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An unusual mechanism of isopeptide bond formation attaches the collagenlike glycoprotein BclA to the exosporium of Bacillus anthracis.

Tan L, Li M, Turnbough CL - MBio (2011)

Bottom Line: Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain.This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT

Unlabelled: The outermost exosporium layer of spores of Bacillus anthracis, the causative agent of anthrax, is comprised of a basal layer and an external hairlike nap. The nap includes filaments composed of trimers of the collagenlike glycoprotein BclA. Essentially all BclA trimers are tightly attached to the spore in a process requiring the basal layer protein BxpB (also called ExsFA). Both BclA and BxpB are incorporated into stable, high-molecular-mass complexes, suggesting that BclA is attached directly to BxpB. The 38-residue amino-terminal domain of BclA, which is normally proteolytically cleaved between residues 19 and 20, is necessary and sufficient for basal layer attachment. In this study, we demonstrate that BclA attachment occurs through the formation of isopeptide bonds between the free amino group of BclA residue A20 and a side chain carboxyl group of an acidic residue of BxpB. Ten of the 13 acidic residues of BxpB can participate in isopeptide bond formation, and at least three BclA polypeptide chains can be attached to a single molecule of BxpB. We also demonstrate that similar cross-linking occurs in vitro between purified recombinant BclA and BxpB, indicating that the reaction is spontaneous. The mechanism of BclA attachment, specifically, the formation of a reactive amino group by proteolytic cleavage and the promiscuous selection of side chain carboxyl groups of internal acidic residues, appears to be different from other known mechanisms for protein cross-linking through isopeptide bonds. Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.

Importance: Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain. Isopeptide bonds generate cross-links within and between proteins that are necessary for proper protein structure and function. In this study, we discovered that BclA, the dominant structural protein of the external nap of Bacillus anthracis spores, is attached to the underlying exosporium basal layer protein BxpB via isopeptide bonds formed through a mechanism fundamentally different from previously described mechanisms of isopeptide bond formation. The most unusual features of this mechanism are the generation of a reactive amino group by proteolytic cleavage and promiscuous selection of acidic side chains. This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

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Related in: MedlinePlus

Exosporium protein complexes containing BclA NTD-eGFP fusion protein(s) attached to BxpB. After separation by SDS-PAGE, protein complexes were visualized by staining with Coomassie blue and analyzed by immunoblotting with anti-GFP and anti-BxpB MAbs. Bands 1, 2, and 3 include complexes with BxpB attached to one, two, and three molecules of the BclA NTD-eGFP fusion protein, respectively. Gel locations and molecular masses of prestained protein standards are shown. The bands in the anti-GFP lane with apparent masses of approximately 30 kDa or less presumably contain free fusion protein or products of fusion protein degradation. The bands in the anti-BxpB lane with apparent masses less than that of band 1 presumably contain BxpB complexes with other basal layer proteins or free BxpB, which has a mass of 17.3 kDa.
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f2: Exosporium protein complexes containing BclA NTD-eGFP fusion protein(s) attached to BxpB. After separation by SDS-PAGE, protein complexes were visualized by staining with Coomassie blue and analyzed by immunoblotting with anti-GFP and anti-BxpB MAbs. Bands 1, 2, and 3 include complexes with BxpB attached to one, two, and three molecules of the BclA NTD-eGFP fusion protein, respectively. Gel locations and molecular masses of prestained protein standards are shown. The bands in the anti-GFP lane with apparent masses of approximately 30 kDa or less presumably contain free fusion protein or products of fusion protein degradation. The bands in the anti-BxpB lane with apparent masses less than that of band 1 presumably contain BxpB complexes with other basal layer proteins or free BxpB, which has a mass of 17.3 kDa.

Mentions: To further investigate the mechanism of BclA attachment to BxpB, we expressed a plasmid-encoded BclA NTD-enhanced green fluorescence protein (eGFP) fusion protein in BclA-deficient B. anthracis strain CLT360 (∆bclA ∆rmlD)/pCLT1525 (13). (Note that the ∆rmlD mutation in this strain prevents rhamnose biosynthesis and stabilizes the fusion protein on the spore surface for unknown reasons.) The BclA NTD directs stable attachment of the fusion protein to the exosporium basal layer of spores produced by this strain (12, 13). Exosporia were purified from these spores, exosporium protein complexes were separated by SDS-PAGE as described above in duplicate gels, and protein bands in the gels were analyzed by immunoblotting with either an anti-BxpB monoclonal antibody (MAb) (13) or a commercially available anti-eGFP MAb. We detected three major eGFP-containing protein bands with apparent molecular masses large enough to contain fusion protein-BxpB complexes, which have a minimum calculated molecular mass of 46.5 kDa. These protein bands had apparent molecular masses of 55, 90, and 130 kDa and were designated bands 1, 2, and 3, respectively (Fig. 2). The relative levels of anti-eGFP MAb staining of these three bands were 1 > 2 >> 3. Using densitometry, we measured the intensities of staining of each band with the anti-BxpB and anti-eGFP MAbs and calculated the relative amounts of BxpB and eGFP in each band. These results indicated that bands 1, 2, and 3 contained one, two, and three fusion proteins per molecule of BxpB, respectively. Based on their apparent molecular masses, and assuming slightly slower gel mobility due to a branched protein structure, our results suggest that the complexes in bands 1, 2, and 3 contain a single molecule of BxpB.


