Ligands For Cell Surface Receptors Research Articles

Interferons and viral infections

Interferons represent a family of cytokines, which is of central importance in the innate immune response to virus infections. All interferons act as secreted ligands of specific cell surface receptors, eliciting the transcription of hundreds of interferon-stimulated genes whose protein products have antiviral activity, as well as antimicrobial, antiproliferative/antitumor, and immunomodulatory effects. Expression of type I and III interferons is induced in virtually all cell types upon recognition of viral molecular patterns, especially nucleic acids, by cytoplasmic and endosomal receptors, whereas type II interferon is induced by cytokines such as IL-12, and its expression is restricted to immune cells such as T cells and NK cells. The effectiveness of the interferon system in counteracting viral infections is reflected by the multitude of inhibitors of interferon induction or interferon action that are encoded by many viruses, preventing their eradication and resulting in the continued coexistence of viruses and vertebrates. The unique biological functions of interferons have led to their therapeutic use in the treatment of diseases such as hepatitis, multiple sclerosis, and certain leukemias.

A cell surface interaction network of neural leucine-rich repeat receptors.

A network of Zebrafish extracellular neuroreceptor interactions are revealed using AVEXIS, a highly stringent interaction assay.

Deactivation of Sphingosine Kinase 1 by Protein Phosphatase 2A

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.

Introducing N-glycans into natural products through a chemoenzymatic approach

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-β- N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment. For those natural products that already carry terminal Glc or GlcNAc residues, direct enzymatic transglycosylation using sugar oxazoline as the donor substrate was achievable to introduce an N-glycan. It was also demonstrated that simultaneous double glycosylation could be fulfilled when the natural product contains two Glc residues. This chemoenzymatic method is concise, site-specific, and highly convergent. Because N-glycans of glycoprotein origin can serve as ligands for diverse lectins and cell-surface receptors, introduction of a defined N-glycan into biologically significant natural products may bestow novel properties onto these natural products for drug discovery and development.

Optimizing Delivery of Multivalent Targeting Constructs for Detection of Secondary Tumors

Targeting drugs or imaging molecules to specific cells by conjugating them to antibodies or ligands for cell surface receptors may allow earlier detection of pathology, better localization for intervention, and fewer side effects. Delivery of these molecules to the target is complicated by construct size, which cannot cross typical endothelial barriers such as the vascular wall, and lack of a priori knowledge of the location of secondary tumor sites to which the construct is targeted. Here we develop mathematical models for diffusive and convection-enhanced delivery of a trivalent construct. Results show that delivery of the construct to the tissue does not yield acceptable contrast or specificity; therefore, unbound construct must be removed from the area of interest by allowing diffusion out of the area or a follow-up injection of fluid containing no construct (e.g., saline). The need for this additional step requires weeks to months for diffusive delivery to yield acceptable contrast, but convection-enhanced delivery may be able to achieve acceptable contrast within several days. Thus, convection-enhanced delivery of multivalent constructs may provide a mechanism to locate secondary tumor sites without prior knowledge of their location which would greatly enhance the ability to detect and treat cancer.

A non-receptor-mediated mechanism for internalization of molecular chaperones

The evolving realization that stress proteins, which have for many years been considered to be exclusively intracellular molecules under normal conditions, can be released from viable cells via a number of potential routes/pathways has prompted interest into their extracellular biology and intercellular signaling properties. That the stress proteins Hsp60, Hsp70 and gp96 can elicit both pro- and anti-inflammatory effects suggests that these molecules play a key role in the maintenance of immunological homeostasis, and a better understanding of the immunobiology of extracellular stress proteins might reveal new and more effective approaches for controlling and managing infectious disease, inflammatory disease and cancer. A number of cell surface receptors for stress proteins have been identified, and the intracellular consequences of these cell surface receptor–ligand interactions have been characterized. To date, studies into the intercellular signaling properties of stress proteins and their interactions with antigen presenting cells have focused on specific receptor-mediated uptake, and have not considered the fact that such cells can also take up proteins via non-specific endocytosis/pinocytosis. Herein we present a methodological approach for assessing receptor-mediated and non-receptor-mediated uptake of gp96 by rat bone marrow-derived dendritic cells.

