Targeting heparanase to the mammary epithelium enhances mammary gland development and promotes tumor growth and metastasis.

Heparanase is an endoglucuronidase that uniquely cleaves the heparan sulfate side chains of heparan sulfate proteoglycans. This activity ultimately alters the structural integrity of the ECM and basement membrane that becomes more prone to cellular invasion by metastatic cancer cells and cells of the immune system. In addition, enzymatically inactive heparanase was found to facilitate the proliferation and survival of cancer cells by activation of signaling molecules such as Akt, Src, signal transducer and activation of transcription (Stat), and epidermal growth factor receptor. This function is thought to be executed by the C-terminal domain of heparanase (8c), because over expression of this domain in cancer cells accelerated signaling cascades and tumor growth. We have used the regulatory elements of the mouse mammary tumor virus (MMTV) to direct the expression heparanase and the C-domain (8c) to the mammary gland epithelium of transgenic mice. Here, we report that mammary gland branching morphogenesis is increased in MMTV-heparanase and MMTV-8c mice, associating with increased Akt, Stat5 and Src phosphorylation. Furthermore, we found that the growth of tumors generated by mouse breast cancer cells and the resulting lung metastases are enhanced in MMTV-heparanase mice, thus supporting the notion that heparanase contributed by the tumor microenvironment (i.e., normal mammary epithelium) plays a decisive role in tumorigenesis. Remarkably, MMTV-8c mice develop spontaneous tumors in their mammary and salivary glands. Although this occurs at low rates and requires long latency, it demonstrates decisively the pro-tumorigenic capacity of heparanase signaling.

Establishment of a tobacco BY2 cell line devoid of plant-specific xylose and fucose as a platform for the production of biotherapeutic proteins.

Plant-produced glycoproteins contain N-linked glycans with plant-specific residues of ß(1,2)-xylose and core α(1,3)-fucose, which do not exist in mammalian-derived proteins. Although our experience with two enzymes that are used for enzyme replacement therapy does not indicate that the plant sugar residues have deleterious effects, we made a conscious decision to eliminate these moieties from plant-expressed proteins. We knocked out the ß(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes, using CRISPR/Cas9 genome editing, in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. In total, we knocked out 14 loci. The knocked-out lines were stable, viable and exhibited a typical BY2 growing rate. Glycan analysis of the endogenous proteins of these lines exhibited N-linked glycans lacking ß(1,2)-xylose and/or α(1,3)-fucose. The knocked-out lines were further transformed successfully with recombinant DNaseI. The expression level and the activity of the recombinant protein were similar to that of the protein produced in the wild-type BY2 cells. The recombinant DNaseI was shown to be totally free from any xylose and/or fucose residues. The glyco-engineered BY2 lines provide a valuable platform for producing potent biopharmaceutical products. Furthermore, these results demonstrate the power of the CRISPR/Cas9 technology for multiplex gene editing in BY2 cells.

Large-scale production of pharmaceutical proteins in plant cell culture-the Protalix experience.

Protalix Biotherapeutics develops recombinant human proteins and produces them in plant cell culture. Taliglucerase alfa has been the first biotherapeutic expressed in plant cells to be approved by regulatory authorities around the world. Other therapeutic proteins are being developed and are currently at various stages of the pipeline. This review summarizes the major milestones reached by Protalix Biotherapeutics to enable the development of these biotherapeutics, including platform establishment, cell line selection, manufacturing process and good manufacturing practice principles to consider for the process. Examples of the various products currently being developed are also presented.

Heparanase enhances the generation of activated factor X in the presence of tissue factor and activated factor VII.

