微生物细胞表面工程研究进展*

微生物细胞表面工程是近年来发展起来的,它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白。微生物细胞表面工程可用于细胞催化剂、细胞吸附剂、活疫苗、生物传感器的开发等。微生物细胞表面工程具有广阔的应用前景,但是国内对这一领域的研究尚刚起步。在介绍了细胞表面工程的基础上,对微生物细胞表面工程技术进展进行了综述,并对该技术的发展给予展望。     

微生物细胞表面呈现技术研究进展

微生物细胞表面工程是近年来发展起来的,它利用细胞表面展示技术使外源蛋白固定化于细胞表面,从而生产微生物细胞表面蛋白。微生物细胞表面工程可用于细胞催化剂、细胞吸附剂、活疫苗、生物传感器的开发等。微生物细胞表面工程具有广阔的应用前景,但是国内对这一领域的研究刚起步。在介绍细胞表面工程的基础上,对微生物细胞表面工程技术进展进行了综述,展望了对该技术的发展。     

Cell-surface discoidin in aggregating cells of Dictyostelium discoideum.

Both discoidin I and discoidin II have been detected on the surface of aggregating (10 h developmental stage) cells of Dictyostelium discoideum NC4 by radioiodination of the cell-surface followed by immunoprecipitation and sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis. Approx. 92% of cell-surface discoidin I and 72% of cell-surface discoidin II can be eluted with 0.5 M-galactose, showing that most of each endogenous lectin is not present as integral membrane protein but rather is bound to cell-surface discoidin receptors. Two-dimensional polyacrylamide-gel-electrophoretic analysis of discoidin I suggests that the native tetramer may be a hetero-multimer composed of both Ia and Ib subunits. Cell-surface discoidin I also contains both types of subunit, but it is not clear whether both subunits have corresponding cell-surface receptors.     

Glycan-modifying bacteria-derived soluble factors from Bacteroides thetaiotaomicron and Lactobacillus casei inhibit rotavirus infection in human intestinal cells

Rotaviruses attach to intestinal cells in a process that requires glycan recognition. Some bacteria from the gut microflora have been shown to modify cell-surface glycans. In this study, human intestinal cultured cells were incubated with bacteria-derived soluble factors and infected with rotavirus. Results show that only bacterial soluble factors that increase cell-surface galactose namely, those of Bacteroides thetaiotaomicron and Lactobacillus casei were able to efficiently block rotavirus infections. Increasing cell-surface galactose using galactosyltransferase resulted in a similar blockage of rotavirus infections. These results indicate that manipulation of cell-surface intestinal glycans by bacterial soluble factors can prevent rotavirus infection in a species-specific manner, and should now be considered a potential therapeutic approach against rotavirus infection.     

Chronic treatment with insulin selectively down-regulates cell-surface GLUT4 glucose transporters in 3T3-L1 adipocytes

A new method for photoaffinity labeling of glucose transporters has been used to compare the effects of glucose-starvation, acute-insulin, and chronic-insulin treatments on the cell-surface glucose transporters in 3T3-L1 adipocytes. Starvation alone increased the cell-surface levels of GLUT1 and GLUT4 by approximately 4- and approximately 2-fold, respectively. As shown by Calderhead, D, M., Kitagawa, K., Tanner, L.T., Holman, G.D., and Lienhard, G.E. (1990) J. Biol. Chem. 265, 13800-13808) acute-insulin treatment increased cell-surface GLUT1 and GLUT4 by approximately 5- and approximately 15-fold respectively. In contrast to this, chronic-insulin treatment gave a further 3-4-fold increase in both cell-surface and total cellular GLUT1, but availability of GLUT4 at the cell-surface was down-regulated to half the level found in the acute treatment but with no change in the total cellular level. This effect occurred in starved and non-starved cells and suggests that starvation, acute-insulin, and chronic-insulin treatments regulate glucose transporter availability through independent mechanisms. The down-regulation of GLUT4 reached a maximally reduced cell-surface level in 6 h while the rise in GLUT1 reached a maximum after 24-48 h. The rise in GLUT1 appeared to compensate for the decline in cell-surface GLUT4 as glucose transport activity was further increased during the long term treatment with insulin. The down-regulation of GLUT4 due to the chronic-insulin treatment is associated with a marked resistance of the cells to restimulate glucose transport and particularly to recruit further GLUT4 to the cell-surface following an additional insulin treatment. The defect appears to be in the signaling mechanism that is responsible for translocation.     

