Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.6.4 (chondroitinase)
 2,039 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human promonocytes U937 synthesize lysozyme and retain approximately one third of it within lysosomes. Lysozyme is not glycosylated; thus, it cannot be subject to mannose-6-phosphate-dependent targeting to lysosomes. It is a basic protein with a pI of 10.5 and is known to interact with negatively charged macromolecules like proteoglycans. Therefore, we examined whether the latter are involved in the lysosomal targeting of lysozyme in U937 cells. We partially diminished the electronegative charge of newly synthesized proteoglycans by inhibiting their sulfation with chlorate. This increased the rate of secretion of lysozyme. Upon treatment of U937 cells with phorbol esters, the rate of secretion of lysozyme was increased to more than 90%. This coincided with an almost complete redistribution of a [(35)S]sulfate bearing proteoglycan to the secretory pathway. After a brief pulse with [(35)S]sulfate in the control, 80% of the [(35)S]sulfate-bearing proteoglycan was retained within the cells, whereas in the treated cells this proportion was decreased to 13%. The secreted proteoglycan was sensitive to chondroitinase ABC and bound to immobilized lysozyme. This interaction was disrupted by 50-300 mM NaCl. The intracellularly retained proteoglycan was degraded with a half-life of 50-60 minutes and seemed to be directed to lysosomes because in the presence of NH(4)Cl the degradation was strongly inhibited. Our results suggest that the proteoglycan is involved in lysosomal targeting of lysozyme in U937 cells.
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PMID:Chondroitin sulfate is involved in lysosomal transport of lysozyme in U937 cells. 1114 36

Gene therapy may provide a long-term approach to the treatment of mucopolysaccharidoses. As a first step toward the development of an effective gene therapy for mucopolysaccharidosis type IVA (Morquio syndrome), a recombinant retroviral vector, LGSN, derived from the LXSN vector, containing a full-length human wildtype N-acetylgalactosamine-6-sulfate sulfatase (GALNS) cDNA, was produced. Severe Morquio and normal donor fibroblasts were transduced by LGSN. GALNS activity in both Morquio and normal transduced cells was several fold higher than normal values. To measure the variability of GALNS expression among different transduced cells, we transduced normal and Morquio lymphoblastoid B cells and PBLs, human keratinocytes, murine myoblasts C2C12, and rabbit synoviocytes HIG-82 with LGSN. In all cases, an increase of GALNS activity after transduction was measured. In Morquio cells co-cultivated with enzyme-deficient transduced cells, we demonstrated enzyme uptake and persistence of GALNS activity above normal levels for up to 6 days. The uptake was mannose-6-phosphate dependent. Furthermore, we achieved clear evidence that LGSN transduction of Morquio fibroblasts led to correction of the metabolic defect. These results provide the first evidence that GALNS may be delivered either locally or systematically by various cells in an ex vivo gene therapy of MPS IVA.
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PMID:Various cells retrovirally transduced with N-acetylgalactosoamine-6-sulfate sulfatase correct Morquio skin fibroblasts in vitro. 1168 41

A human L-selectin-ZZ fusion protein was used to screen porcine inguinal lymph nodes for the presence of monoclonal antibody (mAb) MECA 79-negative ligands. Fractionation of lymph node-conditioned medium by anion-exchange chromatography revealed two distinct L-selectin-binding fractions, one containing a MECA 79 non-reactive species and the second containing two MECA 79 reactive species of approximately 84 000 and 210 000 molecular weight. The MECA 79 non-reactive species exhibited Ca2+- and lectin-dependent binding to L-selectin-ZZ in a solid-phase capture enzyme-linked immunosorbent assay (ELISA), and this was specifically disrupted by the addition of EDTA, mannose-6-phosphate and the blocking anti-L-selectin mAb, DREG-56. Enzymatic characterization of the ligand by trypsin, O-sialoglycoprotease endopeptidase, heparinases I and III, or chondroitinase ABC lyase digestion indicated that L-selectin binding was predominantly dependent upon a chondroitin sulphate-modified glycoprotein determinant. Although Coomassie Blue staining of sodium dodecyl sulphate (SDS) polyacrylamide gels did not reveal a detectable protein species, carbohydrate-specific staining using GlycoTrack revealed a single, heavily glycosylated protein of high molecular weight (> 220 000). These studies have revealed the existence of a MECA 79 non-reactive, chondroitin sulphate glycosaminoglycan-modified ligand, termed csgp>220, which is secreted by peripheral lymph nodes and may play a role in leucocyte trafficking to the lymph node.
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PMID:Identification and characterization of L-selectin ligands in porcine lymphoid tissues. 1198 64

Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disorder caused by a deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS). The aims of this study were to establish Chinese hamster ovary (CHO) cells overexpressing recombinant human GALNS (rhGALNS) and to assess pharmacokinetics and tissue distribution of purified enzymes by using MPS IVA knock-out mouse (Galns(-/-)). The CHO-cell derived rhGALNS was purified from the media by a two-step affinity chromatography procedure. The rhGALNS was administered intravenously to 3-month-old Galns(-/-) mice at a single dose of 250U/g of body weight. The treated mice were examined by assaying the GALNS activity at baseline and up to 240min to assess clearance of the enzyme from blood circulation. The mice were sacrificed 4h after infusion of the enzyme to study the enzyme distribution in tissues. The rhGALNS was purified 1317-fold with 71% yield. The enzyme was taken up by Galns(-/-) chondrocytes (150U/mg/15h). The uptake was inhibited by mannose-6-phosphate. The enzyme activity disappeared from circulation with a half-life of 2.9min. After enzyme infusion, the enzyme was taken up and detected in multiple tissues (40.7% of total infused enzymes in liver). Twenty-four hours after a single infusion of the fluorescence-labeled enzymes into MPS IVA mice, biodistribution pattern showed the amount of tagged enzyme retained in bone, bone marrow, liver, spleen, kidney, and heart. In conclusion, we have shown that the phosphorylated rhGALNS is delivered to multiple tissues, including bone, and that it functions bioactively in Galns(-/-) chondrocytes implying a potential enzyme replacement treatment.
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PMID:Characterization and pharmacokinetic study of recombinant human N-acetylgalactosamine-6-sulfate sulfatase. 1733 63