BACKGROUND: Heparanase is an endo-ß-D-glucuronidase dominantly involved in tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Our hypothesis was that heparanase is directly involved in activation of the coagulation cascade. DESIGN AND METHODS: Activated factor X and thrombin were studied using chromogenic assays, immunoblotting and thromboelastography. Heparanase levels were measured by enzyme-linked immunosorbent assay. A potential direct interaction between tissue factor and heparanase was studied by co-immunoprecipitation and far-western assays. RESULTS: Interestingly, addition of heparanase to tissue factor and activated factor VII resulted in a 3- to 4-fold increase in activation of the coagulation cascade as shown by increased activated factor X and thrombin production. Culture medium of human embryonic kidney 293 cells over-expressing heparanase and its derivatives increased activated factor X levels in a non-enzymatic manner. When heparanase was added to pooled normal plasma, a 7- to 8-fold increase in activated factor X level was observed. Subsequently, we searched for clinical data supporting this newly identified role of heparanase. Plasma samples from 35 patients with acute leukemia at presentation and 20 healthy donors were studied for heparanase and activated factor X levels. A strong positive correlation was found between plasma heparanase and activated factor X levels (r=0.735, P=0.001). Unfractionated heparin and an inhibitor of activated factor X abolished the effect of heparanase, while tissue factor pathway inhibitor and tissue factor pathway inhibitor-2 only attenuated the procoagulant effect. Using co-immunoprecipitation and far-western analyses it was shown that heparanase interacts directly with tissue factor. CONCLUSIONS: Overall, our results support the notion that heparanase is a potential modulator of blood hemostasis, and suggest a novel mechanism by which heparanase increases the generation of activated factor X in the presence of tissue factor and activated factor VII.

Heparanase: busy at the cell surface.

Heparanase activity is strongly implicated in structural remodeling of the extracellular matrix, a process which can lead to invasion by tumor cells. In addition, heparanase augments signaling cascades leading to enhanced phosphorylation of selected protein kinases and increased gene transcription associated with aggressive tumor progression. This function is apparently independent of heparan sulfate and enzyme activity, and is mediated by a novel protein domain localized at the heparanase C-terminus. Moreover, the functional repertoire of heparanase is expanded by its regulation of syndecan clustering, shedding, and mitogen binding. Recent reports indicate that modified glycol-split heparin, which inhibits heparanase activity, can profoundly inhibit the progression of tumor xenografts produced by myeloma and carcinoma cells, thus moving anti-heparanase therapy closer to reality.

Structure-function approach identifies a COOH-terminal domain that mediates heparanase signaling.

Heparanase is an endo-beta-d-glucuronidase capable of cleaving heparan sulfate, activity that is strongly implicated in cellular invasion associated with tumor metastasis, angiogenesis, and inflammation. In addition, heparanase was noted to exert biological functions apparently independent of its enzymatic activity, enhancing the phosphorylation of selected protein kinases and inducing gene transcription. A predicted three-dimensional structure of constitutively active heparanase clearly delineates a TIM-barrel fold previously anticipated for the enzyme. Interestingly, the model also revealed the existence of a COOH-terminal domain (C-domain) that apparently is not an integral part of the TIM-barrel fold. We provide evidence that the C-domain is critical for heparanase enzymatic activity and secretion. Moreover, the C-domain was found to mediate nonenzymatic functions of heparanase, facilitating Akt phosphorylation, cell proliferation, and tumor xenograft progression. These findings support the notion that heparanase exerts enzymatic activity-independent functions, and identify, for the first time, a protein domain responsible for heparanase-mediated signaling. Inhibitors directed against the C-domain, combined with inhibitors of heparanase enzymatic activity, are expected to neutralize heparanase functions and to profoundly affect tumor growth, angiogenesis, and metastasis.

Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli.

BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.

Modulation of monomer conformation of the BglG transcriptional antiterminator from Escherichia coli.

