Structured Review

IBEX Technologies heparitinase
HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with <t>heparitinase</t> to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.
Heparitinase, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?"

Article Title: Structure and epitope distribution of heparan sulfate is disrupted in experimental lung hypoplasia: a glycobiological epigenetic cause for malformation?

Journal: BMC Developmental Biology

doi: 10.1186/1471-213X-11-38

HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.
Figure Legend Snippet: HS structure is abnormal in hypoplastic nitrofen treated rat lungs . HSPG levels, identified by 3G10, are reduced in hypoplastic rat lungs, particularly at E15.5 and E17.5 and in epithelial basement membranes (A). Analysis of specific HS epitopes with 'phage display antibodies revealed an abnormality in HS fine structure. A number of epitopes are reduced or lost from the epithelium e.g., AO4B08V and HS3A8V, respectively (B). In addition, a number of epitopes, e.g., HS4E4V, are reduced in the lung mesenchyme (C) and all epitopes are reduced in epithelial basement membranes (B, C). Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with 3G10 after initial digestion of lung HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs. Bound antibody was then detected with FITC conjugated goat anti-mouse IgG. As a negative control, sections were incubated with heparitinase buffer alone without enzyme, leaving the 3G10 neo-epitope concealed. Incubation of lung sections with HS 'phage display antibodies was followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (ep) epithelium, (bm) basement membrane, (me) mesenchyme.

Techniques Used: Negative Control, Incubation

Airway epithelial basement membranes are abnormal in hypoplastic lungs . Epithelial basement membranes appear thinner in nitrofen treated lungs, with reduced levels of HSPGs, identified by 3G10 antibody (A) and HS epitopes identified by 'phage display HS antibodies, e.g., HS4E4V and HS3B7V (B, C). Discontinuities in basement membrane HS staining were also observed with HS antibody staining (B, C, arrowheads). This was not apparent with 3G10 immunohistochemistry, identifying all HSPGs (A). To visualise the general structure of basement membranes and assess whether the observed abnormalities are HS specific or a general defect in basement membrane structure, lungs were probed with an antibody to laminin (D). Staining with anti-laminin revealed thinner basement membranes, however, no discontinuities were observed. Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with HS antibodies, 3G10 (after digestion of endogenous HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs) or anti-laminin antibody. Bound HS antibodies were detected with rabbit VSV-G tag antibody followed by FITC conjugated goat anti-rabbit IgG, 3G10 was detected with FITC conjugated goat anti-mouse IgG and anti-laminin was detected with FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (aw) airway, (bm) basement membrane, (me) mesenchyme, (ep) epithelium.
Figure Legend Snippet: Airway epithelial basement membranes are abnormal in hypoplastic lungs . Epithelial basement membranes appear thinner in nitrofen treated lungs, with reduced levels of HSPGs, identified by 3G10 antibody (A) and HS epitopes identified by 'phage display HS antibodies, e.g., HS4E4V and HS3B7V (B, C). Discontinuities in basement membrane HS staining were also observed with HS antibody staining (B, C, arrowheads). This was not apparent with 3G10 immunohistochemistry, identifying all HSPGs (A). To visualise the general structure of basement membranes and assess whether the observed abnormalities are HS specific or a general defect in basement membrane structure, lungs were probed with an antibody to laminin (D). Staining with anti-laminin revealed thinner basement membranes, however, no discontinuities were observed. Hypoplastic lungs from rats with nitrofen-induced left sided CDH and control lungs from rats fed olive oil alone were probed with HS antibodies, 3G10 (after digestion of endogenous HS with heparitinase to reveal the 3G10 neo-epitope on all HSPGs) or anti-laminin antibody. Bound HS antibodies were detected with rabbit VSV-G tag antibody followed by FITC conjugated goat anti-rabbit IgG, 3G10 was detected with FITC conjugated goat anti-mouse IgG and anti-laminin was detected with FITC conjugated goat anti-rabbit IgG. Scale bars represent 10 μm. (aw) airway, (bm) basement membrane, (me) mesenchyme, (ep) epithelium.

