Isolation and characterization of the highly immunogenic cell wall-associated protein of Mycobacterium leprae.
In a recent study, we demonstrated that certain reactivities crucial to the immune response in leprosy are due to protein associated with the cell wall peptidoglycan "core" of Mycobacterium leprae. We now describe a primary method for the isolation of a highly immunogenic, large molecular-size, cell wall protein (CW-P) complex from M. leprae, freed of soluble proteins, bound mycolates, arabinogalactan, and much of the peptidoglycan. The complex is of apparent relative molecular size 2 x 10(6) to 20 x 10(6) Da, is distinguished by a high content of Ala, Gly, Leu, Asx, and Glx, and some peptidoglycan, and represents up to 7% of the bacterial mass. It is stable to a variety of dissociation and reductive processes and, in accord with its size, is not resolvable by polyacrylamide gel electrophoresis. The mAb to the CW-P complex also react with the heat shock 65-kDa protein of M. leprae. Conversely, antibodies that recognize internal epitopes within the polypeptide chain of the heat shock protein also react with CW-P; however, antibodies that recognize the N and C termini of the 65-kDa protein fail to react with CW-P, and some anti-CW-P mAb do not recognize any of the soluble proteins of M. leprae. Alternate methods to derive the large peptidoglycan-associated protein result in lower yield and less of the associated heat shock protein, implying that the 65-kDa protein may not be crucial to the immunogenicity of the complex. In an accompanying paper, we demonstrate that T cell clones raised to CW-P also selectively recognize soluble proteins, primarily of 7-kDa and 16-kDa size. Thus, the image of the CW-P complex of M. leprae is of a few immunoreactive polypeptides in avid association with a modicum of peptidoglycan to which the 65-kDa polypeptide may be variably attached, perhaps due to involvement in assembly of the complex.
Hunter
S W
SW
Department of Microbiology, Colorado State University, Fort Collins 80523.
McNeil
M
M
Modlin
R L
RL
Mehra
V
V
Bloom
B R
BR
Brennan
P J
PJ
D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification*; D015153:Blotting, Western; D002473:Cell Wall / Q000032:analysis*; D006360:Heat-Shock Proteins / Q000032:analysis; D009166:Mycobacterium leprae / Q000032:analysis*; D010457:Peptidoglycan / Q000032:analysis; D012995:Solubility
D000596:Amino Acids; D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D010457:Peptidoglycan