Differential effects of 20-hydroxyecdysone on cell interactions and surface proteins in Drosophila cell lines

20-Hydroxyecdysone induces different cellular and biochemical responses in the Drosophila cell lines L3 and S3. The hormonal response in S3 cells includes mitotic arrest and aggregation, whereas L3 cells undergo mitotic arrest without aggregation. The possible involvement of 20-OH-ecdysone-modulated cell-surface proteins in mediating aggregation prompted us to compare the effects of hormonal stimulation on cell-surface proteins in these two cell lines. Radiolabeling of the cell-surface proteins revealed seven polypeptides modulated by 20-OH-ecdysone in S3 cells and three polypeptides so modulated in L3 cells. Increased and decreased labeling, as well as changes in migration of specific polypeptides on two-dimensional gels, were caused by the hormone. Analysis of radiolabelled cell-surface proteins by SDS-polyacrylamide gel electrophoresis revealed nine bands which were affected by 20-OH-ecdysone in S3 cells, whereas only three bands were altered by 20-OH-ecdysone in L3 cells. These observations are compared to earlier reports on the 20-OH-ecdysone-dependent modulation of cell-surface proteins in imaginal discs and other cell lines of Drosophila. We suggest that at least some of the cell-surface proteins which are modulated by 20-OH-ecdysone specifically in S3 cells may be mediators of the increase in cell-cell adhesions which occurs during hormone exposure.     

An AFM-based pit-measuring method for indirect measurements of cell-surface membrane vesicles

Circulating membrane vesicles, which are shed from many cell types, have multiple functions and have been correlated with many diseases. Although circulating membrane vesicles have been extensively characterized, the status of cell-surface membrane vesicles prior to their release is less understood due to the lack of effective measurement methods. Recently, as a powerful, micro- or nano-scale imaging tool, atomic force microscopy (AFM) has been applied in measuring circulating membrane vesicles. However, it seems very difficult for AFM to directly image/identify and measure cell-bound membrane vesicles due to the similarity of surface morphology between membrane vesicles and cell surfaces. Therefore, until now no AFM studies on cell-surface membrane vesicles have been reported. In this study, we found that air drying can induce the transformation of most cell-surface membrane vesicles into pits that are more readily detectable by AFM. Based on this, we developed an AFM-based pit-measuring method and, for the first time, used AFM to indirectly measure cell-surface membrane vesicles on cultured endothelial cells. Using this approach, we observed and quantitatively measured at least two populations of cell-surface membrane vesicles, a nanoscale population (<500 nm in diameter peaking at ∼250 nm) and a microscale population (from 500 nm to ∼2 μm peaking at ∼0.8 μm), whereas confocal microscopy only detected the microscale population. The AFM-based pit-measuring method is potentially useful for studying cell-surface membrane vesicles and for investigating the mechanisms of membrane vesicle formation/release.     

Control of AKR fibroblast phenotype by fibronectin: Regulation of cell-surface fibronectin binding receptor by fibronectin

Results of previous studies show that the expression of fibronectin and its cell-surface fibronectin binding receptor is coregulated in 3-methylchloranthrene transformation of normal AKR-2B cells to form AKR-MCA cells and in N, N,-dimethylformamide (DMF) induction of differentiation of transformed AKR-MCA cells (1990, J. Cell. Physiol., 143:445). In this study, we tested the corgulation hypothesis by transfection experiments using an antisense fibronectin expression vector. We determined the effect of antisense fibronectin RNA expression on untransformed AKR-2B cells, and on the responses of transformed AKR-MCA cells to DMF treatment. Expression of antisense fibronectin RNA in AKR-2B cells down-modulated fibronectin production, reduced adhesion to extracellular fibronectin, and altered cellular morphology Saturation binding and Scatchard analyses using radiolabelled fibronectin revealed a concurrent down-modulation of cell-surface fibronectin binding sites, but the binding affinity of the receptor for the ligand was not affected. Immunoblotting and immunostaining revealed down-modulation of the expression of α5β1 integrins. Expression of antisense fibronectin RNA in AKR-MCA cells down-modulated the ability of DMF to restore normal fibronectin production, cell-surface fibronectin binding receptor, adhesion to extracellular fibronectin, and cellular morphology. These studies show that both fibronectin and its cell-surface fibronectin binding receptor were tightly regulated during transformation and induction of differentiation in these cells, that the ligand and its cell-surface fibronectin binding receptor worked together to bring about phenotypic changes, and that fibronectin production regulated the expression of its cell-surface fibronectin binding receptor. © 1994 Wiley-Liss, Inc.     

