In this study, we have identified the cell wall binding and self-assembly domains in the S-layer protein SlpA of L. brevis ATCC 8287, a strain phylogenetically distant from L. acidophilus group organisms, the S-layer proteins of which have previously been functionally characterized. Two new putative S-layer proteins, Q03P39 and Q03NT3, were identified in the recently sequenced genome of L. brevis ATCC 367  and compared with SlpA and other L. brevis S-layer proteins characterized thus far. Q03P39 is almost identical (99% identity) with SlpD of L. brevis ATCC 14869 and relatively dissimilar (<40% identity) from the SlpA, SlpB and SlpC sequences. Q03NT3 is highly similar with SlpC of L. brevis ATCC 14869 (87% identity) whereas not that similar with the other characterized L. brevis S-layer proteins (<44% identity). Similarity of the mature forms of the new putative S-layer proteins or the mature forms of SlpA, SlpB, SlpC or SlpD proteins with L. acidophilus group S-layer proteins is negligible.
Analysis of the six L. brevis S-layer protein sequences deposited in databanks indicated the subdivision of each sequence into two regions: a conserved N-terminal region characterized by a high predicted pI and potential carbohydrate binding motifs, and a more variable C-terminal region with an acidic predicted pI, with the N-terminal region corresponding for 40–75% of the sequence lengths. The observed high predicted overall pI values of Lactobacillus S-layer proteins  thus seem to be due to the concentration of basic amino acids to a defined region, as is also the case in the S-layer proteins of L. acidophilus group, which have cationic, cell wall binding C-terminal regions.
The presence of a conserved N-terminal region with a high predicted pI in L. brevis S-layer proteins strongly suggested an N-terminal cell wall binding domain. This was confirmed for SlpA of L. brevis ATCC 8287 by interaction studies performed with truncated rSlpA proteins and LiCl-treated L. brevis cells or isolated L. brevis cell wall fragments. In these studies, truncated proteins encompassing the whole positively charged region of SlpA bound to the cell wall; however, the first 145 residues in mature SlpA contained sufficient information for cell wall binding. An assay suitable for measuring the binding strength would be needed to detect the putative difference between the cell wall binding affinities of SlpA1–145 and SlpA1–189. In S-layer proteins of lactobacilli, no SLH motifs have been detected. Instead, interactions between a positively charged S-layer protein region and negatively charged secondary cell wall polymers have been shown to mediate the cell wall binding in the case of SA of L. acidophilus ATCC 4356  and CbsA of L. crispatus JCM 5810 . SA and CbsA were shown to bind teichoic acids, and CbsA bound also to lipoteichoic acids purified from Staphylococcus aureus and Streptococcus faecalis, but not to the teichuronic acid/polysaccharide fraction of the cell wall of L. crispatus JCM 5810. In contrast, the results of this study suggest the involvement of another cell wall structure than teichoic acid or lipoteichoic acid in the interaction between SlpA and the cell wall, as the purification process of the CWF used efficiently removed LTAs, and the extraction of CWF with TCA at +4°C to remove teichoic acids had no effect on the binding of SlpA.
The chemical nature of the cell wall component interacting with the S-layer protein has been determined in Geobacillus stearothermophilus strains [47–49], in Lysinibacillus sphaericus  and in Aneurinibacillus thermoaerophilus . In G. stearothermophilus and L. sphaericus S-layers, which possess SLH-domains, the component is a pyruvylated GlcNac and GalNac-containing polysaccharide not groupable as a teichoic or lipoteichoic acid. In other G. stearothermophilus strains the component is a negatively charged mannuronic acid-containing cell wall polymer, and in A. thermoaerophilus the cell wall receptor is a neutral biantennary oligosaccharide.
The secondary cell wall polymers of lactobacilli are poorly characterized. The detailed structure of a wall polysaccharide of L. casei has been determined , but no precise structures for polysaccharides of L. brevis strains are available at present. In early studies, the cell walls of L. buchneri  and L. brevis ATCC 8287  were shown to contain neutral polysaccharides, which were suggested to be involved in the anchoring of the S-layer protein to the cell wall through hydrogen bonding [54, 55]. These results are in agreement with the data presented in this study, which suggest a non-teichoic acid polysaccharide, either neutral or anionic, involved in the cell wall binding of SlpA, but the detailed structure of this polysaccharide remains to be elucidated.
