Increased expression of extracellular proteins as a hallmark of human endothelial cell in vitro senescence.

A convenient way to study processes of aging in distinct human tissues consists of a molecular analysis of cells from the tissue in question, that were explanted and grown in vitro until they reach senescence. Using human umbilical vein endothelial cells (HUVEC), we have established an in vitro senescence model for human endothelial cells. A major hallmark of HUVEC in vitro senescence is the increased frequency of apoptotic cell death, which occurs as a determining feature of HUVEC senescence. Senescent endothelial cells are also found in vivo in atherosclerotic lesions, suggesting that the presence of such cells may contribute to the development of vascular pathology. To elucidate mechanisms underlying endothelial cell senescence and age-associated apoptosis, gene expression analyses were carried out. In these experiments, we observed the up-regulation of genes coding for extracellular proteins in senescent HUVEC. In particular, a significant upregulation of interleukin-8, VEGI, and the IGF-binding proteins 3 and 5 was observed. Upregulation of these genes was confirmed by both RT-PCR and Western blot. In the case of interleukin-8, a roughly 50-fold upregulation of the protein was also found in cellular supernatants. The extracellular proteins encoded by these genes are well known for their ability to modulate the apoptotic response of human cells, and in the case of interleukin-8, clear links to the establishment of atherosclerotic lesions have been defined. The results described here support a new model, where changes in the secretome of human endothelial cells contribute to vascular aging and vascular pathology.

A novel dipstick developed for rapid Bet v 1-specific IgE detection: recombinant allergen immobilized via a monoclonal antibody to crystalline bacterial cell-surface layers.

The incidence of allergy to airborne proteins derived from tree and grass pollen, feces of mites, spores of molds, and pet dander has been increasing over the last decades. Since precise diagnosis is a prerequisite for successful immunotherapy, there is a rising demand for rapid, reliable, and inexpensive screening methods such as dipstick assays. With the purified recombinant major birch-pollen allergen rBet v 1a as model protein, crystalline bacterial cell-surface layers (S-layers) were tested for their applicability as an immobilization matrix for dipstick development. For this purpose, S-layers were deposited on a mechanically stable microporous support, cross-linked with glutaraldehyde, and free carboxylic acid groups of the S-layer protein were activated with carbodiimide. In the present test system, rBet v 1a was immobilized via the monoclonal mouse antibody BIP 1, which, unlike the allergen, is too large to enter the pores of the S-layer lattice, and which therefore formed a closed monolayer on the outermost surface of the crystal lattice. Moreover, BIP 1 is known to modulate IgE binding to the allergen. After incubation of the dipsticks in serum, washing of the reaction zone under tap water, and binding of an anti-IgE alkaline phosphatase conjugate, 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium was used as substrate, forming an IgE concentration-dependent colored precipitate on the S-layer surface. The investigation of patient sera previously tested with the CAP system confirmed the specificity of the S-layer-based dipstick assay. Since the dipstick is easy to handle and the whole test procedure takes only 90 min, this test system should be applicable for rapid determination of specific IgE and for first screening in the doctor's practice.

Applications of S-layers.

The wealth of information existing on the general principle of S-layers has revealed a broad application potential. The most relevant features exploited in applied S-layer research are: (i) pores passing through S-layers show identical size and morphology and are in the range of ultrafiltration membranes; (ii) functional groups on the surface and in the pores are aligned in well-defined positions and orientations and accessible for binding functional molecules in very precise fashion; (iii) isolated S-layer subunits from many organisms are capable of recrystallizing as closed monolayers onto solid supports at the air-water interface, on lipid monolayers or onto the surface of liposomes. Particularly their repetitive physicochemical properties down to the subnanometer scale make S-layers unique structures for functionalization of surfaces and interfaces down to the ultimate resolution limit. The following review focuses on selected applications in biotechnology, diagnostics, vaccine development, biomimetic membranes, supramolecular engineering and nanotechnology. Despite progress in the characterization of S-layers and the exploitation of S-layers for the applications described in this chapter, it is clear that the field lags behind others (e.g. enzyme engineering) in applying recent advances in protein engineering. Genetic modification and targeted chemical modification would allow several possibilities including the manipulation of pore permeation properties, the introduction of switches to open and close the pores, and the covalent attachment to surfaces or other macromolecules through defined sites on the S-layer protein. The application of protein engineering to S-layers will require the development of straightforward expression systems, the development of simple assays for assembly and function that are suitable for the rapid screening of numerous mutants and the acquisition of structural information at atomic resolution. Attention should be given to these areas in the coming years.

2-D protein crystals as an immobilization matrix for producing reaction zones in dipstick-style immunoassays.

In the present study, the applicability of crystalline bacterial cell-surface layers (S-layers) as novel immobilization matrices and reaction zones for dipstick-style immunoassays was investigated. For this purpose, S-layer-carrying cell-wall fragments from Bacillus sphaericus CCM 2120 were deposited on a microporous support, and the S-layer protein was cross-linked with glutaraldehyde. For developing appropriate test systems, either human IgG was directly linked to the carboxylic acid groups from the S-layer protein or it was immobilized using Protein A or, after biotinylation, using streptavidin. A clear correlation was obtained between the amount of anti-human IgG applied and the absorbance values in the immunoassays. S-layers with covalently bound recombinant major birch pollen allergen were used for quantitative and semiquantitative determination of an antibody raised against it. Using S-layers as an immobilization matrix in comparison to amorphous polymers has advantages in that the closed monolayers of functional macromolecules on their outermost surface allows for strong signals in immunoassays, almost completely eliminates background and prevents diffusion.

Evidence for an S-layer protein pool in the peptidoglycan of Bacillus stearothermophilus.

Intact cells of Bacillus stearothermophilus PV72 revealed, after conventional thin-sectioning procedures, the typical cell wall profile of S-layer-carrying gram-positive eubacteria consisting of a ca. 10-nm-thick peptidoglycan-containing layer and a ca. 10-nm-thick S layer. Cell wall preparations obtained by breaking the cells and removing the cytoplasmic membrane by treatment with Triton X-100 revealed a triple-layer structure, with an additional S layer on the inner surface of the peptidoglycan. This profile is characteristic for cell wall preparations of many S-layer-carrying gram-positive eubacteria. Among several variants of strain PV72 obtained upon single colony isolation, we investigated the variant PV72 86-I, which does not exhibit an inner S layer on isolated cell walls but instead possesses a profile identical to that observed for intact cells. In the course of a controlled mild autolysis of isolated cell walls, S-layer subunits were released from the peptidoglycan of the variant and assembled into an additional S layer on the inner surface of the walls, leading to a three-layer cell wall profile as observed for cell wall preparations of the parent strain. In comparison to conventionally processed bacteria, freeze-substituted cells of strain PV72 and the variant strain revealed in thin sections a ca. 18-nm-wide electron-dense peptidoglycan-containing layer closely associated with the S layer. The demonstration of a pool of S-layer subunits in such a thin peptidoglycan layer in an amount at least sufficient for generating one coherent lattice on the cell surface indicated that the subunits must have occupied much of the free space in the wall fabric of both the parent strain and the variant. It can even be speculated that the rate of synthesis and translation of the S-layer protein is influenced by the packing density of the S-layer subunits in the periplasm of the cell wall delineated by the outer S layer and the cytoplasmic membrane. Our data indicate that the matrix of the rigid wall layer inhibits the assembly of the S-layer subunits which are in transit to the outside.