Importance of S-layer proteins in probiotic activity of Lactobacillus acidophilus M92.

AIMS: To investigate the functional role of surface layer proteins (S-layer) in probiotic strain Lactobacillus acidophilus M92, especially its influence on adhesiveness to mouse ileal epithelial cells. METHODS AND RESULTS: Sodium dodecyl sulphate polyacrylamide gel electrophoresis of cell surface proteins revealed the presence of potential surface layer (S-layer) proteins, ca at 45 kDa in L. acidophilus M92. Southern blot with pBK1 plasmid, containing slpA gene, gave a positive signal, suggesting that L. acidophilus M92 has a slpA gene coding for the S-layer proteins. S-layer proteins of this strain are present during all phases of growth. The S-layer proteins appeared when cells treated with 5 mol l(-1) LiCl were allowed to grow again. Removal of the S-layer proteins reduced adhesion of L. acidophilus M92 to mouse ileal epithelial cells. Furthermore, the viability of cells without S-layer were reduced in simulated gastric juice at low pH range (2, 2.5, 3) and simulated pancreatic juice with bile salts (1.5 and 3 g l(-1)). S-layer proteins of L. acidophilus M92 were resistant to pepsin and pancreatin, in contrast, the treatment with proteinase K led to a significant proteolysis of the S-layer proteins. CONCLUSIONS: These results demonstrated functional role of S-layer; it is responsible for adhesiveness of Lactobacillus acidophilus M92 to mouse ileal epithelial cells and has a protective role for this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: S-layer proteins have an important role in the establishment of probiotic strain Lactobacillus acidophilus M92 in the gastrointestinal tract.

Role of NaOH-extractable cell wall proteins Ccw5p, Ccw6p, Ccw7p and Ccw8p (members of the Pir protein family) in stability of the Saccharomyces cerevisiae cell wall.

The Saccharomyces cerevisiae cell wall contains more than 20 identified mannoproteins. Some of them can be released from the wall by hot SDS/mercaptoethanol treatment and are, therefore, considered as disulphide-linked or non-covalently attached to wall structural components. A number of covalently linked cell wall proteins are released after SDS extraction. They can be divided into these extractable by glucanases and those which can be released with 30 mM NaOH. The SDS-extractable proteins either possess enzymatic activities or are homologues of enzymes, mainly glucanases. Nothing is known, however, about the function of covalently linked proteins. In order to investigate the role of NaOH-extractable cell wall proteins, genes encoding all four identified members of this family of Pir proteins, CCW5, CCW6, CCW7 and CCW8, were disrupted and the phenotype of the mutants obtained was examined. They grew somewhat more slowly, were larger and irregularly shaped, and showed pronounced susceptibility to cell wall synthesis inhibitors like Calcofluor white and Congo red. In addition, the triple and the quadruple deletants had a decreased mating ability. All these properties were more obvious the more of these genes were disrupted, indicating that probably all members of this protein family are at least functionally equivalent in the cell wall.

Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae.

The incorporation of radioactive orthophosphate into the cell walls of Saccharomyces cerevisiae was studied. 33P-labeled cell walls were extensively extracted with hot sodium dodecyl sulfate (SDS). Of the remaining insoluble radioactivity more than 90% could be released by laminarinase. This radioactive material stayed in the stacking gel during SDS-polyacrylamide gel electrophoresis but entered the separating gel upon treatment with N-glycosidase F, indicating that phosphate was linked directly or indirectly to N-mannosylated glycoproteins. The phosphate was bound to covalently linked cell wall proteins as mannose-6-phosphate, the same type of linkage shown previously for soluble mannoproteins (L. Ballou, L. M. Hernandez, E. Alvarado, and C. E. Ballou, Proc. Natl. Acad. Sci. USA 87:3368-3372, 1990). From the phosphate-labeled glycoprotein fraction released by laminarinase, three cell wall mannoproteins, Ccw12p, Ccw13p and Ccw14p, were isolated and identified by N-terminal sequencing. For Ccw13p (encoded by DAN1 [also called TIR3]) and Ccw12p the association with the cell wall has not been described before; Ccw14p is identical with cell wall protein Icwp (I. Moukadiri, J. Armero, A. Abad, R. Sentandreu, and J. Zueco, J. Bacteriol. 179:2154-2162, 1997). In ccw12, ccw13, or ccw14 single or double mutants neither the amount of radioactive phosphate incorporated into cell wall proteins nor its position in the stacking gel was changed. However, the triple mutant brought about a shift of the 33P-labeled glycoprotein components from the stacking gel into the separating gel. The disruption of CCW12 results in a pronounced sensitivity of the cells to calcofluor white and Congo red. In addition, the ccw12 mutant shows a decrease in mating efficiency and a defect in agglutination.

New potential cell wall glucanases of Saccharomyces cerevisiae and their involvement in mating.

