Multiple myeloma (MM) is characterized by the clonal expansion and metastatic spread of malignant plasma cells to multiple sites in the bone marrow (BM). Recently, we implicated the sialyltransferase ...ST3Gal-6, an enzyme critical to the generation of E-selectin ligands, in MM BM homing and resistance to therapy. Since E-selectin is constitutively expressed in the BM microvasculature, we wished to establish the contribution of E-selectin ligands to MM biology. We report that functional E-selectin ligands are restricted to a minor subpopulation of MM cell lines which, upon expansion, demonstrate specific and robust interaction with recombinant E-selectin in vitro. Moreover, an increase in the mRNA levels of genes involved in the generation of E-selectin ligands was associated with inferior progression-free survival in the CoMMpass study. In vivo, E-selectin ligand-enriched cells induced a more aggressive disease and were completely insensitive to Bortezomib. Importantly, this resistance could be reverted by co-administration of GMI-1271, a specific glycomimetic antagonist of E-selectin. Finally, we report that E-selectin ligand-bearing cells are present in primary MM samples from BM and peripheral blood with a higher proportion seen in relapsed patients. This study provides a rationale for targeting E-selectin receptor/ligand interactions to overcome MM metastasis and chemoresistance.
Human peripheral cannabinoid receptor CB2, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural ...and functional studies on CB2 may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB2 and immobilization at solid interfaces. Expression of CB2 as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB2 was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB2 was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB2 from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB2 and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB2.
•Expressed and purified functional recombinant cannabinoid receptor CB2•Immobilized CB2 in oriented fashion onto antibody-coated Biacore CM4 chip•Characterized functional recombinant CB2 by ligand binding in detergent micelles•Characterized binding of Rho-tagged CB2 to surface-immobilized 1D4 antibody by SPR•Characterized binding of monoclonal antibody to surface-immobilized CB2
► Cannabinoid CB2 receptor is expressed in Escherichia coli as a fusion with HaloTag. ► CB2 expressed as a fusion with C-terminal HaloTag retains functional activity. ► Fusion CB2-HaloTag can be ...immobilized and purified on a HaloLink resin. ► N-terminal HaloTag results in misfolding of the receptor in E. coli.
Expression of milligram quantities of functional, stable G protein-coupled receptors (GPCR) for high-resolution structural studies remains a challenging task. The goal of this work was to evaluate the usefulness of the HaloTag system (Promega) for expression and purification of the human cannabinoid receptor CB2, an important target for development of drugs for treatment of immune disorders, inflammation, and pain.
Here we investigated expression in Escherichia coli cells of the integral membrane receptor CB2 as a fusion with the 34kDa HaloTag at N- or C-terminal location, either in the presence or in the absence of the N-terminal maltose-binding protein (MBP). The CB2 was flanked at both ends by the tobacco etch virus (TEV) protease cleavage sites to allow for subsequent removal of expression partners. Expression by induction with either IPTG (in E. coli BL21(DE3) cell cultures) or by auto-induction (in E. coli KRX cells) were compared.
While the N-terminal location of the HaloTag resulted in high levels of expression of the fusion CB2, the recombinant receptor was not functional. However, when the HaloTag was placed in the C-terminal location, a fully active receptor was produced irrespective of induction method or bacterial strain used. For purification, the fusion protein was captured onto HaloLink resin in the presence of detergents. Treatment with specific TEV protease released the CB2 upon washing. To our knowledge, this study represents the first example of expression, surface immobilization and purification of a functional GPCR using HaloTag technology.
There is increasing evidence that E-selectin and its ligands play an important role in the progression of multiple myeloma (MM) and drug resistance. We reported that the sialyltransferase ST3GAL6 ...influences homing and survival in MM, and postulated that it may function in the synthesis of E-selectin ligands (Glavey et al Blood, 2014). We also found that a small subpopulation of cells (~ 5%) from MM cell lines express functional E-selectin ligands, which could be expanded under hypoxic conditions typical of the bone marrow (BM) microenvironment. These cells were identified by reactivity with an antibody (HECA452), which binds the same carbohydrate epitope required for binding to E-selectin. Rolling of MM1S cells on E-selectin was blocked by a small molecule glycomimetic antagonist to E-selectin (GMI-1271). Moreover, GMI-1271 significantly enhanced the anti-myeloma activity of bortezomib (BTZ) in an in vivo murine transplant model (Natoni et al Blood, 2014). We now extend these observations to obtain a more complete understanding of the role E-selectin plays in MM biology and chemotherapy resistance for its potential clinical relevance.
