title: Identification of an endogenous membrane anchor-cleaving enzyme for group A streptococcal M protein. Its implication for the attachment of surface proteins in gram-positive bacteria.
abstract: How streptococcal M protein or other surface proteins of gram-positive bacteria are anchored to the cell is poorly understood. Previously, we reported that M protein released after cell wall removal with a muralytic enzyme lacked the COOH terminal hydrophobic amino acids and charged tail predicted from DNA sequence. An endogenous membrane anchor-cleaving enzyme has now been identified with the ability to release M protein from isolated streptococcal protoplasts. At pH 5.5 in the presence of 30% raffinose, the streptococcal cell wall may be removed with a muralytic enzyme without releasing M protein from the resulting protoplasts indicating that the M molecule is attached through the bacterial cytoplasmic membrane. Release of M molecules occurs when the M protein-charged protoplasts are placed in raffinose buffer at pH 7.4. Although Zn2+, Cd2+, Ca2+, PHMB, and pHMPS inhibit the activity of the releasing enzyme, the blocking activity of Zn2+, Cd2+, and Ca2+ are reversible while PHMB and pHMPS are irreversible. PHMB-treated protoplasts are unable to release M protein at pH 7.4. However, M protein is liberated from these protoplasts when mixed with those prepared from M- streptococci serving as an enzyme source. The supernatant from M- protoplasts is unable to release M protein from PHMB-inactivated M+ protoplasts, confirming that the anchor-cleaving enzyme is membrane bound. Thus, the M protein releasing activity appears to be the result of a thiol-dependent anchor-cleaving enzyme. Streptococcal membranes treated with sodium carbonate and Triton X-114 still retain the M protein verifying that it is an integral membrane molecule. Evidence also is presented indicating significant sequence similarity between M protein and certain GPI-anchored proteins in the region responsible for protein anchoring.
authors.affiliation: Rockefeller University, New York, New York 10021.
authors.forename: V
authors.initials: V
authors.lastname: Pancholi
authors.forename: V A
authors.initials: VA
authors.lastname: Fischetti
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism*; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D006017:Glycolipids / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000032:analysis; D010716:Phosphatidylinositols / Q000378:metabolism; D011523:Protoplasts / Q000378:metabolism; D013291:Streptococcus / Q000201:enzymology*
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010716:Phosphatidylinositols; C014689:streptococcal M protein