Expression, purification and characterization of SARS-CoV-2 spike RBD in ExpiCHO cells.

Reliable diagnosis is critical to identify infections of SARS-CoV-2 as well as to evaluate the immune response to virus and vaccines. Consequently, it becomes crucial the isolation of sensitive antibodies to use as immunocapture elements of diagnostic tools. The final bottleneck to achieve these results is the availability of enough antigen of good quality. We have established a robust pipeline for the production of recombinant, functional SARS-CoV-2 Spike receptor binding domain (RBD) at high yield and low cost in culture flasks. RBD was expressed in transiently transfected ExpiCHO cells at 32 °C and 5% CO2 and purified up to 40 mg/L. The progressive protein accumulation in the culture medium was monitored with an immunobinding assay in order to identify the optimal collection time. Successively, a two-step chromatographic protocol enabled its selective purification in the monomeric state. RBD quality assessment was positively evaluated by SDS-PAGE, Western Blotting and Mass Spectrometry, while Bio-Layer Interferometry, flow cytometer and ELISA tests confirmed its functionality. This effective protocol for the RBD production in transient eukaryotic system can be immediately extended to the production of RBD mutants.

Involvement of mammalian bilitranslocase-like protein(s) in chlorophyll catabolism of Pisum sativum L. tissues.

Putative pea bilin and cyclic tetrapyrrole transporter proteins were identified by means of an antibody raised against a bilirubin-interacting aminoacidic sequence of mammalian bilitranslocase (TC No. 2.A.65.1.1). The immunochemical approach showed the presence of several proteins mostly in leaf microsomal, chloroplast and tonoplast vesicles. In these membrane fractions, electrogenic bromosulfalein transport activity was also monitored, being specifically inhibited by anti-bilitranslocase sequence antibody. Moreover, the inhibition of transport activity in pea leaf chloroplast vesicles, by both the synthetic cyclic tetrapyrrole chlorophyllin and the heme catabolite biliverdin, supports the involvement of some of these proteins in the transport of linear/cyclic tetrapyrroles during chlorophyll metabolism. Immunochemical localization in chloroplast sub-compartments revealed that these putative bilitranslocase-like transporters are restricted to the thylakoids only, suggesting their preferential implication in the uptake of cyclic tetrapyrrolic intermediates from the stroma during chlorophyll biosynthesis. Finally, the presence of a conserved bilin-binding sequence in different proteins (enzymes and transporters) from divergent species is discussed in an evolutionary context.

Development and characterization of a novel mAb against bilitranslocase - a new biomarker of renal carcinoma.

BACKGROUND: Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues. MATERIALS AND METHODS: Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase. RESULTS: On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use. CONCLUSIONS: In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

Bilitranslocase is involved in the uptake of bromosulfophthalein in rat and human liver.

Hepatic disposition of bromosulfophthalein (BSP), bilirubin and bile salts partially overlap, as these anions share both uptake and excretion mechanisms. Multiple organic anion transporters mediate hepatic BSP uptake, i.e. members of the SLCO and SLC22 gene families and bilitranslocase (TCDB #2.A.65.1.1). This study aimed at evaluating the relative contribution of bilitranslocase in BSP uptake in precision-cut human and rat liver slices. To this purpose, two different anti-sequence bilitranslocase antibodies were used as specific, functional inhibitors of bilitranslocase. The intact liver physiology was accurately reproduced in this BSP uptake assay, since uptake was strongly temperature-dependentand inhibited by hepatotropic organic anions, such as 50 nM bilirubin, 1 µM nicotinic acid, 2 µM digoxin, 5 µMindocyanine green and 100 µM taurocholate. The bilitranslocase antibodies inhibited BSP uptake both in rat and human liver slices. The combined use of bilitranslocase antibodies and taurocholate caused additive-type inhibition, confirming that bilitranslocase is not a bile salt transporter; by contrast, bilirubin caused no additive-type inhibition. In conclusion this data, indicate the role of the bilirubin transporter bilitranslocase as one of the transporters involved in the uptake of anions like BSP in parallel with other organic anion carriers. Moreover this data indicate the value of precision-cut liver slices for phenotypic drug uptake studies.

Expression of bilitranslocase in the vascular endothelium and its function as a flavonoid transporter.

AIMS: Ingestion of flavonoid-rich beverages acutely affects endothelial function, causing vasodilation. This effect might be dependent on flavonoid transport into the endothelium. We investigated flavonoid uptake into vascular endothelial cells and whether this was mediated by bilitranslocase (TC 2.A.65.1.1), a bilirubin-specific membrane carrier that also transports various dietary flavonoids. METHODS AND RESULTS: Human and rat aortic primary endothelial cells as well as Ea.hy 926 cells were found to express bilitranslocase, as assessed by immunocytochemistry and immunoblotting analysis using anti-sequence bilitranslocase antibodies targeting two distinct extracellular epitopes of the carrier. Bilitranslocase function was tested by measuring the rate of bromosulfophthalein (a standard bilitranslocase transport substrate) uptake into endothelial cells and was inhibited not only by bilitranslocase antibodies but also by quercetin (a flavonol). Similarly, uptake of both quercetin and malvidin 3-glucoside (an anthocyanin) were also found to be antibody-inhibited. Quercetin uptake into cells was inhibited by bilirubin, suggesting flavonoid uptake via a membrane pathway shared with bilirubin. CONCLUSION: The uptake of some flavonoids into the vascular endothelium occurs via the bilirubin-specific membrane transporter bilitranslocase. This offers new insights into the vascular effects of both flavonoids and bilirubin.

