Avidin-induced lysis of biotinylated erythrocytes by homologous complement via the alternative pathway depends on avidin's ability of multipoint binding with biotinylated membrane.

It was reported that avidin and streptavidin induce lysis of prebiotinylated red blood cells via the alternative pathway of both homologous and heterologous complement. Both of these proteins have four biotin-binding sites, providing a polyvalent interaction with biotinylated components of the erythrocyte membrane. We have compared the effects of mono- and multipoint avidin attachment on the sensitivity of biotinylated erythrocytes to lysis by the complement system. In the presence of anti-avidin antibody, avidin-bearing biotinylated erythrocytes were rapidly lysed by heterologous serum. This lysis was independent from the mode of avidin attachment, implying that complement activation by the classical pathway triggered by interaction between C1 and avidin-bound antibody on the erythrocyte surface is independent from the avidin's ability of polyvalent (multipoint) binding with biotinylated membrane components. In the absence of anti-avidin antibody, biotinylated erythrocytes bearing polyvalently attached avidin were lysed by homologous complement better than cells bearing avidin, which possesses reduced ability for multipoint binding with biotinylated erythrocyte. Two independent approaches to reduce avidin's ability of multipoint binding were used: decrease in surface density of biotin on the erythrocyte membrane and blockage of biotin-binding sites of avidin. Both methods result in reduced lysis of avidin-bearing erythrocytes as compared with erythrocytes bearing an equal amount of polyvalent-bound avidin. Thus the activation of homologous complement via the alternative pathway depends on avidin's ability to 'cross-link' to the biotinylated components of the erythrocyte membrane.     

Membrane proteins in senescent erythrocytes.

The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components.     

Biotinylated synthetic chemokines: their use for the development of nonradioactive whole-cell binding assays

A chemokine binding assay on whole cells was developed using biotinylated synthetic CCL22 as a model ligand. CCL22 analogues were produced by a chemical route, resulting in > 97% homogeneous and defined polypeptides. First, the 5 biotinylated CCL22 analogues synthesized were captured by agarose-immobilized streptavidin, indicating that the biotin molecules introduced in positions G1, K27, K49, K61, and K66 of CCL22 were accessible for binding. Then, it was established using a migration assay that the biotinylated chemokines were at least as biologically active as the unmodified CCL22 form. Subsequently, the biotinylated chemokines were evaluated in an FACS-based whole-cell binding assay. Surprisingly, only the CCL22 analogue with the biotin in position K66 constituted a suitable staining reagent for CCR4-positive cells. Finally, binding characteristics and reproducibility of the binding assay were outlined for the CCL22 analogue with the biotin in position K66. These results exemplified that biotinylated synthetic chemokines constitute promising ligands for the development of chemokine receptor-binding assays on whole cells, provided the position of the biotin moiety introduced along the sequence is adequately chosen.     

Biotinylation facilitates the uptake of large peptides by Escherichia coli and other gram-negative bacteria

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.     

Role of membrane sialic acid content in the adhesiveness of aged erythrocytes to human cultured endothelial cells

Following our previous observation that the oldest normal red blood cells were the most adherent to human cultured endothelial cells, we attempted to simulate this age-related adherence. Among all the membrane modifications experienced by erythrocytes during their life-span, loss of sialic acids has attracted considerable attention. Using two different preparations of neuraminidase, we performed a sialic acid depletion on the youngest erythrocytes to reach a sialic acid content similar to that observed in physiologically aged erythrocytes. These pretreated youngest cells displayed limited increase in the adhesiveness to endothelial cells, lower than that found with intact oldest cells. To obtain an adhesiveness of pretreated cells similar to that of naturally aged cells, it was necessary to exceed 80% of sialic acid depletion. At this extent of desialation, modifications of the electrophoretic pattern of glycophorins were observed as well as the appearance of peanut agglutinin reactivity which were never found in physiologically aged erythrocytes. Therefore, the sialic acid loss cannot be considered as being a single determinant factor of the naturally aged red cell adhesiveness.     

