Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria

Abstract

Aspartylglycosaminuria (AGU) is the most common disorder of glycoprotein degradation. AGU patients are deficient in glycosylasparaginase (GA), which results in accumulation of aspartylglucosamine in body fluids and tissues. Human glycosylasparaginase was stably overexpressed in NIH-3T3 mouse fibroblasts, in which the unusual posttranslational processing and maturation of the enzyme occurred in a high degree. The recombinant enzyme was isolated as two isoforms, which were both phosphorylated, and actively transported into AGU fibroblasts and lymphoblasts through mannose-6-phosphate receptor-mediated endocytosis. The rate of uptake into fibroblasts was half-maximal when the concentration of GA in the medium was 5 x 10(-8) M. Immunofluorescence microscopy suggested compartmentalization of the recombinant enzyme in the lysosomes. Supplementation of culture medium with either isoform cleared AGU lymphoblasts of stored aspartylglucosamine when glycosylasparaginase activity in the cells reached 3-4% of that in normal lymphoblasts. A relatively small amount of recombinant GA in the culture medium was sufficient to reverse pathology in the target cells, indicating high corrective quality of the enzyme preparations. The combined evidence indicates that enzyme replacement therapy with the present recombinant glycosylasparaginase might reverse pathology at least in somatic cells of AGU patients.

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

“…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).…”

“…Earlier studies have shown that GA is actively transported into glycosylasparaginase-deficient lymphoblasts, in which it locates mostly in the cytoplasm and lysosomes. 4 The location of ErAII in the lymphoblasts was not studied in this work. In theory, the observed L-asparaginase activity in lysed lymphoblasts could result from ErAII attached to the cell membrane or from enzyme that has entered the cells, or both.…”

“…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.…”

“…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.…”

“…4 The equal efficacy of phosphorylated and dephosphorylated GA in induction of apoptosis of Asn-dependent leukemia cells may indicate either the importance of degradation of extracellular Asn stores by dephosphorylated GA in the apoptosis of cells or the presence of other, mannose-6-phosphate-independent mechanisms for GA to enter the cells. An important extracellular source of L-asparagine for acute lymphoblastic leukemia blasts includes bone marrow mesenchymal cells, which excrete and increase L-asparagine concentration in the leukemic cell microenvironment and cause resistance to bacterial L-asparaginase therapy.…”