Growth Inhibition of Human Multiple Myeloma Cells by a CD40 Ligand (CD40L, CD154) Transgene - Oncolytic Virus Construct.

Human multiple myeloma (MM) remains incurable despite recent advances in induction therapy. To explore biotherapeutic approaches that may potentially enhance the efficacy of conventional treatment, we examined the growth regulatory properties of CD40L (CD154), the natural ligand for the myeloma cell surface receptor, CD40. Based on our previous findings that the recombinant CD40L protein effectively inhibited human MM cell growth, a conditional-replicative adenoviral construct, AdEHCD40L was used for targeted delivery of the CD40L transgene. AdEHCD40L incorporates tumor/tissue specific promoters that limit viral and transgene expression to HIF (hypoxia inducing factor)-1α overexpressing cells, which are prevalent in the human bone marrow compartment. Conditional expression of the early adenoviral E1A gene and the CD154 transgene was validated in the IL-6 independent MM line RPMI 8226 (62% and 66%, respectively), and the IL-6 dependent cell line Kas-6/1 (32.68% and 30%, respectively). Further, treatment with AdEHCD40L at a multiplicity of infection (MOI) of 1 resulted in pronounced growth inhibition for both cell lines (95.5±2.1% and 80.5±9.8%, mean±SD, respectively). AdEHCD40L treatment was more effective than the parental construct without the CD154 transgene (AdEHNULL) in both cell lines (p=0.04). Both AdEHNULL and AdEHCD40L were minimally cytotoxic to normal peripheral blood mononuclear cells (0% at 48hrs) and normal fibroblast cells IMR-90 (2.8±0.3%). The in vivo antitumor activity of AdEHCD40L was examined with a subcutaneous RPMI 8226 heterotransplant model in SCID mice. Intratumoral injection of AdEHCD40L (5x107 pfu, x 5) reduced xenograft growth by 53% at day 29 (4.8±0.9 mm, vs. 10.5±1.2 mm in mock-treated animals; p=0.002), and was more effective than AdEHNULL (7.6±1.1 mm; p=0.03). Adenoviral hexon and CD40L expression was detectable at 29 days post-viral treatment, based on immunohistochemical analysis. Hence intratumoral treatment with AdEHCD40L likely involved oncolytic viral replication. To further characterize cellular events that accompany MM cell growth inhibition, apoptotic activity was measured by annexin V and propidium iodide incorporation. A marked elevation of the annexin V+ subset (21.3±6.5%, vs. 7.7±1.9% in untreated culture; p=0.007) was accompanied by decreased cell viability (52.8±1%, vs. 76.1±11% in untreated cultures; p = 0.04) following AdEHCD40L treatment. Cell cycle distribution analysis demonstrated a corresponding increase in the subG0/G1 compartment (AdEHCD40L, 23.9±3.6%; vs. untreated, 9.1±2.3%) that was consistent with elevated apoptosis. Further, AdEHCD40L increased S phase accumulation by 72 hrs (68.0±2.4%, vs. 54.0±5.5% in AdEHNULL and 48.9±4.6% in untreated culture; p<0.003). Currently, gene expression array analyses are underway to define molecular events that are pertubated by CD154 transgene activity and by viral oncolysis. These findings will further elucidate the mechanism of action of the CD154+oncolytic viral approach for experimental gene therapy of human MM.

Adenovirus Fiber Shaft Contains a Trimerization Element That Supports Peptide Fusion for Targeted Gene Delivery

