delivers a larger fraction of enzyme to MPR-expressing tissues, thus enhancing the effectiveness of MPRtargeted ERT.␤-glucuronidase deficiency ͉ immune tolerance ͉ lysosomal storage disease T he mucopolysacchridoses (MPS) are a group of lysosomal storage diseases (LSDs) in which a deficiency of one or more lysosomal enzymes interferes with the degradation of glycosaminoglycans (GAGs), resulting in their storage in the lysosome. Accumulated GAG storage in lysosomes results in progressive cellular and organ dysfunction (1). In recent years, advances have been made in the treatment of several LSDs (2-5). In these diseases, lysosomal storage can be partially or completely reversed in many target tissues by i.v. infusion of the missing lysosomal enzyme. Traditionally, it was assumed that lysosomal enzymes would be delivered to target tissues so long as the enzymes contained the mannose 6-phosphate (M6P) recognition marker (6). However, other factors can influence the delivery of lysosomal enzymes to target tissues. Among these is the presence of nonphosphorylated oligosaccharides on the enzymes and the extent to which these have been processed from high mannose to complex-type oligosaccharides (7). The latter determines the number of exposed mannose residues on the enzyme, which can target the enzyme preferentially to the mannose receptor (MR). In addition, the distribution of the MR and the mannose 6-phosphate receptor (MPR) and their relative abund...

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“…Most ERT studies showing beneficial effects on the CNS reached only the sub‐endogenous enzyme activity level 32, 46, 48. In contrast, with the applied ERT regimen, we fully restored endogenous LAMAN activity levels in all CNS regions investigated.…”

Section: Discussionmentioning

confidence: 69%

Chronic enzyme replacement therapy ameliorates neuropathology in alpha‐mannosidosis mice

Damme

Lüdemann

Rothaug

et al. 2015

Ann Clin Transl Neurol

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ObjectiveThe lysosomal storage disease alpha‐mannosidosis is caused by the deficiency of the lysosomal acid hydrolase alpha‐mannosidase (LAMAN) leading to lysosomal accumulation of neutral mannose‐linked oligosaccharides throughout the body, including the brain. Clinical findings in alpha‐mannosidosis include skeletal malformations, intellectual disabilities and hearing impairment. To date, no curative treatment is available. We previously developed a beneficial enzyme replacement therapy (ERT) regimen for alpha‐mannosidase knockout mice, a valid mouse model for the human disease. However, humoral immune responses against the injected recombinant human alpha‐mannosidase (rhLAMAN) precluded long‐term studies and chronic treatment.MethodsHere, we describe the generation of an immune‐tolerant alpha‐mannosidosis mouse model that allowed chronic injection of rhLAMAN by transgenic expression of a catalytically inactive variant of human LAMAN in the knockout background.ResultsChronic ERT of rhLAMAN revealed pronounced effects on primary substrate storage throughout the brain, normalization of lysosomal enzyme activities and morphology as well as a decrease in microglia activation. The positive effect of long‐term ERT on neuronal lysosomal function was reflected by an improvement of cognitive deficits and exploratory activity. in vivo and in vitro uptake measurements indicate rapid clearance of rhLAMAN from circulation and a broad uptake into different cell types of the nervous system.InterpretationOur data contribute to the understanding of neurological disorders treatment by demonstrating that lysosomal enzymes such as rhLAMAN can penetrate into the brain and is able to ameliorate neuropathology.

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Enzyme replacement therapy in a mouse model of aspartylglycosaminuria

Cited by 65 publications

References 20 publications

Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

Depletion of L-asparagine supply and apoptosis of leukemia cells induced by human glycosylasparaginase

Enzyme therapy in mannose receptor-null mucopolysaccharidosis VII mice defines roles for the mannose 6-phosphate and mannose receptors

Chronic enzyme replacement therapy ameliorates neuropathology in alpha‐mannosidosis mice

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