Biotinylation of intact Saccharomyces cerevisiae cells with a nonpermeant reagent (Sulfo-NHS-LC-Biotin) allowed the identification of seven cell wall proteins that were released from intact cells by dithiothreitol (DTT). By N-terminal sequencing, three of these proteins were identified as the known proteins beta-exoglucanase 1 (Exg1p), beta-endoglucanase (Bgl2p), and chitinase (Cts1p). One protein was related to the PIR protein family, whereas the remaining three (Scw3p, Scw4p, and Scw10p [for soluble cell wall proteins]) were found to be related to glucanases. Single knockouts of these three potential glucanases did not result in dramatic phenotypes. The double knockout of SCW4 and the homologous gene SCW10 resulted in slower growth, significantly increased release of proteins from intact cells by DTT, and highly decreased mating efficiency when these two genes were disrupted in both mating types. The synergistic behavior of the disruption of SCW4 and SCW10 was partly antagonized by the disruption of BGL2. The data are discussed in terms of a possible counterplay of transglucosidase and glucosidase activities.

Specific labelling of cell wall proteins by biotinylation. Identification of four covalently linked O-mannosylated proteins of Saccharomyces cerevisiae.

Intact Saccharomyces cerevisiae cells were biotinylated with the non-permeable sulfosuccinimidyl-6-(biotinamido) hexanoate reagent. Twenty specifically labelled cell wall proteins would be extracted and visualized on SDS gels via streptavidin/horseradish peroxidase. Nine cell wall proteins were released by SDS extraction under reducing conditions and were designated Scw1-9p for (soluble cell wall proteins); five proteins were released from SDS-extracted cell walls by laminarinase (Ccw1-5p for covalently linked cell wall proteins) and six with mild (30 mM-NaOH, 4 degrees C, 14 h) alkali treatment (Ccw6-11p). N-terminal sequences of the Ccw proteins 6, 7, 8 and 11 showed that these cell wall proteins are members of the PIR gene family (predicted proteins with internal repeats), CCW6 being identical to PIR1 and CCW8 to PIR3. Single gene disruptions of all four genes did not yield a phenotype. In the CCW11 disruption the Ccw11p as well as the laminarinase-extracted Ccw5 protein was missing. The new cell wall proteins are O-mannosylated, contain a Kex2 processing site, but no C-terminal GPI anchor sequence.

Detection of exo-beta-1,3-glucanase activity in polyacrylamide gels after electrophoresis under denaturing or nondenaturing conditions.

A method for the visualization of exo-beta-1,3-glucanase activity in polyacrylamide gels is presented. The procedure consists of the enzyme reaction in the gel with the substrate alpha-naphthylglucopyranoside, and a subsequent staining of the obtained alpha-naphthol with dyes Fast Red B, or Fast Blue BB, respectively. A mixture of exoglucanases produced by the fungus Polyporus squamosus was used for the optimization of the method. The procedure is applicable for the standard Laemmli discontinuous electrophoresis system, even in the presence of sodium dodecyl sulfate, as well as for electrophoresis in linear gradients of the polyacrylamide concentration. The staining method was used for the analysis of exoglucanases secreted by several yeast genera. All yeasts tested produced two types of exoglucanases, a high molecular mass species heterogeneous in size, and one or two smaller homogeneous enzymes.

Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase.

One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity. The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F. Klebl and W. Tanner, J. Bacteriol. 171:6259-6264, 1989). The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain. Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated. The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells.

Binding of Saccharomyces cerevisiae extracellular proteins to glucane.

Interactions of Saccharomyces cerevisiae cell wall proteins with purified yeast glucane were studied. Using the beta-glucanase (BGL2 gene product) as the model cell wall protein, strong binding to glucane was demonstrated at pH lower than 7, while at pH higher than 8 the reaction did not occur. NaCl (2 M) did not influence the binding, while urea in concentrations higher than 4 M affected the interactions. It was also found that most other cell wall proteins, as well as intracellular proteins, reacted with glucane in the same way, showing that the interactions of proteins with glucane are rather nonspecific. Soluble periplasmic proteins invertase and acid phosphatase failed to react with glucane under the same conditions, indicating that these proteins have certain structural features preventing their interactions with glucane.

Saccharomyces cerevisiae a- and alpha-agglutinin: characterization of their molecular interaction.

An O-glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR. An open reading frame codes for a protein of 69 amino acids. A minimum of five serine and five threonine residues of the mature protein are glycosylated. alpha-Agglutinin is a highly N-glycosylated protein of approximately 250 kDa. Both purified agglutinins form a specific 1:1 complex in vitro. Pretreatment of alpha-agglutinin, but not of alpha-agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha-agglutinin in the presence of alpha-agglutinin protects the former from DEPC inactivation. By carboxy terminal shortening of the alpha-agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha-agglutinin for interaction with alpha-agglutinin has been defined. Neither the N- nor the O-linked saccharides of the two agglutinins seem to be essential for their interaction.

