Ο-Sialoglycoprotein Endopeptidase

Ο-sialoglycoprotein endopeptidaseis a neutral metalloprotease purified from Mannheimia haemolytica (formerlyknown as Pasteurella haemolytica). This unique proteolytic enzymespecifically cleaves proteins bearing clusters of negatively charged sugars,makingΟ-sialoglycoproteins and sulfatedglycoproteins good substrates. The enzyme is inhibited at highconcentrations of EDTA (above 10 mM), by sialate analogues (above 5 mM) and bythe putative metal ion activator Zn²⁺ at concentrations around 50 μM. No directactivation byzinc ionshas been observed at any concentration. The enzyme is not effected by serineprotease inhibitors, aspartyl protease inhibitors or thiol inhibitors.

Numerous Ο-sialoglycoproteins have been shown tobe cleaved by this enzyme, but no N-linked sialoglycoproteins orunglycosylated 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.

Applications:

§ Ideal for characterizing cell surface glycoproteins.

§ Useful in glycoprotein epitope-mapping studies.

§ Can be used to modify the adhesion properties of cells, includingrolling behavior of neutrophils.

§ Can be used to degrade Ο-sialoglycoproteins to enable peptidesequencing of the resultant fragments.

§ Used for the immunomagnetic separation of human stem cellsbearing the CD34 antigen, in that it will cleave CD34and release the antibody-magnetic bead complex from the isolated stem cell.

Figure A. Hydrolysis of humanglycophorin A compared with that of bovine K-casein: Hydrolysis of the two ¹²⁵I-labeled substratesas a function of substrate concentration in a 5 min incubation with equivalentamounts of P. haemolytica glycoprotease pH 4.5 precipitate. Hydrolysiswas measured by the disappearance of TCA-soluble products from ¹²⁵I-labeledglycophorin A bands from SDS-PAGE gels and by the appearance of TCA-solubleproducts 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]