Cell-surface H+-ATP synthase as a potential molecular target for anti-obesity drugs

Here we show that the cell-surface expression of the alpha subunit of H(+)-ATP synthase is markedly increased during adipocyte differentiation. Treatment of differentiated adipocytes with small molecule inhibitors of H(+)-ATP synthase or antibodies against alpha and beta subunits of H(+)-ATP synthase leads to a decrease in cytosolic lipid droplet accumulation. Apolipoprotein A-I, which has been shown to bind to the ectopic beta-chain of H(+)-ATP synthase and inhibit the activity of cell-surface H(+)-ATP synthase, also was found to inhibit cytosolic lipid accumulation. These results suggest that the cell-surface H(+)-ATP synthase has a previously unsuspected role in lipid metabolism in adipocytes.     

HIV: a new role for Nef in the spread of HIV.

The HIV Nef protein downregulates the cell-surface expression of the HIV receptor glycoprotein CD4, but the significance of this event has remained obscure. Recent data suggest that Nef reduces cell-surface CD4 to promote the efficient spread of the virus.     

The Escherichia coli Subtilase Cytotoxin A Subunit Specifically Cleaves Cell-surface GRP78 Protein and Abolishes COOH-terminal-dependent Signaling

GRP78, a molecular chaperone with critical endoplasmic reticulum functions, is aberrantly expressed on the surface of cancer cells, including prostate and melanoma. Here it functions as a pro-proliferative and anti-apoptotic signaling receptor via NH₂-terminal domain ligation. Auto-antibodies to this domain may appear in cancer patient serum where they are a poor prognostic indicator. Conversely, GRP78 COOH-terminal domain ligation is pro-apoptotic and anti-proliferative. There is no method to disrupt cell-surface GRP78 without compromising the total GRP78 pool, making it difficult to study cell-surface GRP78 function. We studied six cell lines representing three cancer types. One cell line per group expresses high levels of cell-surface GRP78, and the other expresses low levels (human hepatoma: Hep3B and HepG2; human prostate cancer: PC3 and 1-LN; murine melanoma: B16F0 and B16F1). We investigated the effect of Escherichia coli subtilase cytoxin catalytic subunit (SubA) on GRP78. We report that SubA specifically cleaves cell-surface GRP78 on HepG2, 1-LN, and B16F1 cells without affecting intracellular GRP78. B16F0 cells (GRP78^low) have lower amounts of cleaved cell-surface GRP78. SubA has no effect on Hep3B and PC3 cells. The predicted 28-kDa GRP78 COOH-terminal fragment is released into the culture medium by SubA treatment, and COOH-terminal domain signal transduction is abrogated, whereas pro-proliferative signaling mediated through NH₂-terminal domain ligation is unaffected. These experiments clarify cell-surface GRP78 topology and demonstrate that the COOH-terminal domain is necessary for pro-apoptotic signal transduction occurring upon COOH-terminal antibody ligation. SubA is a powerful tool to specifically probe the functions of cell-surface GRP78.     

Evaluating cell-surface expression and measuring activation of mammalian odorant receptors in heterologous cells

A fundamental question in olfaction is which odorant receptors (ORs) are activated by a given odorant. A major roadblock to investigating odorant-OR relationships in mammals has been the inability to express ORs in heterologous cells suitable for screening active ligands for ORs. The discovery of the receptor-transporting protein family has facilitated the effective cell-surface expression of ORs in heterologous cells. The establishment of a robust heterologous expression system for mammalian ORs facilitates the high-throughput 'deorphanization' of these receptors by matching them to their cognate ligands. This protocol details the method used for evaluating the cell-surface expression and measuring the functional activation of ORs of transiently expressed mammalian ORs in HEK293T cells. The stages of OR cell-surface expression include cell culture preparation, transfer of cells, transfection, immunocytochemistry or flow cytometry, odorant stimulation and luciferase assay. This protocol can be completed in a period of 3 d from the transfer of cells to cell-surface expression detection and/or measurement of functional activation.     

Antibodies to cell-surface antigens of plant protoplasts

Both mouse and rabbit polyclonal antibodies to plant cell-surface antigens were developed by immunization with cell membrane material from oat (Avena sativa L. cv. Garry) roots. We were able to quickly assess the activity of antisera by monitoring the degree of protoplast agglutination and by using an indirect immunofluorescence assay. Using polyclonal antibodies to cell-surface antigens, we have found that oat root protoplasts share common surface antigens with protoplasts from other plant tissues and species. From experiments with antisera treated with excess oat leaf or oat root protoplasts before our immunoassays, we have obtained evidence for the existence of organ-specific cell-surface antigens in higher plants.     

