Search

This article has been withdrawn by the authors as part of this review overlapped with the contents of Pietrangelo A and Ridgway ND. 2018. Cellular and Molecular Life Sciences. 75; 3079-98., (Published under license by The American Society for Biochemistry and Molecular Biology, Inc.)

We describe the use of two heparin-binding proteins, avidin and lactoferrin, as probes for monitoring the amount of heparin immobilized to plastic surfaces. The proteins were derivatized with either fluorescent labels or europium chelates, enabling sensitive, fast, reproducible, and robust assays, and were used to measure the amount of protein bound to heparinized microplates, with particular attention to plates that have been coated with bovine serum albumin (BSA)-heparin conjugate. This direct method unequivocally shows that BSA-heparin affords an economical, convenient, and reliable method for coating both polystyrene microtiter plates and magnetic beads with heparin. We demonstrate that assays using directly labeled proteins overcome the problems of dissociation of the heparin-protein complex, which can occur during incubation and washing steps associated with antibody-based detection methods, and the loss in binding capacity caused by certain blocking regimes. We suggest that labeled avidin and lactoferrin are convenient probes for heparinized surfaces with the potential for much wider applicability than that presented here.

Surface plasmon resonance (SPR) biosensors such as the BIAcore 2000 are a useful tool for the analysis of protein-heparin interactions. Generally, biotinylated heparin is captured on a streptavidin-coated surface to create heparinized surfaces for subsequent binding analyses. In this study we investigated three commonly used techniques for the biotinylation of heparin, namely coupling through either carboxylate groups or unsubstituted amines along the heparin chain, or through the reducing terminus of the heparin chain. Biotinylated heparin derivatives were immobilized on streptavidin sensor chips and several heparin-binding proteins were examined. Of the surfaces investigated, heparin attached through the reducing terminus had the highest binding capacity, and in some cases had a higher affinity for the proteins tested. Heparin immobilized via intrachain bare amines had intermediate binding capacity and affinity, and heparin immobilized through the carboxylate groups of uronic acids had the lowest capacity for the proteins tested. These results suggest that immobilizing heparin to a surface via intrachain modifications of the heparin molecule can affect the binding of particular heparin-binding proteins.

An expressed luciferase viability assay (ELVA) has been developed for cell viability and cell number based on detecting the expression of luciferase transfected into the cells. Stable transfectants were produced that expressed luciferase constitutively. Like many endogenous enzymes, luciferase is rapidly degraded following cell death, so that the enzyme can be used as a measure of cell viability. A modified luciferase assay was used in which the reagents were added directly to the cells in a microplate. The main advantages compared to other cell viability assays are the wide dynamic range, high sensitivity, low background, and the absence of any requirement to wash or harvest the cells. Stable transfectants of three factor-dependent cell lines (B13, Ba/F3 and CTLL) were produced and used in cytokine assays. Three strategies of selection after electroporation were tested: (1) using a plasmid containing both the genes encoding firefly luciferase and a selectable marker (neo), (2) cotransfection of a plasmid containing luciferase and a plasmid containing a selectable marker (puromycin resistance), and (3) cotransfection of a plasmid containing luciferase and a plasmid containing the human IL-5Ralpha-chain, and selecting in IL-5. This latter strategy produces an IL-5 responsive cell line expressing luciferase in a single step without the need for antibiotic selection.

Hematopoiesis is regulated by two sets of signals, those generated by cytokines and those generated when precursor cells interact with bone marrow (BM) stroma. The intimate contact between precursors and stroma appears to be mediated by multiple, different receptor-ligand binding events. To identify receptor-ligand pairs mediating the adhesion of hematopoietic precursor cells to stroma, an in vitro model of hematopoiesis was used. This involved coculturing the BM-derived, interleukin-3 (IL-3)-dependent, multipotential cells, FCDP-mix A4 (A4) with a stromal equivalent embryonic mesenchymal cell line, Swiss 3T3 (3T3). In coculture, A4 cells survive, proliferate, and differentiate in the absence of exogenous IL-3, providing they are attached to the 3T3 cell surface. By using detergent lysates of surface-biotinylated A4 cells, A4 cell molecules that bind to the stroma could be detected by either fluorescein isothiocyanate (FITC)-streptavidin or FITC-antibody staining and flow cytometry. Using this approach the beta 2 integrin, Mac-1, and CD45, a receptor-type tyrosine phosphatase, were identified as molecules on the A4 cell surface that bind 3T3 cells. Various glycosaminoglycans (GAGs), particularly heparin and heparan sulfate, blocked binding of A4 cell surface molecules to the 3T3 cells. The binding of CD45 and Mac-1 to the 3T3 cells was similarly blocked by these GAGs. Removal of heparin-binding molecules from A4 cell lysates diminished binding to the 3T3 cells and digestion of the 3T3 cell surface with heparinase abolished the binding of CD45 and Mac-1. The data suggest that heparan sulfate on the 3T3 cell surface is a ligand for both CD45 and Mac-1, but the two molecules recognize different heparan sulfate structural motifs.

The platelet-endothelial cell adhesion molecule-1 (PE-CAM-1), defined by the CD31 monoclonal antibody (MoAb), was initially described as a cell-cell adhesion molecule mediating both homotypic and heterotypic adhesion. In this report, we show that enriched CD34+ human hematopoietic progenitor cell populations, containing early myeloid, erythroid, and multipotential progenitor cells, are CD31+. Analyses of CD34+ cell lines representing early myeloid, multipotential, and pre-pre-B-lymphoid progenitors indicate that precursors of both myeloid and B-lymphoid cells express PECAM-1 at high levels. Three-color flow-cytometric analyses also show that normal human bone marrow CD31+ CD34+ subsets coexpress myeloid (CD33) or B-lymphoid (CD19, CD10) markers. Except for the monocytic cell line, U937, all CD34- cell lines tested, which represent more mature stages of the myeloid, erythroid, and lymphoid lineages, expressed substantially lower or negligible levels of PECAM-1. Western blotting studies indicated that the CD31 MoAb, JC/70A, detected molecules in the 120- to 140-kD molecular weight range on the monocytic CD34- CD33+ CD31+ cell line, U937; on the CD34+ CD31+ CD33+ CD19- multipotential/lymphomyeloid precursor cell lines, KG1 and KG1B; on the CD34+ CD31+ CD19+ CD10+ CD33- precursor pre-pre-B-cell line, MIK-ALL; and on a CD34(+)-enriched precursor cell population from normal human bone marrow. A single molecular weight species was generally observed with enriched membrane preparations, whereas two PECAM-1 molecules were present in whole-cell lysates of cell lines and the CD34+ bone marrow cell subset. Preliminary studies show that a proportion of the PECAM-1 molecules on the lymphomyeloid/multipotential progenitor cell line, KG1, and on the monocytic cell line, U937, binds to heparin-sepharose. A soluble form of PECAM-1 also binds heparin-sepharose. The high level of expression of PECAM-1 on CD34+ cells suggests that this glycoprotein may function as a heterotypic adhesion molecule, possibly mediating multipotential, myeloid, and early-B-lymphoid precursor cell interactions with stromal cells and extracellular matrix molecules via heparan sulfate proteoglycans. It may also act as a homotypic adhesion molecule by interacting with PECAM-1 on bone marrow stromal macrophage-like cells and endothelial cells or on endothelial cells during stem/progenitor cell migration. Thus, this molecule has the potential importance of directing both lineage commitment and trafficking of early hematopoietic progenitor cells.