The parental, heterogeneous MM cell lines MM1S and RPMI8226 (MM1Spar, RPMI8226par, respectively) were sequentially sorted to obtain cell lines highly enriched (>85% and 80%) for the expression of cell surface carbohydrates bound by HECA452, and designated MM1SHECA452 and RPMI8226HECA452. The cell lines could be passaged in vitro and were stable for enriched E-selectin ligand expression. In contrast to parental cells, both MM1SHECA452 and RPMI8226HECA452 showed strong binding to E-selectin in static adhesion assays. Both MM1SHECA452 and RPMI8226HECA452 exhibited strong rolling on E-selectin under shear stress. MM1Spar or RPMI8226par failed to roll well on E-selectin. The addition of GMI-1271 during culture conditions led to a marked reduction in adhesion of MM1SHECA452 and profoundly inhibited rolling on E-selectin of both HECA452 enriched MM cell lines.
The significance of these in vitro findings was studied in vivo. MM1Spar or MM1SHECA452 cells were injected i.v. into SCID beige mice. Beginning 5 days post tumor injection, the survival impact of treatment with saline control, GMI-1271, BTZ or a combination of both was determined. Mice transplanted with MM1SHECA452 had more aggressive disease with significantly shorter survival compared to those transplanted with MM1Spar. In contrast to MM1Spar cells, mice engrafted with MM1SHECA452 demonstrated a marked resistance to BTZ treatment. Whereas GMI-1271 treatment alone had no impact on survival, the combination of GMI-1271 and BTZ led to a highly significant improvement in survival of MM1Spar engrafted mice (P=0.0363), and more importantly broke the resistance and restored the anti-myeloma activity of BTZ in MM1SHECA452 engrafted mice (P=0.0123) (figure 1). The number of peripheral blood (PB) human CD138+ cells was increased in MM1SHECA452-engrafted mice within 60 min following a single injection of GMI-1271, and persisted for at least 24 hours (2.37% v. 0.03%, p <0.001). This effect was consistent with GMI-1271 disrupting the tumor microenvironment and mobilizing MM1SHECA-452 cells from the BM niche.
Given these findings we wished to see if samples from patients with MM express E-selectin ligands and whether higher levels are seen with disease progression. BM and/or PB were obtained following informed consent from patients with MM and plasma cells (CD38+/CD138+) were analyzed for E-selectin ligand expression by flow cytometry using the HECA452 antibody. To date all primary MM samples (n=25) contained HECA452-reactive cell populations (median 22%). A consistently higher proportion of circulating MM cells express HECA452 when compared with paired BM samples (n=14), with a median difference of 33% (Wilcoxon signed rank test, p=0.02). HECA452 expression of MM in PB was significantly higher (on average 40% higher) in samples taken at relapse vs. diagnosis, (unpaired t test, p = 0.0008)
These data provide compelling evidence that E-selectin ligand bearing cells play an important role in disease progression and drug resistance in MM, and a strong rationale for clinical strategies incorporating GMI-1271 to improve patient outcome.
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Smith:GlycoMimetics, Inc.: Employment. Locatelli-Hoops:GlycoMimetics, Inc.: Employment. Oliva:GlycoMimetics, Inc.: Employment. Fogler:GlycoMimetics, Inc.: Employment. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. O'Dwyer:Celgene: Honoraria, Research Funding.
Human peripheral cannabinoid receptor CB₂, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural ...and functional studies on CB₂ may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB₂ and immobilization at solid interfaces. Expression of CB₂ as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB₂ was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB₂ was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB₂ from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB₂ and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB₂.
Tandem affinity purification has been increasingly applied to isolation of recombinant proteins. It relies on two consecutive chromatographic steps that take advantage of the affinity tags placed at ...opposing ends of the target protein. This allows for efficient removal of contaminating proteins, including products of proteolytic degradation of the fusion that lack either N- or C-terminal tags. Here, we describe the use of two small affinity tags, a poly-histidine tag and a Strep-tag for expression and purification of the human cannabinoid receptor CB2, an integral membrane G protein-coupled receptor.
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