Intracellular Ice Formation Is Affected by Cell Interactions

Cell-to-cell and cell-to-surface interactions are important to the structure and function of tissues. These interactions are also important determinants of low-temperature responses in tissues. Four in vitro models using hamster fibroblast cells in tissue culture were used to investigate the influence of cell-cell and cell-surface interactions on intracellular ice formation in these systems. The four models were: (a) single cells in suspension; (b) cells individually attached to glass with only cell-to-surface adhesion; (c) colonies of cells attached to glass with both cell-cell and cell-surface interactions; and (d) multicellular spheroids with extensive cell-cell contacts. Cryomicroscopy was used to monitor the prevalence and kinetics of intracellular ice formation after ice nucleation in the extracellular solution. The temperature for intracellular freezing in 50% of the cells was significantly affected by both cell-cell and cell-surface interactions. There was also evidence of intercellular nucleation through cell-cell interactions. The results indicate that cell-cell and cell-surface interactions play a significant role in the low-temperature response of tissue systems.     

Oxytocin-induced prostaglandin E2 (PGE2) synthesis is regulated by progesterone via oxytocinase in Ishikawa cells.

Cell-surface oxytocinase inactivates oxytocin and regulates oxytocin stimulation. We reported that oxytocinase in human endometrial epithelial cells was secreted from the cell membrane in the mid-secretory phase and disappeared from the cell surface. On the other hand, the production in human endometrium of prostaglandins, which play important roles in the reproductive process, has been reported to be upregulated by oxytocin. We investigated whether progesterone affects cell-surface oxytocinase and oxytocin-induced prostaglandin E2 (PGE2) production in vitro. Progesterone induced secretion of oxytocinase into the culture medium, which resulted in a decrease in cell-surface oxytocinase. Production of PGE2 was increased slightly by oxytocin without progesterone, and significantly with progesterone. The inhibition of oxytocinase activity by amastatin had a similar effect to the loss of cell-surface oxytocinase caused by progesterone. It is therefore likely that the cell-surface oxytocinase of endometrial epithelial cells modified by progesterone plays an important role in the function of the human endometrium through PGE2.     

The lipoprotein lipase of white adipose tissue. Changes in the adipocyte cell-surface content of enzyme in response to extracellular effectors in vitro.

An indirect labelled-second-antibody cellular immunoassay for adipocyte surface lipoprotein lipase was used to assess the changes that occurred during the incubation of cells in the presence and absence of effectors. In the absence of any specific effectors, the amount of immunodetectable lipoprotein lipase present at the surface of adipocytes remained constant throughout the 4 h incubation period at 37 degrees C. Under such conditions total cellular enzyme activity also remained constant, with no activity appearing in the medium. In the presence of heparin, cell-surface immunodetectable lipoprotein lipase increased by up to 20%, whereas in the presence of cycloheximide they decreased by up to 60%. Thus the obvious turnover of enzyme from this cell-surface site was found to be relatively rapid and dependent for its replenishment, at least in part, on protein synthesis. In the presence of insulin alone, a substantial increase in cell-surface lipoprotein lipase protein occurred, only part of which was dependent on protein synthesis. The total cellular activity of lipoprotein lipase was unaffected by the presence of insulin. The insulin-dependent increase in cell-surface enzyme was potentiated somewhat in the presence of dexamethasone, which was not shown to exert any independent effect. Glucagon, adrenaline and theophylline all produced a significant decline in the cell-surface immunodetectable lipoprotein lipase, which in the case examined (adrenaline) was partially additive with regard to the independent effect of cycloheximide. Cell-surface immunodetectable lipoprotein lipase amounts were decreased significantly when cells were incubated in the presence of either colchicine or tunicamycin. The concerted way in which cell-surface lipoprotein lipase altered during the incubations of adipocytes in the presence of effectors suggested that the translocation of enzyme to and from this cellular site was dependent on hormonal action and the integrity of intracellular protein-transport mechanisms.     

Gene disruption identifies a 290 kDa cell-surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii

The C-terminal coding region of the gene (denoted cshA) encoding a high-molecular-mass (290 kDa) cell-surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3' end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell-surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus-anchored cell-surface protein determinant of hydrophobicity and coaggregation in S. gordonii.     

