166
PH. BULET and S. UTTENWEILER-JOSEPH
To summarize, 4 out of the 24 DIMs could be identified by their molecular
mass as already known antimicrobial peptides (the two glycoforms of drosocin,
metchnikowin and drosomycin). The 20 other DIMs represent novel molecules
involved in the immune response of Drosophila which have been characterized by
their molecular mass.
MALDI- TOP MS analysis of complex biological mixtures often permits the
detection of peptide compounds. The mass accuracy of MALDI- TOP MS is sufficient to allow the identification of molecules by their molecular mass if they are
already known. Identified peptides can then serve as internal calibrants to measure a more accurate molecular mass for unknown pep tides. Consequently, these
accurate molecular masses characterize the unknown compounds.
6
Time Course of Induction and Degradation Process of the DIMs
In order to know if the DIMs are elements of immediate defense reactions such
as coagulation and melanization (see Introduction) or involved in slower reactions, we investigated by MALDI MS their appearance in the hemolymph of
immune-induced Drosophila at various time intervals post infection (from Ih up
to 3 weeks). The most significant results were reported on Fig. 1l.4 (for further
details see Uttenweiler-Joseph et al. 1998). All the DIMs begin to be detectable in
the hemolymph 6h after immunization and reach a maximum of intensity after
24h in a very reproducible fashion. In contrast, individual variations appear
between flies after one day of immunization. However, as a general rule, the level
of DIMs slowly decreases after 24h and they become undetectable 2 or 3 weeks
after immune-challenge (Fig. 11.4).
Regarding the antimicrobial peptides (drosocin, metchnikowin and drosomycin), their stability in the hemolymph of infected flies could be determined precisely
as their transcription profIles have already been reported (Charlet et al. 1996, Levashin a et al. 1995, Fehlbaum et al. 1994). Briefly, Northern blot analysis has revealed a
decrease in transcriptional activity of genes encoding the antimicrobial peptides
24-36h after challenge. However, MALDI-TOF MS analysis shows that drosocin-2S
(DIM 11) disappears 2 weeks after the immune-challenge, whereas drosocin-IS
(DIM 9) is undetectable only after 3 weeks. The stability course observed for metchnikowin (DIM 17) is identical to the one observed for DIM 9 while drosomycin
(DIM 19) remains detectable for up to 3 weeks in most flies (Fig. 1l.4). The in vivo
stability of the antimicrobial peptides can be explained by two main structural features: (1) an overrepresentation in proline residues for drosocin and metchnikowin
and (2) a compact three-dimensional structure for drosomycin (Landon et al. 1997).
The changes in the relative ratio between the two glycoforms of drosocin suggest
that a circulating exoglycosidase cleaves the distal sugar moiety (namely galactose).
MALDI- TOP MS analysis of complex biological mixtures at different time intervals after stimulation of a physiological process allows to directly study the life
time (induction, persistence and disappearance) of a molecule that has been characterized by its molecular mass. If the transcription profile of the gene encoding a
certain peptide is known, the in vivo stability of this peptide can be investigated by
direct MALDI analysis of the biological sample.
PH. BULET and S. UTTENWEILER-JOSEPH
To summarize, 4 out of the 24 DIMs could be identified by their molecular
mass as already known antimicrobial peptides (the two glycoforms of drosocin,
metchnikowin and drosomycin). The 20 other DIMs represent novel molecules
involved in the immune response of Drosophila which have been characterized by
their molecular mass.
MALDI- TOP MS analysis of complex biological mixtures often permits the
detection of peptide compounds. The mass accuracy of MALDI- TOP MS is sufficient to allow the identification of molecules by their molecular mass if they are
already known. Identified peptides can then serve as internal calibrants to measure a more accurate molecular mass for unknown pep tides. Consequently, these
accurate molecular masses characterize the unknown compounds.
6
Time Course of Induction and Degradation Process of the DIMs
In order to know if the DIMs are elements of immediate defense reactions such
as coagulation and melanization (see Introduction) or involved in slower reactions, we investigated by MALDI MS their appearance in the hemolymph of
immune-induced Drosophila at various time intervals post infection (from Ih up
to 3 weeks). The most significant results were reported on Fig. 1l.4 (for further
details see Uttenweiler-Joseph et al. 1998). All the DIMs begin to be detectable in
the hemolymph 6h after immunization and reach a maximum of intensity after
24h in a very reproducible fashion. In contrast, individual variations appear
between flies after one day of immunization. However, as a general rule, the level
of DIMs slowly decreases after 24h and they become undetectable 2 or 3 weeks
after immune-challenge (Fig. 11.4).
Regarding the antimicrobial peptides (drosocin, metchnikowin and drosomycin), their stability in the hemolymph of infected flies could be determined precisely
as their transcription profIles have already been reported (Charlet et al. 1996, Levashin a et al. 1995, Fehlbaum et al. 1994). Briefly, Northern blot analysis has revealed a
decrease in transcriptional activity of genes encoding the antimicrobial peptides
24-36h after challenge. However, MALDI-TOF MS analysis shows that drosocin-2S
(DIM 11) disappears 2 weeks after the immune-challenge, whereas drosocin-IS
(DIM 9) is undetectable only after 3 weeks. The stability course observed for metchnikowin (DIM 17) is identical to the one observed for DIM 9 while drosomycin
(DIM 19) remains detectable for up to 3 weeks in most flies (Fig. 1l.4). The in vivo
stability of the antimicrobial peptides can be explained by two main structural features: (1) an overrepresentation in proline residues for drosocin and metchnikowin
and (2) a compact three-dimensional structure for drosomycin (Landon et al. 1997).
The changes in the relative ratio between the two glycoforms of drosocin suggest
that a circulating exoglycosidase cleaves the distal sugar moiety (namely galactose).
MALDI- TOP MS analysis of complex biological mixtures at different time intervals after stimulation of a physiological process allows to directly study the life
time (induction, persistence and disappearance) of a molecule that has been characterized by its molecular mass. If the transcription profile of the gene encoding a
certain peptide is known, the in vivo stability of this peptide can be investigated by
direct MALDI analysis of the biological sample.