Cloning, Golgi Localization, and Enzyme Activity of the Full-length Heparin/Heparan Sulfate-Glucuronic Acid C5-epimerase

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis–Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The Km for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca2+ were inhibitory in the absence of Mg2+ but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3β-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.

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Chemoenzymatically Prepared Heparan Sulfate Containing Rare 2-O-Sulfonated Glucuronic Acid Residues

ACS Chemical Biology ◽

10.1021/acschembio.5b00071 ◽

2015 ◽

Vol 10 (6) ◽

pp. 1485-1494 ◽

Cited By ~ 11

Author(s):

Rio S. Boothello ◽

Aurijit Sarkar ◽

Vy My Tran ◽

Thao Kim Nu Nguyen ◽

Nehru Viji Sankaranarayanan ◽

...

Keyword(s):

Heparan Sulfate ◽

Glucuronic Acid

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Immunogenicity of liposome-bound hyaluronate in mice. At least two different antigenic sites on hyaluronate are identified by mouse monoclonal antibodies.

Journal of Experimental Medicine ◽

10.1084/jem.168.3.971 ◽

1988 ◽

Vol 168 (3) ◽

pp. 971-982 ◽

Cited By ~ 16

Author(s):

H M Fillit ◽

M Blake ◽

C MacDonald ◽

M McCarty

Keyword(s):

Ascorbic Acid ◽

Heparan Sulfate ◽

Acid Treatment ◽

Glucuronic Acid ◽

Antigenic Site ◽

Electrostatic Forces ◽

Igg Antibody ◽

Antigenic Sites ◽

Mode Of Presentation ◽

Phosphorylated Compounds

Hyaluronate (HA) was previously demonstrated to be immunogenic in rabbits. The immunogenicity of HA in mice was studied. Hyaluronidase-digested streptococcal HA (IA1) covalently linked to liposomes (IA1-liposomes) were produced for immunization. Mice immunized with IA1-liposomes developed measurable serum antibodies to IA1, while mice immunized with IA1 in Freund's adjuvant did not. mAbs produced by two stable hybridomas (10G6 and 5F11) from mice immunized with IA1-liposomes produced IgG antibody reactive with HA in ELISA. 10G6 had a much higher avidity for liposome-bound IA1 than free IA1, while 5F11 did not, suggesting that the mode of presentation of IA1 is important in HA immunogenicity and antigenicity. Both mAbs recognized terminal HA immunodeterminants exposed by hyaluronidase treatment. Sonication had no effect on HA reactivity for either mAb. However, ascorbic acid treatment significantly reduced the antigenicity of HA for mAb 5F11, but not 10G6. Only 10G6 was inhibited by glucuronic acid. Electrostatic forces appear to play a role in the binding site of 5F11, but not 10G6. 5F11 crossreacts with heparan sulfate and phosphorylcholine, while 10G6 did not crossreact with any glycosaminoglycans or phosphorylated compounds tested. These results confirm that HA is immunogenic. They suggest that the mode of presentation of HA is important for the induction of the immune response, and in HA antigenicity. At least two different antigenic sites on HA were demonstrated. 10G6 recognizes a terminal HA antigenic site expressed on IA1-liposomes that contains glucuronic acid in its immunodominant site. 5F11 recognizes an HA antigenic site in which electrostatic forces appear to play a role, is sensitive to ascorbic acid treatment, and is crossreactive with heparan sulfate. The use of mAbs should facilitate immunologic studies of HA.

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Heparan Sulfate Separation, Sequencing, and Isomeric Differentiation: Ion Mobility Spectrometry Reveals Specific Iduronic and Glucuronic Acid-Containing Hexasaccharides

Analytical Chemistry ◽

10.1021/ac902186h ◽

2009 ◽

Vol 81 (24) ◽

pp. 10179-10185 ◽

Cited By ~ 43

Author(s):

Matthew R. Schenauer ◽

John K. Meissen ◽

Youjin Seo ◽

James B. Ames ◽

Julie A. Leary

Keyword(s):

Heparan Sulfate ◽

Ion Mobility Spectrometry ◽

Ion Mobility ◽

Glucuronic Acid ◽

Isomeric Differentiation

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Enoxaparin and Pentosan Polysulfate Bind to the SARS-CoV-2 Spike Protein and Human ACE2 Receptor, Inhibiting Vero Cell Infection

Biomedicines ◽

10.3390/biomedicines10010049 ◽

2021 ◽

Vol 10 (1) ◽

pp. 49

Author(s):

Maria Ennemoser ◽

Julia Rieger ◽

Eva Muttenthaler ◽

Tanja Gerlza ◽

Kurt Zatloukal ◽

...