Biotinylation Facilitates the Uptake of Large Peptides by Escherichia coli and Other Gram-Negative Bacteria

Gram-negative bacteria such as Escherichia coli can normally only take up small peptides less than 650 Da, or five to six amino acids, in size. We have found that biotinylated peptides up to 31 amino acids in length can be taken up by E. coli and that uptake is dependent on the biotin transporter. Uptake could be competitively inhibited by free biotin or avidin and blocked by the protonophore carbonyl m-chlorophenylhydrazone and was abolished in E. coli mutants that lacked the biotin transporter. Biotinylated peptides could be used to supplement the growth of a biotin auxotroph, and the transported peptides were shown to be localized to the cytoplasm in cell fractionation experiments. The uptake of biotinylated peptides was also demonstrated for two other gram-negative bacteria, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa. This finding may make it possible to create new peptide antibiotics that can be used against gram-negative pathogens. Researchers have used various moieties to cause the illicit transport of compounds in bacteria, and this study demonstrates the illicit transport of the largest known compound to date.     

Cloning and characterization of Scavidin, a fusion protein for the targeted delivery of biotinylated molecules.

We have constructed a novel fusion protein "Scavidin" consisting of the macrophage scavenger receptor class A and avidin. The Scavidin fusion protein is transported to plasma membranes where the avidin portion of the fusion protein binds biotin with high affinity and forms the basis for the targeted delivery of biotinylated molecules. Subcellular fractionation analysis, immunostaining, and electron microscopy demonstrated endosomal localization of the fusion protein. According to pulse-labeling and cross-linking studies Scavidin is found as monomers (55 kDa), dimers, and multimers, of which the 220-kDa form was the most abundant. The biotin binding capacity and active endocytosis of the biotinylated ligands were demonstrated in rat malignant glioma. Local Scavidin gene transfer to target tissues could have general utility as a universal tool to deliver biotinylated molecules at systemic low concentrations for therapeutic and imaging purposes, whereby high local concentration is achieved.     

Biotinylation of low density lipoproteins via free amino groups without loss of receptor binding activity

Low density lipoproteins were biotinylated via free amino groups using carbodiimide-activated biotin or D-biotin-N-hydroxysuccinimide ester. The receptor binding activity of the biotinylated LDL was determined by their ability to displace 125I-labeled LDL from the rat hepatic LDL receptor in the liposome filtration assay. LDL biotinylated with either of the two reagents was able to compete effectively with 125I-labeled LDL providing less than twenty biotin moieties were incorporated per lipoprotein particle. When more than twenty biotins were linked there was a marked loss of activity. The following conditions were adopted as standard for the biotinylation of LDL via free amino groups: 0.3 mumol of D-biotin-N-hydroxysuccinimide ester was incubated with 2 mg of LDL for 1 hr at room temperature. These conditions reproducibly yielded 11.3 +/- 0.6 biotins per LDL particle. With the biotinylated LDL and a performed streptavidin-biotinylated peroxidase complex, the hepatic LDL receptor from rats treated with 17 alpha-ethinyl estradiol was visualized as a single band on electroblots. Finally, the biotinylated LDL was used in an enzyme-linked sorbent assay for the LDL receptor. When solubilized liver membrane proteins from rats treated with 17 alpha-ethinyl estradiol were fixed to the wells with 1.6% paraformaldehyde, a specific binding greater than 0.4 absorbance units was observed which was about ninefold higher than with solubilized proteins from normal rats. We therefore suggest that D-biotin-N-hydroxysuccinimide ester can be used to biotinylate LDL reliably without destroying the lipoprotein's ability to bind specifically to its high affinity receptor.     

Erythrocyte aging: a comparison of model systems for simulating cellular aging in vitro

Senescent cell antigen (SCANT) is a "neo antigen" that appears on the surface of normal old cells and initiates IgG binding and cellular removal. To investigate the mechanism by which SCANT is generated from its parent molecule, band 3, we subjected intact human erythrocytes to treatments that have been reported to result in changes in band 3 and/or to mimick aging in vitro. The validity of these treatments as model systems for erythrocyte aging was evaluated using a "red cell aging panel" that provides a biochemical profile of a senescent red cell. Treatments were assessed for their ability to induce in vitro the following changes observed in normal erythrocytes aged in vivo: 1 increased breakdown of band 3 as detected by immunoblotting, 2 decrease in anion transport efficiency as detected with a sulfate self-exchange assay, 3 decrease in total glyceraldehyde 3-phosphate dehydrogenase activity with an increase in membrane-bound activity, and 4 increase in the binding of autologous IgG as detected with a protein A binding assay. Neither incubation with the free radical-generating xanthine oxidase/xanthine system, nor treatment with malondialdehyde, and end product of free radical-initiated lipid (per)oxidation, results in age-specific changes. Loading of the cells with calcium and oxidation with iodate results in increased breakdown of band 3, but does not lead to increased binding of autologous IgG. Only erythrocytes that have been stored for 3-4 weeks show the same structural and functional changes as observed during aging in vivo.     

