Szecheng J. Lo

Application of Recombinant Rhodostomin in Studying Cell Adhesion.

Rhodostomin from venom of Agkistrodon rhodostoma (also called Calloselasma rhodostoma) contains 68 amino acid residues including 6 pairs of disulfide bonds and an arginine-glycine-aspartic acid (RGD) sequence at positions 49-51. It has been known as one of the strongest antagonists to platelet aggregation among the family termed disintegrin. In this review paper, in addition to introducing the characteristics of disintegrin and its related molecules, the advantages of using recombinant DNA technology to produce rhodostomin are described. The recombinant rhodostomin has been demonstrated to facilitate cell adhesion via interaction between the RGD motif of rhodostomin and integrins on the cell surface. This property allowed us to use the recombinant rhodostomin as an extracellular matrix to study cell adhesion and to distinguish attachment efficiency between two melanoma cell lines B16-F1 and B16-F10, the former is a low metastasis cell while the latter is a high metastasis cell. Furthermore, by using the recombinant rhodostomin as a substrate, osteoprogenitor-like cells are able to be selected and enriched within 3 days from rat bone marrow which contains a heterogeneous cell population. Finally, we show that the recombinant rhodostomin can be immobilized on beads and which serve as an affinity column to dissect cell-surface protein(s) binding to the RGD motif of rhodostomin.

Positional importance of Pro53 adjacent to the Arg49-Gly50-Asp51 sequence of rhodostomin in binding to integrin alphaIIbbeta3.

Rhodostomin (RHO), a disintegrin isolated from snake venom, has been demonstrated to inhibit platelet aggregation through interaction with integrin alphaIIbbeta3, but there is a lack of direct evidence for RHO-integrin alphaIIbbeta3 binding. In addition, no study on the length of Arg(49)-Gly(50)-Asp(51) (RGD) loop of RHO influencing on its binding to integrin alphaIIbbeta3 has been reported. In the present study we have developed a highly sensitive dot-blot and glutathione S-transferase-RHO pull-down assays; the latter was coupled with a biotin-avidin-horseradish peroxidase enhanced-chemiluminescence detection system. These were able to demonstrate the direct binding of RHO to integrin alphaIIbbeta3. The pull-down assay further showed that four alanine-insertion mutants upstream of the RGD motif and three insertions downstream of the RGD were able to decrease integrin alphaIIbbeta3 binding activity to only a limited extent. By contrast, two insertions immediately next to RGD and one insertion in front of the Cys(57) caused almost complete loss of binding activity to alphaIIbbeta3. The results of the platelet-aggregation-inhibition assay and platelet-adhesion assay for the insertion mutants were consistent with results of the pull-down assay. It is thus concluded that, although an insertion of a single alanine residue in many positions of the RGD loop has only minor effects on RHO binding to integrin alphaIIbbeta3, the specific position of Pro(53) residue adjacent to the RGD sequence is important for RHO binding to platelet integrin alphaIIbbeta3.

Expression of Foreign Antigens on the Surface of Escherichia coli by Fusion to the Outer Membrane Protein TraT

The traT gene is one of the F factor transfer genes and encodes an outer membrane protein which is involved in interactions between an Escherichia coli and its surroundings. This protein was altered so as to permit the expression of foreign proteins on the outer membrane of E. coli in this study. A 729-bp DNA fragment, including the leader and entire structural gene sequence of traT, was amplified and obtained by PCR. This sequence was then subcloned downstream of the tac promoter of pDR540, resulting in a TraT expression vector, pT2. Here, we report that the expression of TraT protein, fused either with a partial pre-S antigen of hepatitis B virus (60 and 98 amino acids, respectively) or with the snake venom rhodostomin (72 amino acids), was successfully achieved on the outer membrane of E. coli, using the pT2 plasmid. This result was demonstrated using dot blot and immunofluorescence analysis. This finding supports the notion that the pT2 plasmid can be used as an E. coli display system. This system can detect a foreign peptide of about 100 amino acid residues in length on the bacterial surface.

