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Transmembrane Domain Oligomerization Propensity determined by ToxR Assay
Published on: May 26, 2011
Truncated forms of mannose-binding protein multimerize and bind to mannose-rich Salmonella montevideo but fail to
J E Schweinle1, M Nishiyasu, T Q Ding
1Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
Abstract:
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.
Insights
Mannose-binding protein (MBP) binds to Salmonella, but its collagen-like region is essential for enhancing C3 deposition and making bacteria serum-sensitive. The carbohydrate-recognition domain alone binds bacteria but doesn't trigger this immune response.
Area of Science:
- Immunology
- Microbiology
- Structural Biology
Background:
- Human mannose-binding protein (MBP) binds to mannose-rich bacteria like Salmonella montevideo (SH5770).
- MBP binding enhances complement component 3 (C3) deposition, leading to bacterial lysis (serum sensitivity).
Purpose of the Study:
- To investigate the structural features of MBP required for enhancing C3 deposition and bacterial serum sensitivity.
- To determine the role of different MBP domains in its immune functions.
Main Methods:
- Preparation of truncated MBP forms (delta MBP and collagenase-digested MBP, cdMBP) lacking collagen-like and N-terminal regions.
- Detection of MBP forms using immunoblotting and monoclonal antibodies.
- Assessing MBP binding to SH5770 via enzyme-linked immunosorbent assay (ELISA) and radiolabeling.
- Measuring C3 deposition enhancement on SH5770 incubated with different MBP forms.
- Analyzing the molecular form of truncated MBP using sucrose-density gradient centrifugation.
Main Results:
- Both whole recombinant MBP (rMBP) and truncated MBP forms (cdMBP, delta MBP) bound to SH5770.
- rMBP significantly enhanced C3 deposition (8-fold), while cdMBP and delta MBP did not increase it over control levels.
- Binding of truncated MBP was specific, inhibited by mannan and MBP, indicating intact carbohydrate-recognition domains.
- Truncated MBP existed as monomers and multimers in solution, with multimers showing higher binding affinity.
- The collagen-like region was absent in truncated MBP forms.
Conclusions:
- The carbohydrate-recognition domains of MBP are sufficient for binding to mannose-rich bacteria.
- The collagen-like region of MBP is essential for its ability to enhance C3 deposition and confer serum sensitivity to Salmonella.
- MBP's oligomeric structure may influence its binding efficiency, but the collagen-like region is critical for complement activation.
