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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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Acidic residues of BxpB that can serve as sites for covalent attachment of BclA. Formation of >250-kDa BclA/BxpB-containing exosporium protein complexes formed by the indicated strains was detected by immunoblotting with an anti-BclA MAb. The strains examined were Sterne (wild type [WT]), a Sterne mutant lacking bxpB (∆bxpB), and variants of the ∆bxpB mutant that carried a plasmid directing the correctly timed expression of wild-type BxpB (pWT) and the indicated mutant BxpB proteins. In the 10M mutant protein, all acidic residues except D5, D12, and E14 were changed to alanines; in the 10M+D/E mutant proteins, all acidic residues except D5, D12, E14, and the indicated D/E residue were changed to alanines. Only the part of the immunoblot containing bands is shown, and the gel locations and molecular masses of prestained protein standards are indicated. The arrowhead points to the band containing glycosylated monomeric BclA, and the brace marks the >250-kDa BclA/BxpB-containing complexes (13).
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f3: Acidic residues of BxpB that can serve as sites for covalent attachment of BclA. Formation of >250-kDa BclA/BxpB-containing exosporium protein complexes formed by the indicated strains was detected by immunoblotting with an anti-BclA MAb. The strains examined were Sterne (wild type [WT]), a Sterne mutant lacking bxpB (∆bxpB), and variants of the ∆bxpB mutant that carried a plasmid directing the correctly timed expression of wild-type BxpB (pWT) and the indicated mutant BxpB proteins. In the 10M mutant protein, all acidic residues except D5, D12, and E14 were changed to alanines; in the 10M+D/E mutant proteins, all acidic residues except D5, D12, E14, and the indicated D/E residue were changed to alanines. Only the part of the immunoblot containing bands is shown, and the gel locations and molecular masses of prestained protein standards are indicated. The arrowhead points to the band containing glycosylated monomeric BclA, and the brace marks the >250-kDa BclA/BxpB-containing complexes (13).

Mentions: The results shown in Tables 1 and 2 demonstrate that BclA NTD attachment can occur at 10 of the 13 widely scattered acidic residues of BxpB. Attachment to BxpB amino-terminal residues D5, D12, and E14 was not detected, although numerous BxpB fragments including these residues were identified by LC-MS/MS. To further investigate the selection of BclA attachment sites, we constructed a series of plasmids capable of expressing, from the bxpB promoter, wild-type BxpB and BxpB mutant proteins in which selected acidic residues were changed to alanines. The mutations included changing all 13 acidic residues (designated 13M), changing all acidic residues except D5, D12, and E14 (designated 10M), and changing all acidic residues except D5, D12, E14, and 1 of the other 10 D/E residues (designated 10M plus the other retained D/E residue). The expression plasmids were individually introduced by transformation into a ∆bxpB variant of the Sterne strain (CLT307), and the formation of >250-kDa complexes containing BclA and BxpB was examined during sporulation. These complexes were detected by immunoblotting with an anti-BclA MAb (Fig. 3), and the presence of wild-type or mutant BxpB protein was confirmed by immunoblotting with an anti-BxpB MAb (data not shown) (13) or by MS/MS analysis of proteolytic fragments as described above, respectively.


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)

Acidic residues of BxpB that can serve as sites for covalent attachment of BclA. Formation of >250-kDa BclA/BxpB-containing exosporium protein complexes formed by the indicated strains was detected by immunoblotting with an anti-BclA MAb. The strains examined were Sterne (wild type [WT]), a Sterne mutant lacking bxpB (∆bxpB), and variants of the ∆bxpB mutant that carried a plasmid directing the correctly timed expression of wild-type BxpB (pWT) and the indicated mutant BxpB proteins. In the 10M mutant protein, all acidic residues except D5, D12, and E14 were changed to alanines; in the 10M+D/E mutant proteins, all acidic residues except D5, D12, E14, and the indicated D/E residue were changed to alanines. Only the part of the immunoblot containing bands is shown, and the gel locations and molecular masses of prestained protein standards are indicated. The arrowhead points to the band containing glycosylated monomeric BclA, and the brace marks the >250-kDa BclA/BxpB-containing complexes (13).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f3: Acidic residues of BxpB that can serve as sites for covalent attachment of BclA. Formation of >250-kDa BclA/BxpB-containing exosporium protein complexes formed by the indicated strains was detected by immunoblotting with an anti-BclA MAb. The strains examined were Sterne (wild type [WT]), a Sterne mutant lacking bxpB (∆bxpB), and variants of the ∆bxpB mutant that carried a plasmid directing the correctly timed expression of wild-type BxpB (pWT) and the indicated mutant BxpB proteins. In the 10M mutant protein, all acidic residues except D5, D12, and E14 were changed to alanines; in the 10M+D/E mutant proteins, all acidic residues except D5, D12, E14, and the indicated D/E residue were changed to alanines. Only the part of the immunoblot containing bands is shown, and the gel locations and molecular masses of prestained protein standards are indicated. The arrowhead points to the band containing glycosylated monomeric BclA, and the brace marks the >250-kDa BclA/BxpB-containing complexes (13).
Mentions: The results shown in Tables 1 and 2 demonstrate that BclA NTD attachment can occur at 10 of the 13 widely scattered acidic residues of BxpB. Attachment to BxpB amino-terminal residues D5, D12, and E14 was not detected, although numerous BxpB fragments including these residues were identified by LC-MS/MS. To further investigate the selection of BclA attachment sites, we constructed a series of plasmids capable of expressing, from the bxpB promoter, wild-type BxpB and BxpB mutant proteins in which selected acidic residues were changed to alanines. The mutations included changing all 13 acidic residues (designated 13M), changing all acidic residues except D5, D12, and E14 (designated 10M), and changing all acidic residues except D5, D12, E14, and 1 of the other 10 D/E residues (designated 10M plus the other retained D/E residue). The expression plasmids were individually introduced by transformation into a ∆bxpB variant of the Sterne strain (CLT307), and the formation of >250-kDa complexes containing BclA and BxpB was examined during sporulation. These complexes were detected by immunoblotting with an anti-BclA MAb (Fig. 3), and the presence of wild-type or mutant BxpB protein was confirmed by immunoblotting with an anti-BxpB MAb (data not shown) (13) or by MS/MS analysis of proteolytic fragments as described above, respectively.

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