An unusual mechanism of isopeptide bond formation attaches the collagenlike glycoprotein BclA to the exosporium of Bacillus anthracis.

Tan L, Li M, Turnbough CL - MBio (2011)

Exosporium protein complexes containing BclA NTD-eGFP fusion protein(s) attached to BxpB. After separation by SDS-PAGE, protein complexes were visualized by staining with Coomassie blue and analyzed by immunoblotting with anti-GFP and anti-BxpB MAbs. Bands 1, 2, and 3 include complexes with BxpB attached to one, two, and three molecules of the BclA NTD-eGFP fusion protein, respectively. Gel locations and molecular masses of prestained protein standards are shown. The bands in the anti-GFP lane with apparent masses of approximately 30 kDa or less presumably contain free fusion protein or products of fusion protein degradation. The bands in the anti-BxpB lane with apparent masses less than that of band 1 presumably contain BxpB complexes with other basal layer proteins or free BxpB, which has a mass of 17.3 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104494&req=5

f2: Exosporium protein complexes containing BclA NTD-eGFP fusion protein(s) attached to BxpB. After separation by SDS-PAGE, protein complexes were visualized by staining with Coomassie blue and analyzed by immunoblotting with anti-GFP and anti-BxpB MAbs. Bands 1, 2, and 3 include complexes with BxpB attached to one, two, and three molecules of the BclA NTD-eGFP fusion protein, respectively. Gel locations and molecular masses of prestained protein standards are shown. The bands in the anti-GFP lane with apparent masses of approximately 30 kDa or less presumably contain free fusion protein or products of fusion protein degradation. The bands in the anti-BxpB lane with apparent masses less than that of band 1 presumably contain BxpB complexes with other basal layer proteins or free BxpB, which has a mass of 17.3 kDa.
Mentions: To further investigate the mechanism of BclA attachment to BxpB, we expressed a plasmid-encoded BclA NTD-enhanced green fluorescence protein (eGFP) fusion protein in BclA-deficient B. anthracis strain CLT360 (∆bclA ∆rmlD)/pCLT1525 (13). (Note that the ∆rmlD mutation in this strain prevents rhamnose biosynthesis and stabilizes the fusion protein on the spore surface for unknown reasons.) The BclA NTD directs stable attachment of the fusion protein to the exosporium basal layer of spores produced by this strain (12, 13). Exosporia were purified from these spores, exosporium protein complexes were separated by SDS-PAGE as described above in duplicate gels, and protein bands in the gels were analyzed by immunoblotting with either an anti-BxpB monoclonal antibody (MAb) (13) or a commercially available anti-eGFP MAb. We detected three major eGFP-containing protein bands with apparent molecular masses large enough to contain fusion protein-BxpB complexes, which have a minimum calculated molecular mass of 46.5 kDa. These protein bands had apparent molecular masses of 55, 90, and 130 kDa and were designated bands 1, 2, and 3, respectively (Fig. 2). The relative levels of anti-eGFP MAb staining of these three bands were 1 > 2 >> 3. Using densitometry, we measured the intensities of staining of each band with the anti-BxpB and anti-eGFP MAbs and calculated the relative amounts of BxpB and eGFP in each band. These results indicated that bands 1, 2, and 3 contained one, two, and three fusion proteins per molecule of BxpB, respectively. Based on their apparent molecular masses, and assuming slightly slower gel mobility due to a branched protein structure, our results suggest that the complexes in bands 1, 2, and 3 contain a single molecule of BxpB.

Bottom Line: Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain.This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

ABSTRACT

Unlabelled: The outermost exosporium layer of spores of Bacillus anthracis, the causative agent of anthrax, is comprised of a basal layer and an external hairlike nap. The nap includes filaments composed of trimers of the collagenlike glycoprotein BclA. Essentially all BclA trimers are tightly attached to the spore in a process requiring the basal layer protein BxpB (also called ExsFA). Both BclA and BxpB are incorporated into stable, high-molecular-mass complexes, suggesting that BclA is attached directly to BxpB. The 38-residue amino-terminal domain of BclA, which is normally proteolytically cleaved between residues 19 and 20, is necessary and sufficient for basal layer attachment. In this study, we demonstrate that BclA attachment occurs through the formation of isopeptide bonds between the free amino group of BclA residue A20 and a side chain carboxyl group of an acidic residue of BxpB. Ten of the 13 acidic residues of BxpB can participate in isopeptide bond formation, and at least three BclA polypeptide chains can be attached to a single molecule of BxpB. We also demonstrate that similar cross-linking occurs in vitro between purified recombinant BclA and BxpB, indicating that the reaction is spontaneous. The mechanism of BclA attachment, specifically, the formation of a reactive amino group by proteolytic cleavage and the promiscuous selection of side chain carboxyl groups of internal acidic residues, appears to be different from other known mechanisms for protein cross-linking through isopeptide bonds. Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.

Importance: Isopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain. Isopeptide bonds generate cross-links within and between proteins that are necessary for proper protein structure and function. In this study, we discovered that BclA, the dominant structural protein of the external nap of Bacillus anthracis spores, is attached to the underlying exosporium basal layer protein BxpB via isopeptide bonds formed through a mechanism fundamentally different from previously described mechanisms of isopeptide bond formation. The most unusual features of this mechanism are the generation of a reactive amino group by proteolytic cleavage and promiscuous selection of acidic side chains. This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanism in vivo and adapted for protein cross-linking in vitro.

Show MeSH
Related in: MedlinePlus