Identification of a cell-surface antigen produced by a gene on human chromosome 3 (cen-q22) and not expressed by Rhnull cells.

A monoclonal antibody, 1D8, which recognizes a cell-surface antigen expressed by human chromosome 3 in Chinese hamster-human somatic-cell hybrids, has been produced. Testing of hybrids containing various deletions of chromosome 3 determines that the gene encoding the antigen is regionally localized to 3q (cen-22). This regional mapping is distinct from that elsewhere reported for two other cell-surface antigens assigned to chromosome 3--namely, the human transferrin receptor and the p97 melanoma-associated antigen. In addition, biochemical characterization is different from that elsewhere reported for other chromosome 3-encoded cell-surface antigens. When tested against a panel of rare-phenotype red blood cells, the only cells that failed to react were those of the Rhnull phenotype. The antibody reacts only weakly with homozygous -D- and fetal red cells, in contrast with a previously described antibody, R6A, which does not react with Rhnull cells. Furthermore, R6A does not recognize a cell-surface antigen expressed by chromosome 3 in Chinese hamster-human somatic-cell hybrids. Thus, the monoclonal antibody 1D8 recognizes a previously undescribed cell-surface antigen encoded by human chromosome 3 and not expressed on Rhnull cells. The gene on chromosome 3 regulating expression of this antigen may be that defective in Rhnull disease or may require the normal allele at an unlinked Rhnull locus for expression. Linkage studies will be required to further elucidate this matter.     

Internalization of the Kv1.4 potassium channel is suppressed by clustering interactions with PSD-95

The contribution of voltage-dependent ion channels to nerve function depends upon their cell-surface distributions. Nevertheless, the mechanisms underlying channel localization are poorly understood. Two phenomena appear particularly important: the clustering of channels by membrane-associated guanylate kinases (MAGUKs), such as PSD-95, and the regional stabilization of cell-surface proteins by differential suppression of endocytosis. Could these phenomena be related? To test this possibility we examined the effect of PSD-95 on the internalization rate of Kv1.4 K(+) channels in transfected HEK293 cells using cell-surface biotinylation assays. When expressed alone Kv1.4 was internalized with a half-life of 87 min, but, in the presence of PSD-95, Kv1.4 internalization was completely suppressed. Immunochemistry and electrophysiology showed PSD-95 had little effect on total or cell-surface levels of Kv1.4 or on current amplitude, activation, or inactivation kinetics. Clustering was necessary and sufficient to suppress Kv1.4 internalization since C35S-PSD-95, a mutant reported to bind but not cluster Kv1.4, (confirmed by imaging cells co-expressing a functional, GFP-variant-tagged Kv1.4) restored and, surprisingly, enhanced the rate of Kv1.4 internalization (t((1)/(2)) = 16 min). These data argue PSD-95-mediated clustering suppresses Kv1.4 internalization and suggest a fundamentally new role for PSD-95, and perhaps other MAGUKs, orchestrating the stabilization of channels at the cell-surface.     

Degradation of internalized αvβ5 integrin is controlled by uPAR bound uPA: effect on β1 integrin activity and α-SMA stress fiber assembly

Myofibroblasts (Mfs) that persist in a healing wound promote extracellular matrix (ECM) accumulation and excessive tissue contraction. Increased levels of integrin αvβ5 promote the Mf phenotype and other fibrotic markers. Previously we reported that maintaining uPA (urokinase plasminogen activator) bound to its cell-surface receptor, uPAR prevented TGFβ-induced Mf differentiation. We now demonstrate that uPA/uPAR controls integrin β5 protein levels and in turn, the Mf phenotype. When cell-surface uPA was increased, integrin β5 levels were reduced (61%). In contrast, when uPA/uPAR was silenced, integrin β5 total and cell-surface levels were increased (2-4 fold). Integrin β5 accumulation resulted from a significant decrease in β5 ubiquitination leading to a decrease in the degradation rate of internalized β5. uPA-silencing also induced α-SMA stress fiber organization in cells that were seeded on collagen, increased cell area (1.7 fold), and increased integrin β1 binding to the collagen matrix, with reduced activation of β1. Elevated cell-surface integrin β5 was necessary for these changes after uPA-silencing since blocking αvβ5 function reversed these effects. Our data support a novel mechanism by which downregulation of uPA/uPAR results in increased integrin αvβ5 cell-surface protein levels that regulate the activity of β1 integrins, promoting characteristics of the persistent Mf.