Direct binding of peptides to MHC class I molecules on living cells. Analysis at the single cell level.

To directly assess the binding of exogenous peptides to cell surface-associated MHC class I molecules at the single cell level, we examined the possibility of combining the use of biotinylated peptide derivatives with an immunofluorescence detection system based on flow cytometry. Various biotinylated derivatives of the adenovirus 5 early region 1A peptide 234-243, an antigenic peptide recognized by CTL in the context of H-2Db, were first screened in functional assays for their ability to bind efficiently to Db molecules on living cells. Suitable peptide derivatives were then tested for their ability to generate positive fluorescence signals upon addition of phycoerythrin-labeled streptavidin to peptide derivative-bearing cells. Strong fluorescent staining of Db-expressing cells was achieved after incubation with a peptide derivative containing a biotin group at the C-terminus. Competition experiments using the unmodified parental peptide as well as unrelated peptides known to bind to Kd, Kb, or Db, respectively, established that binding of the biotinylated peptide to living cells was Db-specific. By using Con A blasts derived from different H-2 congenic mouse strains, it could be shown that the biotinylated peptide bound only to Db among > 20 class I alleles tested. Moreover, binding of the biotinylated peptide to cells expressing the Dbm13 and Dbm14 mutant molecules was drastically reduced compared to Db. Binding of the biotinylated peptide to freshly isolated Db+ cells was readily detectable, allowing direct assessment of the relative amount of peptide bound to distinct lymphocyte subpopulations by three-color flow cytometry. While minor differences between peripheral T and B cells could be documented, thymocytes were found to differ widely in their peptide binding activity. In all cases, these differences correlated positively with the differential expression of Db at the cell surface. Finally, kinetic studies at different temperatures strongly suggested that the biotinylated peptide first associated with Db molecules available constitutively at the cell surface and then with newly arrived Db molecules.     

Binding properties of biotinylated epidermal growth factor to its receptor on cultured cells and tissue sections

A biotinylated derivative of murine epidermal growth factor (EGF) was prepared by covalent attachment of the terminal amino group of EGF to N-biotinyl-epsilon-aminocaproyl-N-hydroxysuccinimide. The stoichiometry of biotin incorporation was in the range of one biotin moiety per EGF molecule. The biotinylated EGF (biotinyl-epsilon-caproyl-EGF, BioEGF) binds to EGF receptors on intact Ehrlich ascites carcinoma (EAC) cells with an affinity similar to that of native EGF and displays the same mitogenic activity as EGF in a soft agar test system with normal rat kidney (NRK) cells. BioEGF was visualized on cultured cells and tissue sections of a head and neck tumour by commercial streptavidin/avidin detection systems. Cytochemical analyses of certain tumour forms can be easily performed using the BioEGF probe.     

The enzymatic biotinylation of proteins: a post-translational modification of exceptional specificity.

Biotin is a coenzyme essential to all life forms. The vitamin has biological activity only when covalently attached to certain key metabolic enzymes. Most organisms have only one enzyme for attachment of biotin to other proteins and the sequences of these proteins and their substrate proteins are strongly conserved throughout nature. Structures of both the biotin ligase and the biotin carrier protein domain from Escherichia coli have been determined. These, together with mutational analyses of biotinylated proteins, are beginning to elucidate the exceptional specificity of this protein modification.     

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.     

Metabolic biotinylation of cell surface receptors for in vivo imaging

We have developed a versatile, potent technique for imaging cells in culture and in vivo by expressing a metabolically biotinylated cell-surface receptor and visualizing it with labeled streptavidin moieties. The recombinant reporter protein, which incorporates a biotin acceptor peptide (BAP) between an N-terminal signal sequence and a transmembrane domain, (BAP-TM) was efficiently biotinylated by endogenous biotin ligase in mammalian cells with the biotin displayed on the cell surface. Tumors expressing the BAP-TM have high sensitivity for magnetic resonance and fluorescence tomographic imaging in vivo after intravascular injection of streptavidin conjugated to magnetic nanoparticles or fluorochromes, respectively. Moreover, streptavidin-horseradish peroxidase conjugates in conjunction with a peroxidase-sensitive gadolinium agent further increased and prolonged the magnetic resonance signal. This BAP-TM allows noninvasive real-time imaging of any cell type transduced to express this reporter protein in culture or in vivo.     