Full-spreading platelets induced by the recombinant rhodostomin are via binding to integrins and correlated with FAK phosphorylation

We have previously reported that non-activated platelets can be induced by morphological changes from the recombinant fusion protein of GST-rhodostomin [GST-RHO(RGD)], a member of disintegrin with an arginine-glycine-aspartic acid (RGD) motif. In this study, we further characterized the factors involved in platelet shape changes induced by rhodostomin. From less to full-spreading, four cell spreading indexes, p1, p2, s1 and s2, were designated to the platelet shape based on the scanning electron micrographs. Results of peptide competition and antibody blocking confirmed that interaction between the RGD of rhodostomin and the alpha(IIb)beta3 integrins of platelets was required for induction of a higher percentage of s2 cells. When platelets were pretreated with calphostin C, herbimycin A and cytochalasin B, respectively, the percentage of p1 and p2 cells on rhodostomin-coated plates was increased and, concomitantly, the percentage of s1 and s2 cells was decreased. Biochemical analyses indicated that the focal adhesion kinase (FAK or pp125FAK) in platelets that adhered to GST-RHO(RGD) was phosphorylated in contrast to little or no phosphorylation of FAK in cells adhered to fibrinogen or non-activated cells. Furthermore, the degree of FAK phosphorylation was consistently correlated with morphological changes in platelets treated with various drugs. Taking all the results together, we suggested that rhodostomin could directly bind to integrins of platelets and then trigger signal transduction leading to FAK phosphorylation and actin polymerization and finally resulting in platelet full-spreading.

Biogenesis of the hepatitis B viral middle (M) surface protein in a human hepatoma cell line: demonstration of an alternative secretion pathway.

In the serum of hepatitis B virus (HBV)-infected patients, two different types of particles, a 42 nm virion and a 22 nm subviral particle, were identified. The envelope of both particles is composed of three proteins, the large (L), middle (M), and major/small (S) surface proteins but the ratio between these components varies in each. The M protein appears in a lesser amount than the S protein in both virion and subviral particles, although it is translated from the same subgenomic RNA, and this is due to its poor initiation context of translation. In addition, only the glycosylated form of M protein is secreted in contrast to both glycosylated and unglycosylated forms of L and S proteins that are secreted. To investigate the biogenesis of M protein, human hepatoma cells transfected with plasmids containing a mutated HBV DNA were used to produce a high amount of M protein. Electron microscopic observation revealed that despite a higher proportion of the M protein being found in the transfected cells, the secreted surface antigen particles possess similar size and density to 22 nm subviral particles. Detailed biochemical analyses showed the following. (1) The unglycosylated M protein was predominantly present in the microsomal fraction but not present in any other subcellular fractions. (2) The M protein formed 22-nm-like particles in the endoplasmic reticulum (ER) and was retained in the post-ER or pre-Golgi regions. (3) In addition to the complex glycosylated form of M protein, a high-mannose form of M protein could be secreted. (4) Normally, no unglycosylated M protein was secreted. However, glycosylation was not essential for M protein secretion since M protein deprived of glycosylation by tunicamycin treatment was detected in the medium. These findings suggest that (i) the M protein was probably translated and co-translocated into the ER and at least one site was glycosylated before leaving the ER resulting in no secretion of unglycosylated M protein, and (ii) the M protein had two secretion pathways, one through the conventional pathway and the other probably directly through the ER.

Glutathione S-transferase-rhodostomin fusion protein inhibits platelet aggregation and induces platelet shape change

Rhodostomin (RHO) from Agkistrodon rhodostoma venom, consisting of 68 amino acids with an arginine-glycine-aspartic acid (RGD) sequence and 12 cysteine residues, is a potent inhibitor of platelet aggregation. We previously demonstrated that cell culture plates coated with the bacterially produced fusion protein of glutathione S-transferase-RHO [GST-RHO(RGD)] can facilitate human hepatoma cell attachment via intergrin interaction within 15 min. In this study, we further characterized the effect of RHO fusion protein on platelet cells by creating two other related fusion proteins, GST-RHO(RGE) and GST-(PS)RHO. The former was a single amino acid-substituted mutant, in which the aspartic acid residue of RGD was replaced by glutamic acid, and the latter was an insertion mutant, in which a pentapeptide of protein kinase A phosphorylation site was inserted between GST and RHO. These two mutant proteins together with a wild-type of GST-RHO(RGD) and native form of RHO were used to study effects on the inhibition of ADP-induced platelet aggregation. Results indicated that GST-RHO(RGD) inhibited platelet aggregation as potently as the native RHO, while the two other mutants were inactive. Furthermore, when unactivated platelet cells attached on the GST-RHO(RGD)-coated plate, they became a flattened pancake shape. From the results of facilitation of cell attachment on fusion protein-coated plates, we concluded that: (1) the GST-RHO(RGD) fusion protein is equally functional in inhibition of platelet aggregation and facilitation of cell attachment, which is through the interaction of RGD and integrins on the cell membrane; (2) the GST-RHO(RGE) mutant protein is unable to bind with integrins and results in loss of function; (3) the insertion mutant of GST-(PS)RHO may disrupt a proper conformation of RHO and also results in loss of function; (4) the bacterially produced fusion protein GST-RHO(RGD) can be properly used as an antithrombotic agent and an extracellular matrix.

Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts

The envelope of the hepatitis B virus (HBV) is composed of three species of proteins, the large (L), middle (M), and major (S) surface proteins (HBsAgs), each of different molecular weights but sharing a common C-terminus. These three HBsAgs, encoded by two species of viral subgenomic transcripts (2.1 and 2.4 kb), which have a heterogenous 5-terminus, appear in different amounts in both the 42-nm virions and the 22-nm subviral particles. To investigate the involvement of translational control in the differential expression of the L, M, and S proteins, we tested the translational capability of 2.1- and 2.4-kb transcripts in in vitro translation and of 2.1-kb transcripts in in vivo transfection experiments. Results of in vitro translation indicated that a large amount of the L protein and a very small amount of the M and S proteins were synthesized from the 2.4-kb mRNA. Translation of the 2.1-kb mRNA resulted in a 4:1 ratio of the S protein to the M protein. In contrast, translation of a similar 2.1-kb mRNA containing an optimal initiation context (5-ACCATGG-3) of the pre-S2 region resulted in a reversed ratio, four times as much M protein as S protein. This result was also obtained by transfection of hepatoma cells with plasmid DNAs containing the mutated sequence (5-ACCATGG-3) of the pre-S2 region. In considering these results, the production of a large amount of the L protein from the 2.4-kb mRNA and the determination of the level of the M protein by the context of translational initiation, we suggest that a preferential translational initiation is involved in the expression of differential amounts of the L, M, and S proteins.

Cell-adhesion and morphological changes are not sufficient to support anchorage-dependent cell growth via non-integrin-mediated attachment.

Cell‐adhesion and spread are important for cell survival. Although extensive studies have suggested several potential mechanisms of action, it is not yet clear how important cell‐morphological change per se contributes to the cell‐surviving signal. We employed a non‐integrin‐mediated cell‐adhesion system to explore this question. BHK—Japanese encephalitis virus (JEV) cells (BHK21 cells that are persistently infected with JEV) express a large amount of JEV‐envelope protein (JEV E) on their surfaces, and can attach and form pseudopodia on the anti‐JEV E antibody‐coated substrates. However, cells that adhered on the antibody substrate underwent a caspase‐3‐mediated apoptosis together with a down‐regulation of mitogen‐activated protein kinase activity within 20 h after adhesion, which indicates that viral‐protein‐mediated cell‐adhesion and cell‐spread are not sufficient for supporting cell survival. This provides a different perspective for the study of the relationships between the cell‐morphological change and the cell‐survival signal.

Receptor-Mediated Endocytosis as a Selection Force to Enrich Bacteria Expressing Rhodostomin on Their Surface

Previously, we developed a TraT display system to express snake venom rhodostomin (RHO), a disintegrin, on the external surface of Escherichia coli [J Biomed Sci 6:64-70;1999]. To show a new potential use of the TraT display system, we employed a biotin labeling technique coupled with SDS-PAGE and flow cytometry analyses to further demonstrate and confirm the expression of TraT-RHO on the E. coli surface. We also showed that the expression of TraT-RHO on the cell surface not only facilitated the bacteria adhesion to BHK-21 cells but also induced bacterial internalization into BHK-21 cells. This feature allowed us to enrich the TraT-RHO expression bacteria about 10,000-fold starting with a mixture of TraT-RHO bacteria with beta-galactosidase-positive bacteria in a ratio of 10(2):10(7) through four cycles of BHK-21 cell endocytosis and replating of engulfed bacteria on agar plates. We therefore suggest that the TraT display system can be applied to select out bacteria expressing a specific peptide sequence from a large population of display library through the process of receptor-mediated endocytosis and reamplification cycles.

Hepatitis B viral polymerase fusion proteins are biologically active and can interact with the hepatitis C virus core protein in vivo

Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.