Adenoviral (Ad) vectors have been widely used in human gene therapy clinical trials. However, their application has frequently been restricted by the unfavorable expression of cell surface receptors critical for Ad infection. Infections by Ad2 and Ad5 are largely regulated by the elongated fiber protein that mediates its attachment to a cell surface receptor, coxsackie and adenovirus receptor (CAR). The fiber protein is a homotrimer consisting of an N-terminal tail, a long shaft, and a C-terminal knob region that is responsible for high-affinity receptor binding and Ad tropism. Consequently, the modification of the knob region, including peptide insertion and C-terminal fusion of ligands for cell surface receptors, has become a major research focus for targeting gene delivery. Such manipulation tends to disrupt fiber assembly since the knob region contains a stabilization element for fiber trimerization. We report here the identification of a novel trimerization element in the Ad fiber shaft. We demonstrate that fiber fragments containing the N-terminal tail and shaft repeats formed stable trimers that assembled onto Ad virions independently of the knob region. This fiber shaft trimerization element (FSTE) exhibited a capacity to support peptide fusion. We showed that Ad, modified with a chimeric protein by direct fusion of the FSTE with a growth factor ligand or a single-chain antibody, delivered a reporter gene selectively. Together, these results indicate that the shaft region of Ad fiber protein contains a trimerization element that allows ligand fusion, which potentially broadens the basis for Ad vector development.

Hyaluronidase Expression Induces Prostate Tumor Metastasis in an Orthotopic Mouse Model

Molecular mechanisms of prostate cancer progression are frequently studied in mice by orthotopic injection of aggressive cell lines, which yield primary tumors that spontaneously metastasize to lymph nodes. In this report, we characterized the human prostate carcinoma cell line 22Rv1 in an orthotopic system and evaluated the functional relevance of the hyaluronidase Hyal1, a correlate of invasive human prostate cancer, to progression in this model. To provide real-time insights into these processes, we first validated use of an epidermal growth factor-conjugated fluorophore to illuminate orthotopic prostate tumors and their metastases in whole animal imaging. Animals receiving intraprostatic injections were tracked throughout a 6-week period. Tumor sizes were correlated 92% with total fluorescence intensities of 22 prostate tumors. In contrast to the highly tumorigenic and metastatic PC3M-LN4 cells, the 22Rv1 line was orthotopically tumorigenic but not metastatic, despite larger tumor sizes. Lymph node metastasis was successfully imaged in animals with PC3M-LN4 tumors on endpoint dissection. Stable transfection of 22Rv1 cells with Hyal1 did not alter growth kinetics of primary orthotopic tumors, but all animals implanted with Hyal1 transfectants exhibited tumor-positive para-aortic lymph nodes. Hyal1 is implicated as an inducer of prostate cancer metastatic progression.

Adventures in Drug Discovery: Potent Agents Based on Ligands for Cell-Surface Receptors

How does one go about discovering new drugs? This question is addressed by descriptions of drug discovery research in three project areas that pertain to antagonist ligands for cell-surface receptors. The molecular targets of interest are protease-activated receptor-1 (PAR-1), vasopressin receptors (V1a and V2 subtypes), and the fibrinogen receptor (GPIIb/IIIa). I present different approaches to the identification of high-affinity ligands for these receptors, en route to drug candidates. The PAR-1 project resulted in a pharmacological tool compound that facilitated in vivo proof-of-principle studies, whereas the vasopressin and fibrinogen receptor projects resulted in several preclinical development compounds, three of which advanced into human clinical trials.

Peptide ligands targeting integrin α3β1 in non-small cell lung cancer

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.