Techniques Used: Staining, Immunohistochemistry

HS 'phage display antibodies identify distinct epitopes in situ . In fetal rat lungs, HS antibodies display different patterns of staining. HS3B7V exclusively labels epithelial basement membranes, whereas HS4E4V and HS3A8V show a more widespread staining pattern. In addition to epithelial basement membrane staining, HS4E4V labels sub-epithelial mesenchymal cells surrounding smaller distal airways and HS3A8V highlights the entire lung mesenchyme and in addition, stains epithelial cells at E15.5. One antibody, HS4C3V, did not stain fetal rat lungs of any developmental age; however, positive staining of adult rat kidney confirmed the functionality of HS4C3V in immunohistochemistry. E15.5 and E17.5 rat lungs and adult rat kidney were probed with HS antibodies followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Negative controls were omission of HS antibody or digestion of HS with heparitinase prior to antibody incubation (HS4E4V shown, heparitinase digest controls for other antibodies are shown in additional files). Scale bar represents 10 μm and all images are the same magnification. (ep) epithelium, (me) mesenchyme, (bm) basement membrane, (aw) airway, (G) glomerulus, (cap) peritubular capillary.
Figure Legend Snippet: HS 'phage display antibodies identify distinct epitopes in situ . In fetal rat lungs, HS antibodies display different patterns of staining. HS3B7V exclusively labels epithelial basement membranes, whereas HS4E4V and HS3A8V show a more widespread staining pattern. In addition to epithelial basement membrane staining, HS4E4V labels sub-epithelial mesenchymal cells surrounding smaller distal airways and HS3A8V highlights the entire lung mesenchyme and in addition, stains epithelial cells at E15.5. One antibody, HS4C3V, did not stain fetal rat lungs of any developmental age; however, positive staining of adult rat kidney confirmed the functionality of HS4C3V in immunohistochemistry. E15.5 and E17.5 rat lungs and adult rat kidney were probed with HS antibodies followed by rabbit VSV-G tag antibody and FITC conjugated goat anti-rabbit IgG. Negative controls were omission of HS antibody or digestion of HS with heparitinase prior to antibody incubation (HS4E4V shown, heparitinase digest controls for other antibodies are shown in additional files). Scale bar represents 10 μm and all images are the same magnification. (ep) epithelium, (me) mesenchyme, (bm) basement membrane, (aw) airway, (G) glomerulus, (cap) peritubular capillary.

Techniques Used: In Situ, Staining, Immunohistochemistry, Incubation

Related Articles

Blocking Assay:

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. To investigate the role of MPO binding in the efficiency of perlecan oxidation by the MPO-H2 O2 -halide systems, the effect of removal of heparan sulfate with heparinase III and the inclusion of heparin to block binding of MPO to perlecan was examined. .. In these studies, surface-bound perlecan was treated with heparinase III and then pre-incubated with MPO in the presence or absence of heparin under identical conditions to those employed in (in gelatin-blocked wells).

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. Removal of heparan sulfate with heparinase III, which decreased the affinity of MPO binding to perlecan but did not block this interaction , did not significantly protect the perlecan protein core against damage by MPO-H2 O2 -halide systems (oxidant/perlecan molar ratio of 200) ( ). .. However, marked protection was observed in the presence of heparin (100 µg/ml) , which completely blocked binding of MPO to perlecan , with the extent of damage induced by the MPO-H2 O2 -Cl− system and the MPO-H2 O2 -Br− systems being of a similar magnitude to that observed with reagent HOCl and HOBr (the low extents of damage by the reagent oxidants are consistent with gelatin exerting a protective effect by acting as an alternative target for oxidation).

