Subcellular localization of the Streptococcus mutans P1 protein C terminus.
To determine the subcellular location of the Streptococcus mutans P1 protein C-terminal anchor, cell envelope fractionation experiments were conducted in combination with Western immunoblotting, using monoclonal antibody MAb 6-8C specific for an epitope that maps near the C terminus of P1 protein and also a polyclonal antibody preparation directed against the P1 C-terminal 144 amino acids (P1COOH). P1 protein was detected in cell walls but not the membrane purified from S. mutans cells by the monoclonal antibody. In contrast, P1 protein was not detected in the same cell wall preparation using the anti-P1COOH polyclonal antibody. However, proteins released from the cell walls by treatment with mutanolysin contained antigen that was recognized by the anti-P1COOH antibody, suggesting that the epitopes recognized by the antibody were masked by peptidoglycan in the cell wall preparations. When cell walls were treated with boiling trichloroacetic acid to solubilize cell-wall-associated carbohydrate, P1 antigen could not be detected in either the solubilized carbohydrate, or in the remaining peptidoglycan, regardless of whether polyclonal or monoclonal antibody was used. However, when the peptidoglycan was treated with mutanolysin, P1 antigen could be detected in the mutanolysin solubilized fraction by MAb 6-8C. Collectively, these data suggest that the C-terminal 144 amino acids of the P1 protein are embedded within the cell wall, and associated exclusively with the peptidoglycan. Furthermore, the ability of the anti-P1COOH antibody to recognize P1 antigen only after mutanolysin treatment of cell walls suggests these C-terminal 144 amino acids are tightly intercalated within the peptidoglycan strands.
Homonylo-McGavin
M K
MK
Department of Oral Biology, Faculty of Dentistry, University of Manitoba, Winnipeg, Canada.
Lee
S F
SF
Bowden
G H
GH
D001426:Bacterial Proteins / Q000032:analysis*; D015153:Blotting, Western; D002473:Cell Wall / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D008562:Membrane Glycoproteins; D010457:Peptidoglycan / Q000737:chemistry; D013295:Streptococcus mutans / Q000737:chemistry*; D013347:Subcellular Fractions
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010457:Peptidoglycan; C055764:S-layer proteins