Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203.
Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M+ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M+ and M- bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M+ bacteria after treatment with N-acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1-5 and 198-202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M- bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.
Hong
K
K
Department of Bacteriology, Osaka University Medical School, Japan.
Komurasaki
Y
Y
Kobayashi
H
H
Ishikawa
H
H
Inoue
K
K
D000818:Animals; D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000276:immunology; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D002352:Carrier Proteins; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D010587:Phagocytosis / Q000187:drug effects; D011485:Protein Binding / Q000276:immunology; D013297:Streptococcus pyogenes / Q000737:chemistry* / Q000276:immunology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; C014689:streptococcal M protein