Bioavailability of flavonoids: a review of their membrane transport and the function of bilitranslocase in animal and plant organisms.

Fruits and vegetables are rich in flavonoids, and ample epidemiological data show that diets rich in fruits and vegetables confer protection against cardiovascular, neurodegenerative and inflammatory diseases, and cancer. However, flavonoid bioavailability is reportedly very low in mammals and the molecular mechanisms of their action are still poorly known. This review focuses on membrane transport of flavonoids, a critical determinant of their bioavailability. Cellular influx and efflux transporters are reviewed for their involvement in the absorption of flavonoids from the gastro-intestinal tract and their subsequent tissue distribution. A focus on the mammalian bilirubin transporter bilitranslocase (TCDB 2.A.65.1.1) provides further insight into flavonoid bioavailability and its relationship with plasma bilirubin (an endogenous antioxidant). The general function of bilitranslocase as a flavonoid membrane transporter is further demonstrated by the occurrence of a plant homologue in organs (petals, berries) where flavonoid biosynthesis is most active. Bilitranslocase appears associated with sub-cellular membrane compartments and operates as a flavonoid membrane transporter.

Uptake of grape anthocyanins into the rat kidney and the involvement of bilitranslocase.

Anthocyanins are among the most common flavonoids in the human diet. In spite of their very low bioavailability, anthocyanins are indicated as active in preventing the progress of cardiovascular and neurodegenerative diseases, obesity, inflammation, and cancer. Any piece of knowledge concerning absorption, tissue distribution, metabolism, and excretion of dietary anthocyanins is expected to help understanding the apparent paradox between their low concentrations in cells and their bioactivity. The aim of this work was to investigate the renal uptake of dietary anthocyanins and the underlying molecular mechanism. A solution containing anthocyanins extracted from grape (Vitis vinifera) was introduced into the isolated stomach of anesthetized rats; after both 10 and 30 min, plasma, liver, and kidney were analyzed for their anthocyanin contents. While anthocyanins in the liver were at apparent equilibrium with plasma both after 10 and 30 min, kidney anthocyanins were 3- and 2.3-fold higher than in plasma, after 10 and 30 min, respectively. Since the transport activity of the bilitranslocase in kidney basolateral membrane vesicles was competitively inhibited by malvidin 3-glucoside (K(i) = 4.8 +/- 0.2 microM), the anthocyanin uptake from blood into kidney tubular cells is likely to be mediated by the kidney isoform of this organic anion membrane transporter.

Uptake of bilirubin into HepG2 cells assayed by thermal lens spectroscopy. Function of bilitranslocase.

Bilitranslocase is a carrier protein localized at the basolateral domain of the hepatocyte plasma membrane. It transports various organic anions, including bromosulfophthalein and anthocyanins. Functional studies in subcellular fractions enriched in plasma membrane revealed a high-affinity binding site for bilirubin, associated with bilitranslocase. The aim of this work was to test whether the liver uptake of bilirubin depends on the activity of bilitranslocase. To this purpose, an assay of bilirubin uptake into HepG2 cell cultures was set up. The transport assay medium contained bilirubin at a concentration of approximately 50 nm in the absence of albumin. To analyse the relative changes in bilirubin concentration in the medium throughout the uptake experiment, a highly sensitive thermal lens spectrometry method was used. The mechanism of bilirubin uptake into HepG2 cells was investigated by using inhibitors such as anti-sequence bilitranslocase antibodies, the protein-modifying reagent phenylmethanesulfonyl fluoride and diverse organic anions, including nicotinic acid, taurocholate and digoxin. To validate the assay further, both bromosulfophthalein and indocyanine green uptake in HepG2 cells was also characterized. The results obtained show that bilitranslocase is a carrier with specificity for both bilirubin and bromosulfophthalein, but not for indocyanine green.

Hemolytic effects of water-soluble fullerene derivatives.

A series of water-soluble fullerene C(60) derivatives has been investigated for their cytotoxic and hemolytic properties, with the aim to correlate structure with toxicity. We observed that cationic chains induce significant toxicity while the presence of neutral or anionic moieties did not produce any response in our model. A validation of these experimental observations has been performed by theoretical studies in which hydrophilic and hydrophobic surface areas were correlated quantitatively with hemolytic properties.