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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

Positive-ion MS/MS spectrum used to determine the sequence of a branched peptide containing BxpB residues 60 to 69 with AF peptides derived from the NTD of BclA attached to residues D60 and D66. The spectrum was produced by electrospray ionization collision-activated dissociation of (M+ 2 H)2+ ions (m/z = 728.2). Fragmentation endpoints of y ions and b ions are indicated on the peptide sequence. Ion labels and their meanings are as follows: *, loss of ammonia; °, loss of water; F, loss of phenylalanine due to cleavage of the AF peptide bond; AF, loss of AF peptide due to cleavage of the isopeptide bond; multiple * and/or ° symbols, multiple losses of ammonia and/or water.
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f1: Positive-ion MS/MS spectrum used to determine the sequence of a branched peptide containing BxpB residues 60 to 69 with AF peptides derived from the NTD of BclA attached to residues D60 and D66. The spectrum was produced by electrospray ionization collision-activated dissociation of (M+ 2 H)2+ ions (m/z = 728.2). Fragmentation endpoints of y ions and b ions are indicated on the peptide sequence. Ion labels and their meanings are as follows: *, loss of ammonia; °, loss of water; F, loss of phenylalanine due to cleavage of the AF peptide bond; AF, loss of AF peptide due to cleavage of the isopeptide bond; multiple * and/or ° symbols, multiple losses of ammonia and/or water.

Mentions: Many proteolytic fragments containing only BclA, BxpB, ExsY, or CotY sequences were identified. In addition, eight BxpB fragments with one or two attached AF peptides were identified (Table 1). The MS/MS spectrum of one of these fragments is shown in Fig. 1. In each of the eight compound fragments, the AF peptide was attached to an internal acidic (D or E) residue of BxpB, which was accompanied by the loss of mass of one water molecule. This result indicated the formation of an isopeptide bond between the amino group of BclA residue A20 and a side chain carboxyl group of BxpB. The attachment of an AF peptide occurred at 8 of the 13 acidic residues of BxpB, which contains 167 amino acids (9). Comparing independently derived fragments containing the same BxpB residues showed that a particular acidic residue might be involved in an isopeptide bond in one fragment but not in another (Table 1), indicating a somewhat random pattern of AF peptide attachment. On the other hand, none of the acidic residues near the amino terminus of BxpB (i.e., D5, E7, D12, and E14) participated in the formation of an isopeptide bond with BclA.


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)

Positive-ion MS/MS spectrum used to determine the sequence of a branched peptide containing BxpB residues 60 to 69 with AF peptides derived from the NTD of BclA attached to residues D60 and D66. The spectrum was produced by electrospray ionization collision-activated dissociation of (M+ 2 H)2+ ions (m/z = 728.2). Fragmentation endpoints of y ions and b ions are indicated on the peptide sequence. Ion labels and their meanings are as follows: *, loss of ammonia; °, loss of water; F, loss of phenylalanine due to cleavage of the AF peptide bond; AF, loss of AF peptide due to cleavage of the isopeptide bond; multiple * and/or ° symbols, multiple losses of ammonia and/or water.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Positive-ion MS/MS spectrum used to determine the sequence of a branched peptide containing BxpB residues 60 to 69 with AF peptides derived from the NTD of BclA attached to residues D60 and D66. The spectrum was produced by electrospray ionization collision-activated dissociation of (M+ 2 H)2+ ions (m/z = 728.2). Fragmentation endpoints of y ions and b ions are indicated on the peptide sequence. Ion labels and their meanings are as follows: *, loss of ammonia; °, loss of water; F, loss of phenylalanine due to cleavage of the AF peptide bond; AF, loss of AF peptide due to cleavage of the isopeptide bond; multiple * and/or ° symbols, multiple losses of ammonia and/or water.
Mentions: Many proteolytic fragments containing only BclA, BxpB, ExsY, or CotY sequences were identified. In addition, eight BxpB fragments with one or two attached AF peptides were identified (Table 1). The MS/MS spectrum of one of these fragments is shown in Fig. 1. In each of the eight compound fragments, the AF peptide was attached to an internal acidic (D or E) residue of BxpB, which was accompanied by the loss of mass of one water molecule. This result indicated the formation of an isopeptide bond between the amino group of BclA residue A20 and a side chain carboxyl group of BxpB. The attachment of an AF peptide occurred at 8 of the 13 acidic residues of BxpB, which contains 167 amino acids (9). Comparing independently derived fragments containing the same BxpB residues showed that a particular acidic residue might be involved in an isopeptide bond in one fragment but not in another (Table 1), indicating a somewhat random pattern of AF peptide attachment. On the other hand, none of the acidic residues near the amino terminus of BxpB (i.e., D5, E7, D12, and E14) participated in the formation of an isopeptide bond with BclA.

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