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Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287.

Avall-Jääskeläinen S, Hynönen U, Ilk N, Pum D, Sleytr UB, Palva A - BMC Microbiol. (2008)

Bottom Line: The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far.Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, University of Helsinki, Finland. silja.avall@helsinki.fi

ABSTRACT

Background: Lactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group.

Results: In this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179-435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.

Conclusion: This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

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SDS-PAGE analysis of SlpA fragments obtained after trypsin digestion. Lane 1, undigested SlpA. Lane 2, SlpA after digestion with trypsin. Numbers on the left indicate molecular weights in kilodaltons.
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Figure 3: SDS-PAGE analysis of SlpA fragments obtained after trypsin digestion. Lane 1, undigested SlpA. Lane 2, SlpA after digestion with trypsin. Numbers on the left indicate molecular weights in kilodaltons.

Mentions: To gain insight to the domain structure of SlpA, wild type SlpA was digested with trypsin. This revealed two protease resistant fragments with apparent molecular masses of 25 kDa and 23 kDa (Fig. 3). The N-terminal sequences of these peptides were determined to be GFSTTAG (larger peptide) and SVTATND (smaller peptide). These correspond to amino acids starting from 190 and 209 of mature SlpA, respectively. Peptide mass mapping of the protease resistant fragments obtained after trypsin digestion revealed that the peptide encompassing the last 12 amino acids of full length SlpA is lacking from these fragments (data not shown). The protease resistance of the regions 190 to 423 and 209 to 423 in mature SlpA strongly suggested the existence of a compact domain structure most likely representing a region exposed on the outer surface of the S-layer.


Identification and characterization of domains responsible for self-assembly and cell wall binding of the surface layer protein of Lactobacillus brevis ATCC 8287.

Avall-Jääskeläinen S, Hynönen U, Ilk N, Pum D, Sleytr UB, Palva A - BMC Microbiol. (2008)

SDS-PAGE analysis of SlpA fragments obtained after trypsin digestion. Lane 1, undigested SlpA. Lane 2, SlpA after digestion with trypsin. Numbers on the left indicate molecular weights in kilodaltons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2571106&req=5

Figure 3: SDS-PAGE analysis of SlpA fragments obtained after trypsin digestion. Lane 1, undigested SlpA. Lane 2, SlpA after digestion with trypsin. Numbers on the left indicate molecular weights in kilodaltons.
Mentions: To gain insight to the domain structure of SlpA, wild type SlpA was digested with trypsin. This revealed two protease resistant fragments with apparent molecular masses of 25 kDa and 23 kDa (Fig. 3). The N-terminal sequences of these peptides were determined to be GFSTTAG (larger peptide) and SVTATND (smaller peptide). These correspond to amino acids starting from 190 and 209 of mature SlpA, respectively. Peptide mass mapping of the protease resistant fragments obtained after trypsin digestion revealed that the peptide encompassing the last 12 amino acids of full length SlpA is lacking from these fragments (data not shown). The protease resistance of the regions 190 to 423 and 209 to 423 in mature SlpA strongly suggested the existence of a compact domain structure most likely representing a region exposed on the outer surface of the S-layer.

Bottom Line: The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far.Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, University of Helsinki, Finland. silja.avall@helsinki.fi

ABSTRACT

Background: Lactobacillus brevis ATCC 8287 is covered by a regular surface (S-) layer consisting of a 435 amino acid protein SlpA. This protein is completely unrelated in sequence to the previously characterized S-layer proteins of Lactobacillus acidophilus group.

Results: In this work, the self-assembly and cell wall binding domains of SlpA were characterized. The C-terminal self-assembly domain encompassed residues 179-435 of mature SlpA, as demonstrated by the ability of N-terminally truncated recombinant SlpA to form a periodic structure indistinguishable from that formed by full length SlpA. Furthermore, a trypsin degradation analysis indicated the existence of a protease resistant C-terminal domain of 214 amino acids. By producing a set of C-terminally truncated recombinant SlpA (rSlpA) proteins the cell wall binding region was mapped to the N-terminal part of SlpA, where the first 145 amino acids of mature SlpA alone were sufficient for binding to isolated cell wall fragments of L. brevis ATCC 8287. The binding of full length rSlpA to the cell walls was not affected by the treatment of the walls with 5% trichloroacetic acid (TCA), indicating that cell wall structures other than teichoic acids are involved, a feature not shared by the Lactobacillus acidophilus group S-layer proteins characterized so far. Conserved carbohydrate binding motifs were identified in the positively charged N-terminal regions of six Lactobacillus brevis S-layer proteins.

Conclusion: This study identifies SlpA as a two-domain protein in which the order of the functional domains is reversed compared to other characterized Lactobacillus S-layer proteins, and emphasizes the diversity of potential cell wall receptors despite similar carbohydrate binding sequence motifs in Lactobacillus S-layer proteins.

Show MeSH