Enzyme-linked Immunosorbent Assay:

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. Surface-adsorbed perlecan was found to bind MPO and this binding was partially inhibited by prior removal of the heparan sulfate chains by treatment with heparinase III ( ); removal of heparin sulfate by heparinase III was confirmed by ELISA ( ). ..

other:

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: Enzymatic digestion of perlecan was carried out using heparinase III (0.01 U/ml) or chondroitinase ABC (0.05 U/ml) in PBS containing 0.01% BSA for 2 h at 37°C.

Binding Assay:

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. Perlecan binding of FGF-2 and collagen V via its heparan sulfate chains was confirmed and, in line with previous findings ( ; ), this was reversed by prior treatment of the perlecan with heparinase III (data not shown). .. Oxidation of surface-adsorbed perlecan by HOCl and HOBr (oxidant/perlecan molar ratio of 1000) did not affect either FGF-2 ( ) or collagen V binding , demonstrating that the heparan sulfate sequences that bind these ligands ( ; ) are resistant to modification by these oxidants.

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. Enzymatic digestion of surface-adsorbed perlecan was carried out with heparinase III (0.01 U/ml, 50 µl) or chondroitinase ABC (0.05 U/ml, 50 µl) in 0.01% BSA or 0.01% fish gelatin (for MPO binding studies) for 2 h at 37°C. .. Treatment of surface-adsorbed perlecan with HNO2 was performed at pH 3.9 to cleave heparan sulfate specifically at its GlcNH2 residues ( ) by incubation with 4.5 M sodium nitrite (50 µl) and 2 M acetic acid (10 µl) for 10 min at 22°C.

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. To investigate the role of MPO binding in the efficiency of perlecan oxidation by the MPO-H2 O2 -halide systems, the effect of removal of heparan sulfate with heparinase III and the inclusion of heparin to block binding of MPO to perlecan was examined. .. In these studies, surface-bound perlecan was treated with heparinase III and then pre-incubated with MPO in the presence or absence of heparin under identical conditions to those employed in (in gelatin-blocked wells).

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. Surface-adsorbed perlecan was found to bind MPO and this binding was partially inhibited by prior removal of the heparan sulfate chains by treatment with heparinase III ( ); removal of heparin sulfate by heparinase III was confirmed by ELISA ( ). ..

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. Removal of heparan sulfate with heparinase III, which decreased the affinity of MPO binding to perlecan but did not block this interaction , did not significantly protect the perlecan protein core against damage by MPO-H2 O2 -halide systems (oxidant/perlecan molar ratio of 200) ( ). .. However, marked protection was observed in the presence of heparin (100 µg/ml) , which completely blocked binding of MPO to perlecan , with the extent of damage induced by the MPO-H2 O2 -Cl− system and the MPO-H2 O2 -Br− systems being of a similar magnitude to that observed with reagent HOCl and HOBr (the low extents of damage by the reagent oxidants are consistent with gelatin exerting a protective effect by acting as an alternative target for oxidation).

Fluorescence In Situ Hybridization:

Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan
Article Snippet: .. Enzymatic digestion of surface-adsorbed perlecan was carried out with heparinase III (0.01 U/ml, 50 µl) or chondroitinase ABC (0.05 U/ml, 50 µl) in 0.01% BSA or 0.01% fish gelatin (for MPO binding studies) for 2 h at 37°C. .. Treatment of surface-adsorbed perlecan with HNO2 was performed at pH 3.9 to cleave heparan sulfate specifically at its GlcNH2 residues ( ) by incubation with 4.5 M sodium nitrite (50 µl) and 2 M acetic acid (10 µl) for 10 min at 22°C.

Derivative Assay:

Article Title: Cell Density-Dependent Fibroblast Growth Factor-2 Signaling Regulates Syndecan-4 Expression in Cultured Vascular Endothelial Cells
Article Snippet: .. Heparinase II (derived from Flavobacterium heparinum ) and heparinase III (EC 4.2.2.8, derived from F. heparinum) were acquired from IBEX Technologies (Montreal, QC, Canada). .. Diethylaminoethyl (DEAE)-Sephacel was obtained from Sigma-Aldrich (St Louis, MO, USA).