External ATP Required for Viability

In animal cells, adenosine triphosphate (ATP) serves not only as an energy source but also as an extracellular signaling molecule, a ligand for purinergic cell surface receptors. A role for extracellular ATP in plants is also beginning to emerge, and Chivasa et al. report that extracellular ATP is required for plant cell viability and that depletion of ATP may be one mechanism by which the process of pathogen-induced hypersensitive cell death is triggered (see commentary by Eckardt). Arabidopsis cells in culture released ATP into the medium. Cell-impermeant enzymes that consumed or metabolized ATP, such as hexokinase or apyrase, depleted extracellular ATP and caused cell death in cultures of Arabidopsis and maize. The nonhydrolyzable ATP analog βγ-methyleneadenosine 5′-triphosphate (AMP-PCP) also decreased cell viability. Application of apyrase, hexokinase, or AMP-PCP also caused localized cell death in leaves of Arabidopsis , tobacco, and bean plants. The entire Arabidopsis plant was susceptible to death if grown with hexokinase or AMP-PCP in the medium (hydroponically grown plants) or nutrient agar. Fumonisin B1 (FB1) is an elicitor produced by the maize pathogen Fusarium moniliforme , which causes programmed cell death and activation of the plant defense response. Arabidopsis cell cultures grown in the presence of FB1 had decreased extracellular ATP, and the decline preceded loss of membrane integrity. Addition of ATP rescued the cells from FB1-induced death, as did addition of other nucleoside triphosphates, which prevented ATP from decreasing by competing for ATP-degrading enzymes. Exogenous ATP also rescued Arabidopsis plants grown in the presence of FB1 from death, but did not rescue the plants from the growth retardation effects of FB1, suggesting that ATP selectively protects against FB1-mediated cell death. Mass spectrometry revealed that the abundance of several (34) intracellular proteins, including several implicated in the stress response, was altered by depletion of extracellular ATP. Thus, extracellular ATP appears to be one of the signals that inhibits what is known as the "default death pathway" in plants and connects cell viability to the pathogen elicitor-triggered cell death response. The mechanism by which ATP exerts this effect--for example, through phosphorylation of an extracellular protein, through serving as a cofactor in the formation of an extracellular complex, or through serving as a ligand for an extracellular receptor--remains to be determined. S. Chivasa, B. K. Ndimba, W. J. Simon, K. Lindsey, A. R. Slabas, Extracellular ATP functions as an endogenous external metabolite regulating plant cell viability. Plant Cell 17 , 3019-3034 (2005). [Abstract] [Full Text] N. A. Eckardt, Ins and outs of programmed cell death and toxin action. Plant Cell 17 , 2849-2851 (2005). [Full Text]

Selective Small Molecule Peptidomimetic Ligands of TrkC and TrkA Receptors Afford Discrete or Complete Neurotrophic Activities

We designed a minilibrary of 55 small molecule peptidomimetics based on beta-turns of the neurotrophin growth factor polypeptides neurotrophin-3 (NT-3) and nerve growth factor (NGF). Direct binding, binding competition, and biological screens identified agonistic ligands of the ectodomain of the neurotrophin receptors TrkC and TrkA. Agonism is intrinsic to the peptidomimetic ligand (in the absence of neurotrophins), and/or can also be detected as potentiation of neurotrophin action. Remarkably, some peptidomimetics afford both neurotrophic activities of cell survival and neuronal differentiation, while others afford discrete signals leading to either survival or differentiation. The high rate of hits identified suggests that focused minilibraries may be desirable for developing bioactive ligands of cell surface receptors. Small, selective, proteolytically stable ligands with defined biological activity may have therapeutic potential.

A photochemical snapshot of CD22 binding

Identification of endogenous glycan-binding ligands for cell-surface receptors has been difficult. Incorporation of a photoactive sialic acid analog into B-cell surface glycoproteins suggests that CD22 molecules may cluster by binding carbohydrate antigens on neighboring CD22 molecules.

Method for Selecting and Enriching Cells Expressing Low Affinity Ligands for Cell Surface Receptors