The BglG protein positively regulates expression of the bgl operon in Escherichia coli by binding as a dimer to the bgl transcript and preventing premature termination of transcription in the presence of beta-glucosides. BglG activity is negatively controlled by BglF, the beta-glucoside phosphotransferase, which reversibly phosphorylates BglG according to beta-glucoside availability, thus modulating its dimeric state. BglG consists of an RNA-binding domain and two homologous domains, PRD1 and PRD2. Based on structural studies of a BglG homologue, the two PRDs fold similarly, and the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have recently shown that the affinity between PRD1 and PRD2 of BglG is high, and a fraction of the BglG monomers folds in the cell into a compact conformation, in which PRD1 and PRD2 are in close proximity. We show here that both BglG forms, the compact and noncompact, bind to the active site-containing domain of BglF, IIB(bgl), in vitro. The interaction of BglG with IIB(bgl) or BglF is mediated by PRD2. Both BglG forms are detected as phosphorylated proteins after in vitro phosphorylation with IIB(bgl) and are dephosphorylated by BglF in vitro in the presence of beta-glucosides. Nevertheless, genetic evidence indicates that the interaction of IIB(bgl) and BglF with the compact form is seemingly less favorable. Using in vivo cross-linking, we show that BglF enhances folding of BglG into a compact conformation, whereas the addition of beta-glucosides reduces the amount of this form. Based on these results we suggest a model for the modulation of BglG conformation and activity by BglF.

A fraction of the BglG transcriptional antiterminator from Escherichia coli exists as a compact monomer.

Expression of the bgl operon in Escherichia coli, induced by beta-glucosides, is positively regulated by BglG, a transcriptional antiterminator. In the presence of inducer, BglG dimerizes and binds to the bgl transcript to prevent premature termination of transcription. The dimeric state of BglG is determined by BglF, a membrane-bound enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS), which reversibly phosphorylates BglG according to beta-glucoside availability. BglG is composed of an RNA-binding domain followed by two homologous PTS regulation domains (PRD1 and PRD2). The predicted structure of dimeric LicT, a BglG homologue from Bacillus subtilis, suggests that the two PRDs adopt a similar structure and that the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have shown recently that the PRD1 and PRD2 domains of BglG can form a stable heterodimer. We report here, based on in vitro and in vivo cross-linking experiments, that a fraction of BglG is present in the cell in a compact form in which PRD1 and PRD2 are in close proximity. The compact form is present mainly in the BglG monomers. Our results imply that the monomer-dimer transition involves a conformational change. The possible role of the compact form in preventing untimely induction of the bgl operon is discussed.

Interactions between the PTS regulation domains of the BglG transcriptional antiterminator from Escherichia coli.

The E. coli BglG protein inhibits transcription termination within the bgl operon in the presence of beta-glucosides. BglG represents a family of transcriptional antiterminators that bind to RNA sequences, which partially overlap rho-independent terminators, and prevent termination by stabilizing an alternative structure of the transcript. The activity of BglG is determined by its dimeric state, which is modulated by reversible phosphorylation catalyzed by BglF, a PTS permease. Only the non-phosphorylated BglG dimer binds to RNA and allows read-through of transcription. BglG is composed of three domains: an RNA-binding domain followed by two domains, PRD1 and PRD2 (PTS regulation domains), which are similar in their sequence and folding. Based on the three-dimensional structure of dimeric LicT, a BglG homologue from Bacillus subtilis, the interactions within the dimer are PRD1-PRD1 and PRD2-PRD2. We have shown before that PRD2 mediates homodimerization very efficiently. Using genetic systems and in vitro techniques that assay and characterize protein-protein interactions, we show here that the PRD1 dimerizes very slowly, but once it does, the homodimers are stable. These results support our model that formation of BglG dimers initiates with PRD2 dimerization followed by zipping up of two BglG monomers to create the active RNA-binding domain. Moreover, our results demonstrate that PRD1 and PRD2 heterodimerize efficiently in vitro and in vivo. The affinity among the PRDs is in the following order: PRD2-PRD2 > PRD1-PRD2 > PRD1-PRD1. The interaction between PRD1 and PRD2 offers an explanation for the requirement of conserved residues in PRD1 for the phosphorylation of PRD2 by BglF.