Immobilization of Bioactive Fibroblast Growth Factor-2 into Cubic Proteinous Microcrystals (Bombyx mori Cypovirus Polyhedra) That Are Insoluble in a Physiological Cellular Environment

Abstract

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.Hormones and cytokines in circulation represent a typical mode of cell-cell communication via diffusible extracellular signal molecules. Extracellular concentrations and affinities to cell-surface receptors are the major determinants of the potency of signaling proteins. However, with the exception of hematopoietic and/or blood cells, all single cells throughout the various organs receive their signaling cues from the extracellular matrix (ECM).3 The basal lamina underlying the epithelial cell sheets and the specialized ECM in the mesenchyme, such as cartilage and bone, play a crucial role in this regard during the regulation of cellular behavior (1). The ECM provides a supramolecular architecture for the disposition of specific accessory molecules that exist in low abundance, such as growth factors, which are released after proteolytic cleavage to effect a downstream alteration in the behavior of the responding cells. Conversely, cells that regulate remodeling (or degradation under certain conditions) of the ECM do so via the production and secretion of proteases and protease inhibitors (2). Thus, each individual cell sends and receives spatiotemporally restricted signals from its extracellular environment. However, only limited tools are currently available that can generate signaling cues in a spatially restricted manner, at the cellular or subcellular level in vitro, to enable further studies of these events. Previously, we developed a novel protein expression system that enables us to immobilize foreign proteins on insect virus occlusion bodies of protein crystals, termed polyhedra (3, 4). Polyhedra are the main vectors of virus particles from insects and are also the main agents that facilitate the survival of the virus, as they stabilize the virions allowing them to remain viable for very long periods in the environment (5, 6). As polyhedra are quite stable and are virtually insoluble at physio...

Fibroblast Growth Factor-2 Is an Important Factor that Maintains Cellular Immaturity and Contributes to Aggressiveness of Osteosarcoma

“…Collectively, Fgf2 suppressed the osteogenic process in AX cells at the mRNA level and the mechanism might be dependent on the hyperactivation of Erk1/2. To further evaluate the earlier findings in vivo, AX cells were coinjected with Fgf2 or Lif polyhedra which were the cubic protein microcrystals continuously releasing each factor for more than 1 month (13,14,15). Fgf2 and Lif were successfully enriched in the surrounding areas of each polyhedra where Erk also appeared to be hyperphosphorylated ( Supplementary Fig.…”

“…To overexpress Fgf2 and Lif in tumors in vivo, 2 million AX cells were mixed with Fgf2 polyhedra (125 ng of Fgf2 per an injection; refs. 13,14) or Lif polyhedra (25 ng of Lif per an injection; ref. 15) suspended in PBS containing 50 ng/mL Fgf2 or 1,000 U/mL Lif, respectively and subcutaneously injected.…”

Biological Basis of Bone Formation, Remodeling, and Repair—Part I: Biochemical Signaling Molecules

“…Indeed, this sequestration can be essential to their function, as with fibroblast growth factor-2 (FGF-2). 22 Consequently, their activity in vivo reflects a delicate balance between free and bound forms.) The ECM can be subdivided into fibrous proteins, nonfibrous molecules, and enzymes.…”

Electron Tomography Reveals Polyhedrin Binding and Existence of both Empty and Full Cytoplasmic Polyhedrosis Virus Particles inside Infectious Polyhedra

“…In addition to making up the turret/B-spike, VP4, and specifically its N-terminal segment (residues 1 to 79), was shown to "immobilize" foreign proteins onto the polyhedron (8,9), suggesting it may bind polyhedrin within polyhedra. A follow-up biochemical study further suggested the existence of a "polyhedrin recognition signal" within amino acid residues 42 to 93 that is sufficient to immobilize foreign proteins tagged by this fragment (7,13). We took note of the fact that some proteins failed to be immobilized (9), suggesting that there could be alternative interpretations for the immobilization.…”