The affinity of protein-protein interaction in biological systems ranges from extremely strong, such as that between avidin and biotin (Ka = approximately 10 15 M-1) to the very weak, such as E-selectin and E-selectin ligand-1 (Ka = approximately 7.4 × 104 M-1S-1) (1). It is known that the interactions mediated by the surface receptors of immune cells are often of low-affinity and transient in nature (2). Despite weak affinities, cell surface interactions among immune cells have been shown to play an important role in many functions such as surveillance, immunosynapse formation, and activation (2). It is therefore desirable to devise a way to select and enrich cells expressing low-affinity ligands for specific immune cell surface receptors. Here we use the well-studied interaction between CD97 and CD55 as a model system to describe a simple method for this purpose. Both CD97 and CD55 are cell surface proteins expressed predominantly on leukocytes (3–5). The binding of CD97 to CD55 has been demonstrated to be both of low affinity (86 μM) and transient (off-rate; 0.6/s) (6). We and others have previously shown that the interaction is mediated exclusively by the epidermal growth factor (EGF)-like domains of CD97 and the short consensus repeats (SCR) of CD55 in a Ca2+-dependent fashion (6,7). Furthermore, a CD97-like molecule, EMR2, has been shown not to bind CD55 even though it contains highly homologous EGF-like domains (97.5% identity), therefore providing an ideal negative control (8). Our method is based on (i) the efficient isolation and coupling of Fc-fusion proteins to protein AGconjugated paramagnetic beads (Protein A/G Dynabeads®; Dynal A.S., Oslo, Norway) (Figure 1A) and (ii) the simple capture and separation of ligand-positive cells from the Fcfusion protein-coated paramagnetic beads (Figure 1B). The coupling of the Fc-fusion proteins to the protein AG-conjugated paramagnetic beads generates a multivalent protein probe, thereby increasing binding efficiency (Figure 1, A and B). As the major CD55-binding CD97 isoform contains three EGF-domains, we fused the three EGF-domains immediately upstream of the mouse Fc region (Figure 1A). CD97(EGF-1,2,5)-mFc and EMR2(EGF-1,2,5)-mFc fusion proteins were produced as described previously in transiently transfected HEK293T cells (Figure 1C). Protein AG-conjugated paramagnetic beads were washed three times in binding buffer [0.5% bovine serum albumin (BSA) in Hank’s Balanced Salt solution (HBSS)], reconstituted in the binding buffer to the original concentration (10 mg/mL), and incubated at 4°C for 30 min with or without 2 μg mFc-fusion protein/10 μL beads/reaction. Following the removal of unbound proteins by magnetic separation, the fusion protein-bead complexes were then resuspended in 20 μL binding buffer/reaction. K562 cells (CD55+) were washed, adjusted to a cell density of 3 × 106 cells/mL, and incubated at 4°C with the protein-bead

Large-scale identification of proteins expressed in mouse embryonic stem cells

A protein subset expressed in the mouse embryonic stem (ES) cell line, E14-1, was characterized by mass spectrometry-based protein identification technology and data analysis. In total, 1790 proteins including 365 potential nuclear and 260 membrane proteins were identified from tryptic digests of total cell lysates. The subset contained a variety of proteins in terms of physicochemical characteristics, subcellular localization, and biological function as defined by Gene Ontology annotation groups. In addition to many housekeeping proteins found in common with other cell types, the subset contained a group of regulatory proteins that may determine unique ES cell functions. We identified 39 transcription factors including Oct-3/4, Sox-2, and undifferentiated embryonic cell transcription factor I, which are characteristic of ES cells, 88 plasma membrane proteins including cell surface markers such as CD9 and CD81, 44 potential proteinaceous ligands for cell surface receptors including growth factors, cytokines, and hormones, and 100 cell signaling molecules. The subset also contained the products of 60 ES-specific and 41 stemness genes defined previously by the DNA microarray analysis of Ramalho-Santos et al. (Ramalho-Santos et al., Science 2002, 298, 597-600), as well as a number of components characteristic of differentiated cell types such as hematopoietic and neural cells. We also identified potential post-translational modifications in a number of ES cell proteins including five Lys acetylation sites and a single phosphorylation site. To our knowledge, this study provides the largest proteomic dataset characterized to date for a single mammalian cell species, and serves as a basic catalogue of a major proteomic subset that is expressed in mouse ES cells.

The opsonising activity of a pentraxin-like protein isolated from snapper ( Pagrus auratus, Sparidae) serum

In mammals the pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) are important components of the immune response. Although pentraxins have been isolated from a number of fish species few studies detail their functional immunological role. In this paper we report the establishment of a flow cytometry based assay to measure the phagocytic activity of isolated snapper head kidney leukocytes (HKLs). This assay was then used to examine the ability of a pentraxin-like protein isolated from the serum of snapper ( P. auratus) (Sn-PLP) to act as an opsonin. Incubation of snapper head kidney leukocytes (HKL) with FITC-labelled beads resulted in uptake of these particles by approximately 35% of HKLs. Incubation of beads with Sn-PLP enhanced phagocytosis by snapper HKLs in a dose-dependant manner. Enhanced phagocytosis could be inhibited by addition of a rabbit anti-Sn-PLP antibody suggesting that Sn-PLP may act as a ligand for a HKL cell surface receptor. This study provides further evidence toward a functional role for pentraxins in the host defence repertoire of fish.