Interestingly, despite the fact that polysaccharides rather than (lipo)teichoic acids of L. brevis ATCC 8287 are involved in the cell wall binding of SlpA, the C-terminal cell wall binding region of the S-layer protein CbsA of L. crispatus JCM 5810 bound to GHCl-treated L. brevis ATCC 8287 cells . Using the same experimental design we showed that rSlpA and its cell wall binding fragment rSlpA1–145 bind to LiCl-treated L. acidophilus ATCC 4356 cells. The interaction between the S-layer protein and the secondary cell wall component is supposed to be lectin-like and highly specific , and in artificial experimental procedures the lack of a specific interaction between two complementary surfaces may be masked by unspecific charge interactions with lower affinity. To obtain information about specific interactions, competition experiments with fragments of SA, CbsA and SlpA and the corresponding bacterial strains, or direct determinations of the KD values of the interactions, e. g. by surface plasmon resonance studies, are needed.
Amino acid sequence analysis of the L. brevis S-layer proteins revealed motifs with similarity to repeated C-terminal carbohydrate binding sequences detected in clostridial toxins, streptococcal glucosyltransferases and the S-layer proteins of L. acidophilus group organisms [14, 44, 45]. These motifs are supposed to play a general role in protein-carbohydrate interactions by acting as initial attachment sites and thus enabling the specific interactions to occur  and may thus be partly responsible for the observed positive cross-binding results between SlpA and L. acidophilus ATCC 4356 cells, and between the cell wall binding domain of CbsA and L. brevis ATCC 8287 cells (see above). The sequences of the potential L. brevis carbohydrate-binding motifs deviated to some extent from the consensus sequences determined for clostridial toxins and streptococcal transferases [44, 45]. The divergence of the sequences in distantly related organisms and different macromolecular structures is apparently allowed as long as the basic function of the motif, bringing the interacting partners to initial contact, is preserved.
The self-assembly domain of SlpA was shown to comprise residues 179–435 in mature SlpA, as truncated proteins encompassing this region were able to form a periodic structure indistinguishable from that formed by full length SlpA, as detected by electron microscopy. The length of the truncated protein was critical, since rSlpA167–435 and rSlpA149–435 as well as N-terminal truncations shorter than rSlpA179–435 were unable to form regular lattices. Apparently, the region, or part of the region, encompassing amino acids 149–178 disturbs the lattice formation of the truncated proteins either by steric hindrance and/or by preventing the acquisition of a native conformation. Trypsin degradation experiments revealed two protease resistant peptides encompassing residues 190–423 and 209–423 in mature SlpA supporting the hypothesis about a morphologically separate, compact C-terminal unit, which most probably corresponds to the round, periodically arranged structures seen in electron microscope pictures of self-assembly products of SlpA (Fig. 4). Similar trypsin degradation experiments with whole L. brevis cells resulted in identical fragments but at a very low efficiency (data not shown), indicating poor accessibility of the enzyme to the N-terminal domain through the pores in the S-layer, and further supporting the presumption about a protease sensitive, more flexible N-terminal domain shielded from the environment beneath the C-terminal self-assembly domain. A protease-resistant, surface-located self-assembly domain has also been observed in the N-terminal part of the S-layer protein SA of L. acidophilus ATCC 4356 . The results of the present study are supported by the report of Hynönen et al , in which an antiserum specific for the recombinant peptide SlpA66–215, originating from the cell wall binding region, was not able to recognize polymerized SlpA on Lactobacillus brevis ATCC 8287 cells. In the same report, whole S-layered L. brevis ATCC 8287 cells as well as the N-terminal part of SlpA, residues 66–146 of mature SlpA in minimum, were shown to bind to immobilized fibronectin. Fibronectin is highly glycosylated, and the binding of fibronectin to a region of SlpA shielded beneath the C-terminal domain may be explained by an interaction between the protruding oligosaccharide moieties of fibronectin and the identified N-terminal carbohydrate binding sequences of SlpA. In this respect the identification of human blood group A-trisaccharide as the receptor for the S-layer protein of a human L. brevis isolate  is of interest, especially considering that the nine N-terminal amino acids of the S-layer protein of this strain were identical with the N-terminus of SlpA.