Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.2.2.7 (heparinase)
 1,270 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous studies (Sivaram, P., Choi, S. Y., Curtiss, L. K., and Goldberg, I. J.(1994) J. Biol. Chem. 269, 9409-9412) from this laboratory showed that the NH2-terminal region of apoB (NTAB) has binding domains for lipoprotein lipase (LPL). LPL binding to endothelial cells, we hypothesize, involves interaction both with heparan sulfate proteoglycans and with a protein that has homology to NTAB. To test whether cell-surface NTAB would increase the amount and affinity of LPL binding to cells, we produced stable Chinese hamster ovary cell lines that have NTAB anchored to the cell surface. A cDNA encoding the amino-terminal 17% of apoB (apoB17) was fused to a cDNA coding for the last 37 amino acids of decay-accelerating factor (DAF), which contains the signal for glycosylphosphatidylinositol anchor attachment. The fused construct was sequence-verified and cloned into expression vector pCMV5. The pCMV5-apoB17-DAF plasmid was cotransfected with a neomycin resistance gene into wild-type (WT) cells and mutant heparan sulfate proteoglycan-deficient Chinese hamster ovary cells (745 cells), and stable cell lines were established. Expression of apoB17 on the cell surface was confirmed by the release of apoB17 by phosphatidylinositol-specific phospholipase C. LPL binding to WT and apoB17-DAF-transfected cells was determined. Using 0.8-6 microg of LPL, 1.3-2.2-fold more LPL associated with apoB17-DAF WT cells compared with WT cells; apoB17-DAF also increased LPL binding to 745 cells. After heparinase treatment, LPL binding to apoB17-DAF cells was still greater than to treated WT cells. This increased binding to apoB17-DAF cells was almost abolished by treatment of cells with phosphatidylinositol-specific phospholipase C or anti-apoB monoclonal antibody. LPL dissociated from WT cells with k-1 = 2.55 x 10(-2) min-1, whereas LPL dissociated more slowly from apoB17-DAF-containing cells with k-1 = 1.08 x 10(-2) min-1. Furthermore, almost 95% of the LPL on WT cells was dissociated by 1 M NaCl, while only 65% of the LPL dissociated from apoB17-DAF cells at the same high salt concentration. Similarly, in high salt, more LPL remained associated with apoB17-DAF cells than with nontransfected 745 cells. These data show that NTAB on cell surfaces can function as a LPL-binding protein. Moreover, they demonstrate that LPL association with cells can be increased by simultaneously binding to both proteoglycan and non-proteoglycan binding sites.
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PMID:Cell-surface expression of an amino-terminal fragment of apolipoprotein B increases lipoprotein lipase binding to cells. 870 44

Immobilization of biologically active proteins is of great importance to research and industry. Cellulose is an attractive matrix and cellulose-binding domain (CBD) an excellent affinity tag protein for the purification and immobilization of many of these proteins. We constructed two vectors to enable the cloning and expression of proteins fused to the N- or C-terminus of CBD. Their usefulness was demonstrated by fusing the heparin-degrading protein heparinase I to CBD (CBD-HepI and HepI-CBD). The fusion proteins were over-expressed in Escherichia coli under the control of a T7 promoter and found to accumulate in inclusion bodies. The inclusion bodies were recovered by centrifugation, the proteins were refolded and recovered on a cellulose column. The bifunctional fusion protein retained its abilities to bind to cellulose and degrade heparin. C-terminal fusion of heparinase I to CBD was somewhat superior to N-terminal fusion: Although specific activities in solution were comparable, the latter exhibited impaired binding capacity to cellulose. CBD-HepI-cellulose bioreactor was operated continuously and degraded heparin for over 40 h without any significant loss of activity. By varying the flow rate, the mean molecular weight of the heparin oligosaccharide produced could be controlled. The molecular weight distribution profiles, obtained from heparin depolymerization by free heparinase I, free CBD-HepI, and cellulose-immobilized CBD-HepI, were compared. The profiles obtained by free heparinase I and CBD-HepI were indistinguishable, however, immobilized CBD-HepI produced much lower molecular weight fragments at the same percentage of depolymerization. Thus, CBD can be used for the efficient production of bioreactors, combining purification and immobilization into essentially a single step.
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PMID:Immobilization of recombinant heparinase I fused to cellulose-binding domain. 1044 Jun 67

Membrane fusion induced by herpes simplex virus (HSV) is required for both entry and cell-to-cell spread. It is mediated by the viral glycoprotein gB, gD, gH-gL and gD receptors. Although 3-O-sulfated heparan sulfate (3-OS HS) is a receptor for HSV-1 entry, the requirement for heparan sulfate in the fusion process has been ruled out. Here, it is demonstrated that cells expressing 3-OS HS, generated by D-glucosaminyl 3-O-sulfotransferase isoforms-3 and/or -5 (3-OST-3 and 3-OST-5), fused with cells expressing the four glycoproteins. The cell fusion observed exhibited similar requirements but was independent of protein receptors, HVEM or nectin-1. Additionally, removal of 3-OS HS from the cell surface by heparinase-I treatment and, in separate experiments, the presence of soluble 3-OST-3- and 3-OST-5-modified HS, significantly inhibited fusion. Taken together, these results indicate that 3-OS HS can play a crucial role in virus entry and cell fusion.
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PMID:A role for 3-O-sulfated heparan sulfate in cell fusion induced by herpes simplex virus type 1. 1503 23

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
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PMID:Purification and properties of recombinant GST-heparinase III and optimization of cultivation conditions. 2022 73

Rare modification of heparan sulfate (HS) by glucosaminyl 3-O sulfotransferase (3-OST) isoforms generates an entry receptor for herpes simplex virus type-1 (HSV-1). In the zebrafish (ZF) model multiple 3-OST isoforms are differentially expressed. One such isoform is 3-OST-4 which is widely expressed in the central nervous system of ZF. In this report we characterize the role of ZF encoded 3-OST-4 isoform for HSV-1 entry. Expression of ZF 3-OST-4 into resistant Chinese hamster ovary (CHO-K1) cells promoted susceptibility to HSV-1 infection. This entry was 3-O sulfated HS (3-OS HS) dependent as pre-treatment of ZF 3-OST-4 cells with enzyme HS lyases (heparinase II/III) significantly reduced HSV-1 entry. Interestingly, co-expression of ZF 3-OST-4 along with ZF 3-OST-2 which is also expressed in brain rendered cells more susceptible to HSV-1 than 3-OST-4 alone. The role of ZF-3-OST-4 in the spread of HSV-1 was also evaluated as CHO-K1 cells that expressed HSV-1 glycoproteins fused with ZF 3-OST-4 expressing effector CHO-K1 cells. Finally, adding further evidence ZF 3-OST-4 mediated HSV-1 entry was inhibited by anti-3O HS G2 peptide. Taken together our results demonstrate a role for ZF 3-OST-4 in HSV-1 pathogenesis and support the use of ZF as a model to study it.
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PMID:Zebrafish 3-O-sulfotransferase-4 generated heparan sulfate mediates HSV-1 entry and spread. 2449 8