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    IBEX Technologies heparinase iii
    Characterization of endothelial cell-derived perlecan. (A) Native perlecan ( lane 1 ) and <t>heparinase</t> <t>III-treated</t> perlecan ( lane 2 ) were electrophoresed in a 3–8% SDS-PAGE gel under non-reducing conditions, electroblotted to nitrocellulose and probed with antibody CSI-076 against perlecan domain I. (B) Surface-adsorbed perlecan was treated with HNO 2 at pH 3.9 (3.75 M sodium nitrite, 0.33 M acetic acid) or with a control solution (3.75 M sodium chloride, 0.33 M acetic acid / sodium acetate, pH 3.9), then probed by ELISA using antibodies JM403, 10E4 and HepSS-1 against heparan sulfate. Data are means ± SEM (triplicate determinations from a representative experiment) and are expressed as % normal ELISA signal. * = P
    Heparinase Iii, supplied by IBEX Technologies, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/heparinase iii/product/IBEX Technologies
    Average 92 stars, based on 1 article reviews
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    heparinase iii - by Bioz Stars, 2020-09
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    Characterization of endothelial cell-derived perlecan. (A) Native perlecan ( lane 1 ) and heparinase III-treated perlecan ( lane 2 ) were electrophoresed in a 3–8% SDS-PAGE gel under non-reducing conditions, electroblotted to nitrocellulose and probed with antibody CSI-076 against perlecan domain I. (B) Surface-adsorbed perlecan was treated with HNO 2 at pH 3.9 (3.75 M sodium nitrite, 0.33 M acetic acid) or with a control solution (3.75 M sodium chloride, 0.33 M acetic acid / sodium acetate, pH 3.9), then probed by ELISA using antibodies JM403, 10E4 and HepSS-1 against heparan sulfate. Data are means ± SEM (triplicate determinations from a representative experiment) and are expressed as % normal ELISA signal. * = P

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan

    doi: 10.1016/j.matbio.2009.09.005

    Figure Lengend Snippet: Characterization of endothelial cell-derived perlecan. (A) Native perlecan ( lane 1 ) and heparinase III-treated perlecan ( lane 2 ) were electrophoresed in a 3–8% SDS-PAGE gel under non-reducing conditions, electroblotted to nitrocellulose and probed with antibody CSI-076 against perlecan domain I. (B) Surface-adsorbed perlecan was treated with HNO 2 at pH 3.9 (3.75 M sodium nitrite, 0.33 M acetic acid) or with a control solution (3.75 M sodium chloride, 0.33 M acetic acid / sodium acetate, pH 3.9), then probed by ELISA using antibodies JM403, 10E4 and HepSS-1 against heparan sulfate. Data are means ± SEM (triplicate determinations from a representative experiment) and are expressed as % normal ELISA signal. * = P

    Article Snippet: Surface-adsorbed perlecan was found to bind MPO and this binding was partially inhibited by prior removal of the heparan sulfate chains by treatment with heparinase III ( ); removal of heparin sulfate by heparinase III was confirmed by ELISA ( ).

    Techniques: Derivative Assay, SDS Page, Enzyme-linked Immunosorbent Assay

    Binding of MPO by perlecan. Surface-bound perlecan, in gelatin-blocked microplate wells, was incubated with MPO and probed by ELISA using antibody 2C7 against MPO. (A) Binding of MPO to perlecan and effect of prior treatment of perlecan with heparinase III and chondrotinase ABC. (B) Confirmation of heparan sulfate removal by heparinase III by loss of recognition by 10E4 and gain in recognition by 3G10 against heparinase III-derived heparan sulfate stubs. (C) Effect of heparin, heparin sulfate and chondroitin sulfate (100 µg/ml, 10 min, 22°C) on binding of MPO by perlecan. Data are means ± SEM (triplicate determinations from a representative experiment) and are expressed as % normal MPO binding, % normal ELISA signal (10E4 and CSI-076) or % maximal ELISA signal (3G10). In (A) and (B), ELISA signals were corrected for values obtained with non-perlecan coated wells; in (B), ELISA signals were corrected for values obtained with non-perlecan coated wells (blocked with fish gelatin) subject to identical treatments. Treatment with glycosaminoglycans did not significantly reverse background binding of MPO to non-perlecan coated wells (data not shown). * = P

