Deglycosylation of the 45/47-kilodalton antigen complex of Mycobacterium tuberculosis decreases its capacity to elicit in vivo or in vitro cellular immune responses.
A protection against a challenge with Mycobacterium tuberculosis is induced by previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guérin (BCG). The 45/47-kDa antigen complex (Apa) present in culture filtrates of BCG of M. tuberculosis has been identified and isolated based on its ability to interact mainly with T lymphocytes and/or antibodies induced by immunization with living bacteria. The protein is glycosylated. A large batch of Apa was purified from M. tuberculosis culture filtrate to determine the extent of glycosylation and its role on the expression of the immune responses. Mass spectrometry revealed a spectrum of glycosylated molecules, with the majority of species bearing six, seven, or eight mannose residues (22, 24, and 17%, respectively), while others three, four, or five mannoses (5, 9, and 14%, respectively). Molecules with one, two, or nine mannoses were rare (1.5, 3, and 3%, respectively), as were unglycosylated species (in the range of 1%). To eliminate the mannose residues linked to the protein, the glycosylated Apa molecules were chemically or enzymatically treated. The deglycosylated antigen was 10-fold less active than native molecules in eliciting delayed-type hypersensitivity reactions in guinea pigs immunized with BCG. It was 30-fold less active than native molecules when assayed in vitro for its capacity to stimulate T lymphocytes primed in vivo. The presence of the mannose residues on the Apa protein was essential for the antigenicity of the molecules in T-cell-dependent immune responses in vitro and in vivo.
Romain
F
F
Unité de Physiopathologie de l'Infection, Institut Pasteur, 75724 Paris Cedex 15, France.
Horn
C
C
Pescher
P
P
Namane
A
A
Riviere
M
M
Puzo
G
G
Barzu
O
O
Marchal
G
G
D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D005260:Female; D006031:Glycosylation; D006168:Guinea Pigs; D006968:Hypersensitivity, Delayed; D008213:Lymphocyte Activation; D013058:Mass Spectrometry; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D013329:Structure-Activity Relationship; D013601:T-Lymphocytes / Q000276:immunology
D000942:Antigens, Bacterial