Keyword(s):

Heparan Sulfate ◽

Viral Transmission ◽

Full Length ◽

Spike Protein ◽

Target Cells ◽

Pentosan Polysulfate ◽

Cell Infection ◽

Virus Surface ◽

Kd Values

As with many other pathogens, SARS-CoV-2 cell infection is strongly dependent on the interaction of the virus-surface Spike protein with the glycosaminoglycans of target cells. The SARS-CoV-2 Spike glycoprotein was previously shown to interact with cell-surface-exposed heparan sulfate and heparin in vitro. With the aim of using Enoxaparin as a treatment for COVID-19 patients and as prophylaxis to prevent interpersonal viral transmission, we investigated GAG binding to the Spike full-length protein, as well as to its receptor binding domain (RBD) in solution by isothermal fluorescence titration. We found that Enoxaparin bound to both protein variants with similar affinities, compared to the natural GAG ligand heparan sulfate (with Kd-values in the range of 600–680 nM). Using size-defined Enoxaparin fragments, we discovered the optimum binding for dp6 or dp8 for the full-length Spike protein, whereas the RBD did not exhibit a significant chain-length-dependent affinity for heparin oligosaccharides. The soluble ACE2 receptor was found to interact with unfractionated GAGs in the low µM Kd range, but with size-defined heparins with clearly sub-µM Kd-values. Interestingly, the structural heparin analogue, pentosan polysulfate (PPS), exhibited high binding affinities to both Spike variants as well as to the ACE2 receptor. In viral infection experiments, Enoxaparin and PPS both showed a strong inhibition of infection in a concentration range of 50–500 µg/mL. Both compounds were found to retain their inhibitory effects at 500 µg/mL in a natural biomatrix-like human sputum. Our data suggest the early topical treatment of SARS-CoV-2 infections with inhaled Enoxaparin; some clinical studies in this direction are already ongoing, and they further imply an oral or nasal prophylactic inactivation of the virus by Enoxaparin or PPS for the prevention of inter-personal viral transmission.

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Syndecan-1 Promotes Hepatocyte-Like Differentiation of Hepatoma Cells Targeting Ets-1 and AP-1

Biomolecules ◽

10.3390/biom10101356 ◽

2020 ◽

Vol 10 (10) ◽

pp. 1356

Author(s):

Péter Hollósi ◽

Lóránd Váncza ◽

Katalin Karászi ◽

Katalin Dobos ◽

Bálint Péterfia ◽

...

Keyword(s):

Cell Surface ◽

Heparan Sulfate ◽

Molecular Mechanisms ◽

Signal Transduction Pathway ◽

Normal Liver ◽

Surface Expression ◽

Hepatoma Cells ◽

Full Length ◽

Cell Surface Expression ◽

Gene Assay

Syndecan-1 is a transmembrane heparan sulfate proteoglycan which is indispensable in the structural and functional integrity of epithelia. Normal hepatocytes display strong cell surface expression of syndecan-1; however, upon malignant transformation, they may lose it from their cell surfaces. In this study, we demonstrate that re-expression of full-length or ectodomain-deleted syndecan-1 in hepatocellular carcinoma cells downregulates phosphorylation of ERK1/2 and p38, with the truncated form exerting an even stronger effect than the full-length protein. Furthermore, overexpression of syndecan-1 in hepatoma cells is associated with a shift of heparan sulfate structure toward a highly sulfated type specific for normal liver. As a result, cell proliferation and proteolytic shedding of syndecan-1 from the cell surface are restrained, which facilitates redifferentiation of hepatoma cells to a more hepatocyte-like phenotype. Our results highlight the importance of syndecan-1 in the formation and maintenance of differentiated epithelial characteristics in hepatocytes partly via the HGF/ERK/Ets-1 signal transduction pathway. Downregulation of Ets-1 expression alone, however, was not sufficient to replicate the phenotype of syndecan-1 overexpressing cells, indicating the need for additional molecular mechanisms. Accordingly, a reporter gene assay revealed the inhibition of Ets-1 as well as AP-1 transcription factor-induced promoter activation, presumably an effect of the heparan sulfate switch.

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