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: Myeloperoxidase-derived oxidants selectively disrupt the protein core of the heparan sulfate proteoglycan perlecan

    doi: 10.1016/j.matbio.2009.09.005

    Figure Lengend Snippet: Binding of MPO by perlecan. Surface-bound perlecan, in gelatin-blocked microplate wells, was incubated with MPO and probed by ELISA using antibody 2C7 against MPO. (A) Binding of MPO to perlecan and effect of prior treatment of perlecan with heparinase III and chondrotinase ABC. (B) Confirmation of heparan sulfate removal by heparinase III by loss of recognition by 10E4 and gain in recognition by 3G10 against heparinase III-derived heparan sulfate stubs. (C) Effect of heparin, heparin sulfate and chondroitin sulfate (100 µg/ml, 10 min, 22°C) on binding of MPO by perlecan. Data are means ± SEM (triplicate determinations from a representative experiment) and are expressed as % normal MPO binding, % normal ELISA signal (10E4 and CSI-076) or % maximal ELISA signal (3G10). In (A) and (B), ELISA signals were corrected for values obtained with non-perlecan coated wells; in (B), ELISA signals were corrected for values obtained with non-perlecan coated wells (blocked with fish gelatin) subject to identical treatments. Treatment with glycosaminoglycans did not significantly reverse background binding of MPO to non-perlecan coated wells (data not shown). * = P

    Article Snippet: Surface-adsorbed perlecan was found to bind MPO and this binding was partially inhibited by prior removal of the heparan sulfate chains by treatment with heparinase III ( ); removal of heparin sulfate by heparinase III was confirmed by ELISA ( ).

    Techniques: Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Derivative Assay, Fluorescence In Situ Hybridization

    Effects of MEK1/2 inhibitor U0126 on the expression of syndecan-4 core protein expression in the dense culture of vascular endothelial cells. The dense culture of vascular endothelial cells was pretreated with 5 µM MEK1/2 inhibitor U0126 at 37 °C for 3 h and then stimulated with 20 ng/mL FGF-2 for 6 h, and assessed for the syndecan-4 core protein level by western blotting. The bar graph shows the intensity of syndecan-4 in the cell layer in the group treated with heparinase II/III. The values in the bar graphs indicate the means ± S.E. of three samples of the experiments. * Significantly different from the control, p

    Journal: International Journal of Molecular Sciences

    Article Title: Cell Density-Dependent Fibroblast Growth Factor-2 Signaling Regulates Syndecan-4 Expression in Cultured Vascular Endothelial Cells

    doi: 10.3390/ijms21103698

    Figure Lengend Snippet: Effects of MEK1/2 inhibitor U0126 on the expression of syndecan-4 core protein expression in the dense culture of vascular endothelial cells. The dense culture of vascular endothelial cells was pretreated with 5 µM MEK1/2 inhibitor U0126 at 37 °C for 3 h and then stimulated with 20 ng/mL FGF-2 for 6 h, and assessed for the syndecan-4 core protein level by western blotting. The bar graph shows the intensity of syndecan-4 in the cell layer in the group treated with heparinase II/III. The values in the bar graphs indicate the means ± S.E. of three samples of the experiments. * Significantly different from the control, p

    Article Snippet: Heparinase II (derived from Flavobacterium heparinum ) and heparinase III (EC 4.2.2.8, derived from F. heparinum) were acquired from IBEX Technologies (Montreal, QC, Canada).

    Techniques: Expressing, Western Blot