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O-Sialoglycoprotein Endopeptidase

  • 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, making Ο-sialoglycoproteins and sulfated glycoproteins good substrates. 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 effected 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 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.


    § 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 Ο-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.

    Figure A. Hydrolysis of human glycophorin A compared with that of bovine K-casein: Hydrolysis of the two 125I-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 products from 125I-labeled glycophorin A bands from SDS-PAGE gels 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 thedouble reciprocal plot for the same data. Means + SE (n = 3).[19]

Product Name: O-sialoglycoprotein endopeptidase, Lyophilized
Unit Price:
$674.00 CDN
  • References:

    1.   G.L. Otulakowski, P.E. Shewen, A.E. Udoh, A. Mellors and B.N. Wilkie (1983). Proteolysis of sialo-glycoprotein by Pasteurella haemolytica cytotoxic culture supernatants. Infection and Immunity, 42, 64-70. 

    2.   K.M. Abdullah, R.Y.C. Lo, & A. Mellors (1991). Cloning, nucleotide sequence and expression of the P. haemolytica A1 glycoprotease gene. J. Bacteriol. 173, 5597-5603.

    3.   K.M. Abdullah, E.A. Udoh, P.E. Shewen, and A. Mellors (1991). A neutral glycoprotease of P. haemolytica A1 specifically cleaves Ο-sialoglycoproteins. Infect. Immun. 60, 56-62.

    4.   D.R. Sutherland, K.M. Abdullah, P. Cyopick, & A. Mellors (1992). Cleavage of the cell-surface Ο-sialoglycoproteins CD34, CD43, CD44, CD45 by a novel glycoprotease from P. haemolytica. J. Immunol. 148, 1458-1464.

    5.   C.N. Steininger, C.A. Eddy, R.M. Leimgruber, A. Mellors and J.K. Welply (1992). The glycoprotease of P. haemolytica A1 eliminates binding of myeloid cells to P-selectin but not to E-selectin. Biochem. Biophys. Res. Commun. 188, 760-766.

    6.   H. Nakada, M. Inouue, Y. Numata, N. Tanaka, I. Funakoshi, S. Fukui, A. Mellors and I. Yamashina (1993) Epitope structure of Tn glycophorin A for anti-Tn antibody (MLS 128). Proc. Natl. Acad. Sci. USA 90, 2495-2499. (This paper shows how the enzyme can be used to identify specific epitope domains on human erythrocyte glycophorin).

    7.   L. Healy and I. Titley (1993). The P. haemolytica glycoprotease cleaves the Interleukin 7 receptor expressed on human lymphoid cells. Personal communication, with permission to quote the findings.

    8.   R.H. Hu, A. Mellors and V.P. Bhavanandan (1992). Selective cleavage of epitectin from the surface of H.Ep.2 human laryngeal carcinoma cells by a glycoprotease from P. haemolytica. Glycobiology 2, 500.

    9.   E. Yeo, A. Mellors and R. Sutherland (1993). Cleavage of leukocyte surface adhesion receptors for P-selectin: Evidence for an Ο linked sialo-glycoprotein and non-involvement of sialyl-Lewisx, Blood (Supplement) Proc. XIVth Congress of the International Society of Thrombosis and Haemostasis.

    10. J.G. Oriol, K.J. Betteridge, A.J. Clarke and F.J. Sharom (1993). Mucin-like glycoproteins in the equine embryonic capsule. Mol Reprod. Develop. 34, 255-265.

    11. J.C.W. Marsh, D.R. Sutherland, J. Davidson, A. Mellors & A. Keating (1992). Retention of progenitor function in CD34+ cells purified using a novel Ο-sialoglycoprotease. Leukemia 6, 926-934.

    12. D.R. Sutherland, J.C.W. Marsh, J. Davidson, M.A. Baker, A. Keating, and A. Mellors (1992). Differential Sensitivity of CD34 Epitopes to Cleavage by Pasteurella haemolytica Glycoprotease: Implications for Purification of CD34-positive Progenitor Cells. Exp. Hematol. 20, 590-599.

    13. Sutherland D.R. et al. (1996). Identification of CD34+ subsets after glycoprotease selection: Engraftment of CD34+ Thy-1+Lin- stem cells in fetal sheep. Exp. Hematol. 24, 795-806.

    14. A. Mellors and D.R. Sutherland (1994). Tools to cleave glycoproteins. Trends in Biotechnol. 12, 15-18.

    15. W. M. Cladman, M. –A.V. Watt, J. –P. Dini, and A. Mellors (1996). The Pasteurella haemolytica Ο-sialoglycoprotein endopeptidase is inhibited by zinc ions and does not cleave fetuin. Biochem. Biophys. Res. Commun. 220, 141-146.

    16. M. –A.V. Watt, R.Y.C. Lo, and A. Mellors (1997). Refolding of recombinant Pasteurella haemolytica A1 glycoprotease expressed in an E. coli thioredoxin gene fusion system. Cell Stress and Chaperones 2, 180-190.

    17. P. Jiang and A. Mellors (2004). Ο-Sialoglycoprotein endopeptidase. In Handbook of Proteolytic Enzymes: Second Edition ed. A.L. Barrett and J.F. Woessner, p.977-980, Elsevier, London.

    18. M. –A.V. Watt, A. Mellors and R.Y.C. Lo (1997). Comparison of the recombinant and authentic forms of the P. haemolytica A1 glycoprotease. FEMS Microbiology Lett. 47, 37-43.

    19. P. Jiang and A. Mellors (1998). Membrane protein proteolysis assayed by fluorescence quenching: assay of Ο-sialoglycoprotein endopeptidase. Anal. Biochem. 259, 8-15.

    20. A. Mellors and R.Y.C. Lo (1995). The Ο-sialoglycoprotease of Pasteurella haemolytica. Methods in Enzymol. 248, 47:728-740.

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