Truncated forms of mannose-binding protein multimerize and bind to mannose-rich Salmonella montevideo but fail to activate complement in vitro.
Human serum (MBP) and human recombinant (rMBP) mannose-binding protein bind to mannose-rich, serum-resistant Salmonella montevideo (SH5770), enhance C3 deposition, and render the organisms serum-sensitive. We investigated structural features of MBP necessary for this effect. MBP has a cysteine-rich amino-terminal region, a collagen-like region, and a carboxyl-terminal carbohydrate-recognition domain. We prepared carbohydrate-recognition domains lacking the other two domains either by deletion mutagenesis (delta MBP, 16 kDa) or by collagenase digestion of whole rMBP (cdMBP, 16-18 kDa). Whole and truncated MBP were detected on immunoblot by specific monoclonal antibodies that recognize both bound and free MBP. rMBP enhanced C3 deposition on SH5770 8-fold, while cdMBP and delta MBP did not increase C3 deposition over control levels. All forms of MBP bound to SH5770 by enzyme-linked immunosorbent assay and by measuring binding of radiolabeled whole and truncated MBP. Binding by 125I-delta MBP was inhibited by mannan and by MBP. Thus failure of truncated MBPs to enhance C3 deposition was not due to failure to specifically bind carbohydrate residues. To determine the molecular form of truncated MBP in nondenaturing conditions, 125I-delta MBP was centrifuged through a 5-20% sucrose-density gradient. The peak of 125I-delta MBP sedimented to estimated S20,w 2.01, but larger multimers also were present. Multimers bound SH5770 with higher affinity than monomers. We conclude that carbohydrate-recognition regions of MBP produced by collagenase digestion or by deletion mutagenesis are sufficient for ligand binding. However, the collagen-like region is necessary for MBP enhancement of C3 deposition on SH5770.
Schweinle
J E
JE
Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Nishiyasu
M
M
Ding
T Q
TQ
Sastry
K
K
Gillies
S D
SD
Ezekowitz
R A
RA
D001483:Base Sequence; D002352:Carrier Proteins / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D017364:Collagenases / Q000378:metabolism; D003167:Complement Activation; D003176:Complement C3 / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008358:Mannose / Q000378:metabolism*; D037241:Mannose-Binding Lectins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012475:Salmonella / Q000378:metabolism*; D017384:Sequence Deletion
D002352:Carrier Proteins; D003176:Complement C3; D037241:Mannose-Binding Lectins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D017364:Collagenases; D008358:Mannose