Ο-Sialoglycoprotein Endopeptidase Ο-sialoglycoprotein endopeptidase is a neutral metalloprotease purified from Mannheimia haemolytica (formerly known as Pasteurella haemolytica).This unique proteolytic enzyme specifically cleaves proteins bearing clusters of negatively charged sugars: Ο-sialoglycoproteins and sulfated glycoproteins. The enzyme is inhibited at high concentrations of EDTA (above 10 mM), by sialate analogues (above 5 mM) and by the putative metal ion activator Zn2+ at concentrations around 50 μM. No direct activation by zinc ions has been observed at any concentration. The enzyme is not affected by serine protease inhibitors, aspartyl protease inhibitors or thiol inhibitors. Numerous Ο-sialoglycoproteins have been shown to be cleaved by this enzyme, but no N-linked sialoglycoproteins or unglycosylated proteins have been found to be substrates. The list of Ο-sialoglycoprotein endopeptidase substrates includes: human RBC glycophorin A the human antigens CD34, CD43, CD44, CD45 the IL-7 receptor the receptors for E-selectin, L-selectin and P-selectin the human tumor antigens epiglycanin and epitectin the platelet glycoprotein 1ba Ο-sialoglycoprotein the laminin binding proteins, dystroglycan and cranin Product endopeptidase, the lymphocyte adhesion molecule VAP-1 Lyophilized viral glycoproteins; and bone sialoprotein the sulfated glycoprotein CD24 Size 1.2 mg Applications: Cat. # CLE100 Ideal for characterizing cell surface glycoproteins. Useful in glycoprotein epitope-mapping studies. Can be used to modify the adhesion properties of cells, including rolling behaviour of neutrophils. Can be used to degrade Ο-sialoglycoproteins to enable peptide sequencing of the resultant fragments. Used for the immunomagnetic separation of human ISO 9001:2008 and ISO 13485:2003 registered. stem cells bearing the CD34 antigen, in that it will cleave Toll Free: CD34 and release the antibody-magnetic bead complex from the isolated stem cell. In Canada: 1-800-268-5058 In the U.S.A.: 1-800-721-1644 O-Sialoglycoprotein Endopeptidase Catalogue # CLE100, O-sialoglycoprotein endopeptidase, Lyophilized, 1.2 mg, $398 O-sialoglycoprotein endopeptidase is a neutral metalloprotease purified from Mannheimia haemolytica (formerly known as Pasteurella haemolytica). This unique proteolytic enzyme specifically cleaves proteins bearing clusters of negatively charged sugars, so that O-sialoglycoproteins and sulfated glycoproteins are good substrates. The enzyme is inhibited at high concentrations of EDTA (above 10 mM), by sialate analogues (above 5mM) and the putative metal ion activator Zn2+ at concentrations around 50 M. No direct activation by zinc ions has been observed at any concentration. The enzyme is not affected by serine protease inhibitors, aspartyl protease inhibitors or thiol inhibitors. Numerous O-sialoglycoproteins have been shown to be cleaved by this enzyme, but no N-linked sialoglycoproteins or unglycosylated proteins have been found to be substrates. The list of O-sialoglycoprotein substrates includes human RBC glycophorin A; the human antigens CD34, CD43, CD44, CD45; the IL-7 receptor; the receptors for E- selectin, L-selectin and P-selectin; the human tumor antigens epiglycanin and epitectin; the platelet glycoprotein 1ba; the laminin binding proteins, dystroglycan and cranin; the lymphocyte adhesion molecule VAP-1; viral glycoproteins; and bone sialoprotein. A sulfated glycoprotein, CD24, is also cleaved by the enzyme. Applications: - Ideal for characterizing cell surface glycoproteins. - Useful in glycoprotein epitope-mapping studies. - Can be used to modify the adhesion properties of cells, including rolling behavior of neutrophils. - Can be used to degrade O-sialoglycoproteins to enable peptide sequencing of the resultant fragments. - Used for the immunomagnetic separation of human stem cells bearing the CD34 antigen, in that it will cleave CD34 and release the antibody-magnetic bead complex from the isolated stem cell. GlycoproteolyticGlycoproteolytic Activity against Various Activity Substrates: Against Various Substrates: 1800 0.6 1500 1200 mol/liter/min) μ 0.4 Glycophorin A 0.008 900 0.006 0.004 1/F Fluorescence 600 0.2 0.002 κ–Casein 0.000 300 0 2 4 6 [1/S] μg -1 Substrate degraded ( degraded Substrate 0.00 0 5 10 15 2 4 6 8 10 125 FigureFigure 1. A) Hydrolysis A Substrate of human (glycophorinμg) A comparedFigure with that B of bovine [BODIPY-FL-GPA]K-casein: Hydrolysis μg of the two I- labeled substrates as a function of substrate concentration in a 5 min incubation with equivalent amounts of P. haemolytica glycoprotease pH 4.5 precipitate. Hydrolysis was measured by the disappearance of TCA-soluble productsFigure A.from Hydrolysis 125I-labeled of human glycophorin glycophorin AA comparedbands from with thatSDS of-PAGE bovine gels K-casein: and Hydrolysisby the ofappearance the two 125I-labeled of TCA substrates-soluble as a function of substrate125 concentration in[20] a 5 min incubation with equivalent amounts of P. haemolytica glycoprotease pH 4.5 precipitate. productsHydrolysis from was measuredI-labeled by -thecasein. disappearance B) Substrate of TCA-soluble concentration products from dependence 125I-labeled forglycophorin the glycoprotease A bands from hydrolysis SDS-PAGE gels of and by the appearance of TCA-soluble products from 125I-labeled κ-casein.