Influence of glycosylation on the oligomeric structure of yeast acid phosphatase.

Secreted yeast acid phosphatase is found to be an octamer under physiological conditions rather than a dimer, as previously believed. The octameric form of the enzyme dissociates rapidly into dimers at pH below 3 and above 5, or by treatment with guanidine hydrochloride or urea, without further dissociation of dimers. Crosslinking experiments revealed that the dissociation of the octamer occurs through very unstable hexamers and tetramers, showing that the octamer is built of dimeric units. Dissociation to dimer was in all cases accompanied with a loss of most of the enzyme activity. The underglycosylated acid phosphatase, with less than eight carbohydrate chains per subunit, secreted from cells treated with moderate tunicamycin concentrations, contained besides octamers a high proportion of the dimers. With decreasing levels of enzyme glycosylation, the proportion of dimers increases and the amount of octamers correspondingly decreases. Furthermore, underglycosylated octamers were found to be significantly less stable than the fully glycosylated ones. This showed that carbohydrate chains play a significant role in the octamer formation in vivo, and in stabilization of the enzyme octameric form.

Role of glycosylation in secretion of yeast acid phosphatase.

The minimal glycosylation requirement for acid phosphatase secretion and activity was investigated using tunicamycin, an inhibitor of protein glycosylation, and a yeast mutant defective in the synthesis of oligosaccharide outer chains. The results obtained show that outer chain addition is not essential for secretion of active enzyme and that only 4 core chains, out of 8 normally attached to a protein subunit, are sufficient for enzyme transport to the periplasmic space. Enzyme forms with less than 4 chains were retained in membranes of endoplasmic reticulum. Secreted underglycosylated enzyme forms are partially or completely inactive.

Purification, carbohydrate composition and kinetic properties of the constitutive yeast acid phosphatase.

Constitutive acid phosphatase was purified from yeast cells grown in a medium supplied with 100 mM phosphate. Specific activity of the pure enzyme was 63.5 mumol/min X mg. The enzyme contains 42.5% of protein, 55% of mannose and 2.5% of N-acetylglucosamine. The carbohydrate chains are N-glycosidically linked to the protein. The pure enzyme shows non-linearity in the Lineweaver-Burk plot, thus indicating the presence of two enzyme forms with Km values of about 0.65 mM and 8.5 mM. The pH optimum of the enzyme is at pH 3.3. The enzyme is much more sensitive to heat denaturation than the repressible acid phosphatase.

Role of the carbohydrate part of yeast acid phosphatase.

Acid phosphatase, purified from the yeast Saccharomyces cerevisiae, was completely deglycosylated by endo-beta-N-acetylglucosaminidase H or by HF treatment. Three protein bands were obtained on sodium dodecyl sulfate (SDS)-electrophoresis, with molecular weights of 73,000, 71,000 and 61,500. The released carbohydrate chains varied in size from 12 to 142 mannose units. To study the role of carbohydrate chains in the structure and function of acid phosphatase, a comparison of the properties of the partially deglycosylated enzyme with the native one was performed. The 60% deglycosylated enzyme retained the original activity, and CD and fluorescence spectra showed that the native conformation of the enzyme was preserved. The 90% deglycosylated enzyme showed a pronounced loss of enzyme activity, accompanied by the disruption of the three-dimensional structure. The partially deglycosylated enzyme was less soluble and more susceptible to denaturing effects of heat, pH, urea, and guanidine hydrochloride. Under conditions of electrophoresis, the partially deglycosylated enzyme dissociated, indicating a possible role of carbohydrate chains in maintaining the dimeric structure of the enzyme. Susceptibility of acid phosphatase toward proteolysis was drastically increased by deglycosylation.

Structure and function of yeast acid phosphatase.

A pronounced heterogeneity of purified acid phosphatase has been observed. The size heterogeneity which was demonstrated by electrophoretic methods is in agreement with the finding that the enzyme preparation contains families of molecules differing in the carbohydrate content (38% - 70%). The charge heterogeneity was shown by isoelectric focusing indicating the existence of several slightly different protein chains in the enzyme preparation. It was found that the enzyme is a dimer with a mean molecular weight of 252000 Daltons. There are 16 N-glycosidically linked carbohydrate chains, differing in size from 15-150 mannose units, and a very small amount of O-glycosidically linked mannose. Properties of acid phosphatase deglycosilated by treatment with beta-endo-N-acetylglucosaminidase, were compared with the native enzyme and it was found that the carbohydrate chains do not play a direct role in the catalytic activity of the enzyme, but stabilize the tridimensional structure of the molecule. A drastic increase of sensitivity of the deglycosilated enzyme against proteolysis was found.