Identification by microsequencing of lipopolysaccharide-induced proteins secreted by mouse macrophages.
The conditioned medium of the murine macrophage PU5.1.8 was analyzed by two-dimensional gel electrophoresis in order to detect LPS-induced proteins. Spots of interest were identified by microsequencing of internal peptides generated by limited in situ acid hydrolysis. In total conditioned medium, several monokines (TNF-alpha and macrophage inflammatory protein-1 alpha and 1 beta) were identified as LPS-induced spots. Because minor spots could be masked by the complexity of the 2-D pattern, conditioned medium was successively fractionated by zinc precipitation and affinity chromatography (Procion red and Con A agarose). Zinc supernatant fraction, Procion red flow-through, and Con A eluate fractions were further analyzed by 2-D gel electrophoresis for the presence of LPS-induced spots. In these fractions serum amyloid A3, lipocalin 24p3, cathepsin B, and plasminogen activator inhibitor-I were characterized as LPS-induced proteins secreted by macrophages. Lipocalin 24p3 protein was retrieved for the first time. In addition to these proteins that follow a classical secretory pathway, several cellular proteins (mainly ribosomal proteins) were retrieved as LPS-induced proteins in the conditioned medium. Control experiments argue against the obvious explanation that the latter observation is caused solely by cellular leakage.
Meheus
L A
LA
Innogenetics N.V., Industripark Zwijnaarde, Belgium.
Fransen
L M
LM
Raymackers
J G
JG
Blockx
H A
HA
Van Beeumen
J J
JJ
Van Bun
S M
SM
Van de Voorde
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D066298:In Vitro Techniques; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation; D008264:Macrophages / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D000685:Serum Amyloid A Protein / Q000737:chemistry / Q000378:metabolism*
D002352:Carrier Proteins; D008070:Lipopolysaccharides; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D012333:RNA, Messenger; C076498:Saa3 protein, mouse; D000685:Serum Amyloid A Protein