[20] Figure B. Substrate concentration dependence for the glycoprotease hydrolysis of BODIPY-FL–GPA. The 25 μl reaction mixture containing 0.4 μg concentrated culture supernatant glycoprotease, incubated with varying amounts of BODIPY-FL–GPA in 50 mM HEPES buffer at 37°C for 10 min. The reaction was stopped by dilution and the fluorescence was measured. The inset shows the double reciprocal plot for the same data. Means + SE (n = 3).[19] References: 1. G.L. Otulakowski, P.E. Shewen, A.E. Udoh, A. Mellors and B.N. Wilkie (1983). 10. J.G. Oriol, K.J. Betteridge, A.J. Clarke and F.J. Sharom (1993). Mucin-like glycoproteins in the equine Proteolysis of sialo-glycoprotein by Pasteurella haemolytica cytotoxic culture supernatants. embryonic capsule. Mol Reprod. Develop. 34, 255-265. Infection and Immunity, 42, 64-70. 11. J.C.W. Marsh, D.R. Sutherland, J. Davidson, A. Mellors & A. Keating (1992). Retention of progenitor cell 2. K.M. Abdullah, R.Y.C. Lo, & A. Mellors (1991). Cloning, nucleotide sequence and expression of the function in CD34+ cells purified using a novelΟ -sialoglycoprotease. Leukemia 6, 926-934. P. haemolytica A1 glycoprotease gene. J. Bacteriol. 173, 5597-5603. 12. D.R. Sutherland, J.C.W. Marsh, J. Davidson, M.A. Baker, A. Keating, and A. Mellors (1992). 3. K.M. Abdullah, E.A. Udoh, P.E. Shewen, and A. Mellors (1991). A neutral glycoprotease of Differential Sensitivity of CD34 Epitopes to Cleavage by Pasteurella haemolytica Glycoprotease: P. haemolytica A1 specifically cleavesΟ -sialoglycoproteins. Infect. Immun. 60, 56-62. Implications for Purification of CD34-positive Progenitor Cells. Exp. Hematol. 20, 590-599. 4. D.R. Sutherland, K.M. Abdullah, P. Cyopick, & A. Mellors (1992). Cleavage of the cell-surface 13. Sutherland D.R. et al. (1996). Identification of CD34+ subsets after glycoprotease selection: Ο-sialoglycoproteins CD34, CD43, CD44, CD45 by a novel glycoprotease from P. haemolytica. Engraftment of CD34+ Thy-1+Lin- stem cells in fetal sheep. Exp. Hematol. 24, 795-806. J. Immunol. 148, 1458-1464. 14. A. Mellors and D.R. Sutherland (1994). Tools to cleave glycoproteins. Trends in Biotechnol. 12, 15-18. 5. C.N. Steininger, C.A. Eddy, R.M. Leimgruber, A. Mellors and J.K. Welply (1992). The glycoprotease of 15. W. M. Cladman, M. –A.V. Watt, J. –P. Dini, and A. Mellors (1996). The Pasteurella haemolytica P. haemolytica A1 eliminates binding of myeloid cells to P-selectin but not to E-selectin. Ο-sialoglycoprotein endopeptidase is inhibited by zinc ions and does not cleave fetuin. Biochem. Biophys. Res. Commun. 188, 760-766. Biochem. Biophys. Res. Commun. 220, 141-146. 6. H. Nakada, M. Inouue, Y. Numata, N. Tanaka, I. Funakoshi, S. Fukui, A. Mellors and I. Yamashina (1993) 16. M. –A.V. Watt, R.Y.C. Lo, and A. Mellors (1997). Refolding of recombinant Pasteurella haemolytica Epitope structure of Tn glycophorin A for anti-Tn antibody (MLS 128). Proc. Natl. Acad. A1 glycoprotease expressed in an E. coli thioredoxin gene fusion system. Cell Stress and Sci. USA 90, 2495-2499. (This paper shows how the enzyme can be used to identify specific Chaperones 2, 180-190. epitope domains on human erythrocyte glycophorin). 17. P. Jiang and A. Mellors (2004). Ο-Sialoglycoprotein endopeptidase. In Handbook of Proteolytic 7. L. Healy and I. Titley (1993). The P. haemolytica glycoprotease cleaves the Interleukin 7 receptor Enzymes: Second Edition ed. A.L. Barrett and J.F. Woessner, p.977-980, Elsevier, London. expressed on human lymphoid cells. Personal communication, with permission to quote the findings. 18. M. –A.V. Watt, A. Mellors and R.Y.C. Lo (1997). Comparison of the recombinant and authentic forms 8. R.H. Hu, A. Mellors and V.P. Bhavanandan (1992). Selective cleavage of epitectin from the surface of the P. haemolytica A1 glycoprotease. FEMS Microbiology Lett. 47, 37-43. of H.Ep.2 human laryngeal carcinoma cells by a glycoprotease from P. haemolytica. 19. P. Jiang and A. Mellors (1998). Membrane protein proteolysis assayed by fluorescence quenching: Glycobiology 2, 500. assay of Ο-sialoglycoprotein endopeptidase. Anal. Biochem. 259, 8-15. Figure B is reprinted for the 9. E. Yeo, A. Mellors and R. Sutherland (1993). Cleavage of leukocyte surface