Erythrocyte membrane elasticity during in vivo ageing

Changes in the ability of senescent erythrocytes to pass through the micro-circulation may cause them to be trapped in the spleen and removed from the blood. To help understand this process we have measured erythrocyte membrane elasticity, to see whether it changes during in vivo ageing. Human and rabbit red cells were fractionated by isopycnic sedimentation to obtain samples of aged and young cells. These were subjected to micropipette analysis in order to determine their membrane shear elastic modulus. We found that the membrane rigidity did not significantly alter as red cells aged. Previously we have also demonstrated that the changed size and shape of aged cells is unlikely to explain their removal from the circulation (Nash, G.B. and Wyard, S.J. (1981) Biorheology, in the press). Thus we conclude that the lifespan of erythrocytes is not determined by factors related to membrane flexibility or cell shape but may depend on changes in their viscous properties (as suggested by Williams, A.R. and Morris, D.R. (1980), Scand. J. Haematol. 24, 57–62).     

Labelling of Tumour Cells with a Biotinylated Inhibitor of a Cell Surface Protease

Abstract

Our objective has been to prepare a biotinylated affinity probe for the active centre of a protease associated with the surface of tumour cells. We employed three model systems in which easily recognisable tumour cells containing the active protease were uaed as targets for the biotinylated affinity probe. These were: squamous cell carcinoma, leukaemia cells in muscle and outgrowths of prostate carcinoma cells grown in three dimensional collagen gels. The presence of the bound biotinylated affinity probe was demonstrated by its ability to bind Texas-red labelled streptavidin with the results that the tumour cells exhibited red fluorescence. This binding was shown to be competitive with 9-amino acridine, a compound known to bind to the active centre of the target protease. This technique depends upon the affinity of the active centre of an enzyme for a competitive inhibitor and therefore should be applicable to other enzyme systems employing suitable ligands for their activc centres.     

The ultrastructural delineation of cell growth and division processes using the avidin-biotin complex

A novel method for the study of the fate of cell envelope components during growth and division is described. Successive treatment of the budding yeast, Saccharomyces cerevisiae, with sodium periodate and biotin hydrazide results in the covalent attachment of biotin to an unidentified cell surface component(s), without concomitant interference with subsequent growth and/or division. Further treatment of the cells with ferritin-avidin conjugates (FAv) enables the localization of the position of biotinylated surface components. Electron microscopical analysis of the distribution of attached FAv on cells fixed immediately after biotinylation revealed an even distribution of the biotin sites over the entire surface (including buds and scars) of all cells in the population. Labeling of biotinylated cells following a defined growth period revealed a new cell subpopulation completely devoid of label. The absence of biotin sites on the majority of buds and newly formed scars which appeared on the biotinylated yeasts indicate that the labeled cell wall constituents are stationary and not transferred to the newly synthesized cell wall of the daughter cells. The selective interaction of the biotinylated parent cells with avidin or antibiotin antibodies may enable an affinity-based separation of successive generations from a mixed yeast cell population.     

Differential effect of pH on the density and volume of rat reticulocytes and erythrocytes

Rats were injected with⁵⁹Fe-ferrous citrate and bled thereafter at different times (16 h to 49 d). This gave rise to red cell populations in which cells corresponding in age to the time elapsed between injection and bleeding were labeled. The anticoagulant used was either acid-citrate-dextrose (ACD) with a pH adjusted to 7.3 or ACD (pH 5.1). Final pH of the collected blood was about 7.2–7.4 in the former case and 6.4–6.7 in the latter. Red cells were then centrifuged (5) and approximately 7–10% of the packed cells from the top and 7–10% from the bottom of the cell column collected. When reticulocytes are the predominant labeled red cell population, as in blood obtained for about 24 h after isotope injection, a fractionation of these cells and mature erythrocytes is in evidence only when blood is collected at the higher pH. Thus, at pH 7.2–7.4 ratios of specific radioactivities of cells in top fraction/cells in an unfractionated sample are about 3, whereas at pH 6.4–6.7, the analogous ratios are 1 or less. These differences in specific activity ratios, as a function of pH at collection, virtually disappear after about 4 d following isotope injection. The lower pH is known to increase the volume and decrease the density of mature red blood cells. The marked effect of pH on cellular fractionation could be correlated with the smaller change in rat reticulocyte density and volume in acid medium. At pH 6.4–6.7, the densities of mature erythrocytes and reticulocytes are so close that their physical separation by centrifugation is not feasible.     

Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.