Limits...
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.

Show MeSH

Related in: MedlinePlus

Formation of high-molecular-mass complexes containing cross-linked rBclA and rBxpB. Complexes were formed in reaction mixtures containing 20 µM rBclA and 5 µM rBxpB. Samples of purified rBclA and rBxpB and of rBclA-rBxpB cross-linked complexes were separately analyzed in triplicate by SDS-PAGE. The three essentially identical gels were used to detect proteins and protein complexes by immunoblotting with either an anti-BclA or an anti-BxpB MAb or by staining with Coomassie blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3104494&req=5

f4: Formation of high-molecular-mass complexes containing cross-linked rBclA and rBxpB. Complexes were formed in reaction mixtures containing 20 µM rBclA and 5 µM rBxpB. Samples of purified rBclA and rBxpB and of rBclA-rBxpB cross-linked complexes were separately analyzed in triplicate by SDS-PAGE. The three essentially identical gels were used to detect proteins and protein complexes by immunoblotting with either an anti-BclA or an anti-BxpB MAb or by staining with Coomassie blue.

Mentions: To examine the possibility that BclA and BxpB form isopeptide bonds without the participation of other proteins, we synthesized amino-terminally His6-tagged versions of BclA and BxpB in Escherichia coli and purified each recombinant protein by affinity chromatography (9, 10). The His6 tag was removed from recombinant BxpB (rBxpB) (10). The two proteins were combined at µM concentrations in phosphate-buffered saline and incubated at room temperature for 30 min. After separation by SDS-PAGE, stable and high-molecular-mass complexes containing both recombinant BclA (rBclA) and rBxpB were detected by immunoblotting individually with anti-BclA and anti-BxpB MAbs and by staining with Coomassie blue (Fig. 4). These complexes were excised from a polyacrylamide gel and treated in situ with trypsin and chymotrypsin, and the proteolytic fragments were analyzed by LC-MS/MS as described above. A total of 32 branched peptides were identified in which a peptide derived from the amino-terminal region of rBclA (either GSSHHHHHHSSGL or GSSHHHHHHSSGLVPR; residues 2 to 14 or 2 to 17, respectively) was attached to one or two internal acidic residues of a proteolytic fragment of rBxpB (Table 3). Again, this attachment was accompanied by the loss of mass of one water molecule, consistent with isopeptide bond formation. In these branched peptides, isopeptide bonds were apparently formed between the amino group of rBclA residue G2 and the side chain carboxyl groups of any of the 13 acidic residues of rBxpB. Presumably, the initiating methionine residue of rBclA was removed by a methionine aminopeptidase in E. coli.


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)

Formation of high-molecular-mass complexes containing cross-linked rBclA and rBxpB. Complexes were formed in reaction mixtures containing 20 µM rBclA and 5 µM rBxpB. Samples of purified rBclA and rBxpB and of rBclA-rBxpB cross-linked complexes were separately analyzed in triplicate by SDS-PAGE. The three essentially identical gels were used to detect proteins and protein complexes by immunoblotting with either an anti-BclA or an anti-BxpB MAb or by staining with Coomassie blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Formation of high-molecular-mass complexes containing cross-linked rBclA and rBxpB. Complexes were formed in reaction mixtures containing 20 µM rBclA and 5 µM rBxpB. Samples of purified rBclA and rBxpB and of rBclA-rBxpB cross-linked complexes were separately analyzed in triplicate by SDS-PAGE. The three essentially identical gels were used to detect proteins and protein complexes by immunoblotting with either an anti-BclA or an anti-BxpB MAb or by staining with Coomassie blue.
Mentions: To examine the possibility that BclA and BxpB form isopeptide bonds without the participation of other proteins, we synthesized amino-terminally His6-tagged versions of BclA and BxpB in Escherichia coli and purified each recombinant protein by affinity chromatography (9, 10). The His6 tag was removed from recombinant BxpB (rBxpB) (10). The two proteins were combined at µM concentrations in phosphate-buffered saline and incubated at room temperature for 30 min. After separation by SDS-PAGE, stable and high-molecular-mass complexes containing both recombinant BclA (rBclA) and rBxpB were detected by immunoblotting individually with anti-BclA and anti-BxpB MAbs and by staining with Coomassie blue (Fig. 4). These complexes were excised from a polyacrylamide gel and treated in situ with trypsin and chymotrypsin, and the proteolytic fragments were analyzed by LC-MS/MS as described above. A total of 32 branched peptides were identified in which a peptide derived from the amino-terminal region of rBclA (either GSSHHHHHHSSGL or GSSHHHHHHSSGLVPR; residues 2 to 14 or 2 to 17, respectively) was attached to one or two internal acidic residues of a proteolytic fragment of rBxpB (Table 3). Again, this attachment was accompanied by the loss of mass of one water molecule, consistent with isopeptide bond formation. In these branched peptides, isopeptide bonds were apparently formed between the amino group of rBclA residue G2 and the side chain carboxyl groups of any of the 13 acidic residues of rBxpB. Presumably, the initiating methionine residue of rBclA was removed by a methionine aminopeptidase in E. coli.

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