Transferrin-modified liposomes equipped with a pH-sensitive fusogenic peptide: an artificial viral-like delivery system.

Liposomes are one of the most promising systems for selective cellular targeting via introduction of specific ligands for cell-surface receptors. After being taken up by the cells, these liposomes usually follow intracellular pathways of receptor-mediated endocytosis. Control of intracellular trafficking is required for optimized drug delivery. In this study, we elucidated the intracellular fate of transferrin-modified liposomes and succeeded in altering it by introducing the pH-sensitive fusogenic peptide, GALA (WEAALAEALAEALAEHLAEALAEALEALAA). Transferrins that are chemically attached to a liposomal surface (Tf-L) were internalized via receptor-mediated endocytosis more slowly than unmodified transferrins. In contrast to the recyclable nature of transferrin, liposome-attached transferrins together with encapsulated rhodamines were retained in vesicular compartments. When GALA was introduced into liposomal membranes using a cholesteryl moiety for anchoring (Chol-GALA), rhodamines were efficiently released and diffused into the cytosol. The addition of GALA to the Tf-L-containing medium or the encapsulation of GALA in Tf-L did not induce similar effects. These results clearly indicate that GALA must be present on the surface of liposomes to exert its function. In vitro energy transfer and dynamic light scattering experiments suggested that the endosomal escape of the encapsulates in Tf-L equipped with Chol-GALA can be attributed to pH-dependent membrane fusion. With GALA present on the surface, intracellular trafficking of liposomes after receptor-mediated endocytosis could be successfully controlled.

Biologically Active Decorin Is a Monomer in Solution

It has been reported that decorin and its protein core can have molecular masses nearly double the size of those previously published, suggesting a dimeric structure. In this study we tested whether biologically active decorin and its glycoprotein core would form dimers in solution. We used homo- and hetero-bifunctional chemical cross-linking reagents, BS3 and sulfo-SMPB, respectively, as well as glutaraldehyde and found no preferential dimer formation, whether chemical cross-linking was performed in the presence or absence of live cells. Under the same experimental conditions, we easily detected dimers of epidermal growth factor receptor and basic fibroblast growth factor, two glycoproteins known to dimerize. Only at very high cross-linker to decorin molar ratios (2000:1) were trimers and multimers observed, but performing the chemical cross-linking in the presence of a reducing agent abolished these. The elution of decorin protein core in Superose 6 gel chromatography gave masses compatible with monomeric proteins, both before and after denaturation with 2.5 M guanidine HCl. Matrix-assisted laser desorption ionization gave a mass of 44,077 Da for decorin protein core, without any evidence of dimers or oligomers. Extensive oligomerization of the decorin protein core was observed only after dialysis against water and freeze-drying. These oligomers were considered artifacts because they were independent of chemical cross-linking and were resistant to heat denaturation and disulfide-bond reduction. Oligomeric preparations showed markedly reduced biological activity in both phosphorylation and collagen fibrillogenesis assays. Thus, biologically active decorin is a monomer in solution and, as such, is a monovalent ligand for various extracellular matrix proteins, growth factors, and cell surface receptors.

Formation of polyomavirus-like particles with different VP1 molecules that bind the urokinase plasminogen activator receptor.

Icosahedral virus-like particles formed by the self-assembly of polyomavirus capsid proteins (Py-VLPs) can serve as useful nanostructures for delivering nucleic acids, proteins, and pharmaceuticals into animal cells and tissues. Four predominant surface-exposed loops in the VP1 structure offer potential sites to display sequences that might contribute new targeting specificities. Introduction into each of these loops of sequences derived from the amino-terminal fragment of urokinase plasminogen activator (uPA) or a related phage display peptide reduced the solubility of VP1 molecules when expressed in insect cells, and insertions into the EF loop reduced VP1 solubility least. Coexpression in insect cells of the uPA-VP1 molecules and VP1 containing a FLAG epitope in the HI loop permitted the formation of heterotypic Py-VLPs containing uPA-VP1 and FLAG-VP1. These heterotypic VLPs bound to uPAR on the surfaces of animal cells. Heterotypic Py-VLPs containing ligands for multiple cell surface receptors should be useful for targeting specific cells and tissues.