Detection of antibody display phage without clearing of bacterial culture.
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Animals↗
Biomedical subjects
P D Kassner
Publications and source records attributed to P D Kassner.
Genetic selection of phage engineered for receptor-mediated gene transfer to mammalian cells.
Although phage display is a powerful way of selecting ligands against purified target proteins, it is less effective for selecting functional ligands for complex targets like living cells. Accordingly, phage display has had limited utility in the development of targeting agents for gene therapy vectors. By adapting a filamentous bacteriophage for gene delivery to mammalian cells, however, we show here that it is possible to screen phage libraries for functional ligands capable of delivering DNA to cells. For example, when targeted with epidermal growth factor (EGF), M13 bacteriophage were capable of delivering a green fluorescent protein (GFP) gene to EGF receptor bearing cells in a ligand-, time-, and phage concentration-dependent manner. The EGF-targeted phage transduced COS-1 cells in a highly specific manner as demonstrated by competition with excess free EGF or alternatively with anti-EGF receptor antibodies. We further demonstrate that EGF-phage can be selected, by their ability to transduce EGF receptor bearing cells from libraries of peptide display phage. When phage were incubated with COS-1 cells, EGF ligand-encoding sequences were recovered by PCR from FACsorted, GFP-positive cells and the EGF-displaying phage were enriched 1 million-fold by four rounds of selection. These data suggest the feasibility of applying molecular evolution to phage gene delivery to select novel cell-specific DNA-targeting ligands. The same approach could be used to select genetically altered phage that are specifically designed and evolved as gene therapy vectors.
Animals↗
Gene transfer to mammalian cells using genetically targeted filamentous bacteriophage.
We have genetically modified filamentous bacteriophage to deliver genes to mammalian cells. In previous studies we showed that noncovalently attached fibroblast growth factor (FGF2) can target bacteriophage to COS-1 cells, resulting in receptor-mediated transduction with a reporter gene. Thus, bacteriophage, which normally lack tropism for mammalian cells, can be adapted for mammalian cell gene transfer. To determine the potential of using phage-mediated gene transfer as a novel display phage screening strategy, we transfected COS-1 cells with phage that were engineered to display FGF2 on their surface coat as a fusion to the minor coat protein, pIII. Immunoblot and ELISA analysis confirmed the presence of FGF2 on the phage coat. Significant transduction was obtained in COS-1 cells with the targeted FGF2-phage compared with the nontargeted parent phage. Specificity was demonstrated by successful inhibition of transduction in the presence of excess free FGF2. Having demonstrated mammalian cell transduction by phage displaying a known gene targeting ligand, it is now feasible to apply phage-mediated transduction as a screen for discovering novel ligands.
Animals↗
Organizing effects of rapsyn on neuronal nicotinic acetylcholine receptors.
Targeting receptors to appropriate locations on the cell surface is a critical task for neurons. We have examined the possibility that rapsyn controls the distribution of nicotinic receptors on neurons as it does nicotinic receptors on muscle fibers. Cotransfection of QT6 cells with rapsyn and neuronal nicotinic receptor cDNA constructs produced receptor aggregates or clusters that codistributed in part with rapsyn protein. Though all nicotinic receptor subtypes tested were affected by rapsyn, receptors containing the alpha 7 gene product were among the most responsive. In addition, rapsyn caused a portion of the nicotinic receptors containing alpha 7 subunits to become resistant to solubilization with nonionic detergent and to display a marked increase in metabolic stability. The results are consistent with rapsyn linking the receptors to cytoskeletal elements and suggest that it may play an organizing role determining the fate and location of nicotinic receptors on neurons.
Animals↗
Detection of functional nicotinic receptors blocked by alpha-bungarotoxin on PC12 cells and dependence of their expression on post-translational events.
A major class of nicotinic receptors in the nervous system is one that binds alpha-bungarotoxin and contains the alpha7 gene product. PC12 cells, frequently used to study nicotinic receptors, express the alpha7 gene and have binding sites for the toxin, but previous attempts to elicit currents from the putative receptors have failed. Using whole-cell patch-clamp recording techniques and rapid application of agonist, we find a rapidly desensitizing acetylcholine-induced current in the cells that can be blocked by alpha-bungarotoxin. The current amplitude varies dramatically among three populations of PC12 cells but correlates well with the number of toxin-binding receptors. In contrast, the current shows no correlation with alpha7 transcript; cells with high levels of alpha7 mRNA can be negative for toxin binding and yet have other functional nicotinic receptors. Northern blot analysis and reverse transcription-PCR reveal no defects in alpha7 RNA from the negative cells, and immunoblot analysis demonstrates that they contain full-length alpha7 protein, although at reduced levels. Affinity purification of toxin-binding receptors from cells expressing them confirms that the receptors contain alpha7 protein. Transfection experiments demonstrate that PC12 cells lacking native toxin-binding receptors are deficient at producing receptors from alpha7 gene constructs, although the same cells can produce receptors from other transfected gene constructs. The results indicate that nicotinic receptors that bind alpha-bungarotoxin and contain alpha7 subunits require additional gene products to facilitate assembly and stabilization of the receptors. PC12 cells offer a model system for identifying those gene products.
Amino Acid Isomerases↗
Differences in the fate of neuronal acetylcholine receptor protein expressed in neurons and stably transfected cells.
Ligand-gated ion channels are structurally complex transmembrane proteins that all neurons must synthesize for rapid chemical synaptic transmission. The most abundant nicotinic acetylcholine receptor serving as a ligand-gated ion channel in the nervous system is a species that contains alpha7 subunits, binds alpha-bungarotoxin, and has a high relative permeability to calcium. The ability of neurons to make such receptors was compared with that of non-neuronal cells stably transfected with an alpha7 cDNA to determine whether neuron-specific machinery is likely to aid in their assembly or stabilization. Transfected cells expressed alpha7 protein and assembled it into a species that was indistinguishable in size and pharmacology from native receptors, but much of the alpha7 protein they synthesized was rapidly degraded without becoming receptor. Neurons were not only more efficient than the best transfectants at assembling the receptors but also produced a subpopulation of receptors on the cell surface that was relatively stable and resistant to solubilization. This subpopulation, which was absent from transfected cells, may be tethered to cytoskeletal elements in the neurons. The results support the contention that neurons contain components that facilitate the production and stabilization of ligand-gated ion channels.
Animals↗
The integrin VLA-4 supports tethering and rolling in flow on VCAM-1.
Selectins have previously been shown to tether a flowing leukocyte to a vessel wall and mediate rolling. Here, we report that an intergrin, VLA-4, can also support tethering and rolling. Blood T lymphocytes and alpha 4 integrin-transfected cells can tether in shear flow, and then roll, through binding of the intergrin VLA-4 to purified VCAM-1 on the wall of a flow chamber. VLA-4 transfectants showed similar tethering and rolling on TNF-stimulated endothelium. Tethering efficiency, rolling velocity, and resistance to detachment are related to VCAM-1 density. Tethering and rolling did not occur on ICAM-1, fibronectin, or fibronectin fragments, and tethering did not require integrin activation or the presence of an alpha 4 cytoplasmic domain. Arrest of rolling cells on VCAM-1 occurred spontaneously, and/or was triggered by integrin activating agents Mn2+, phorbol ester, and mAb TS2/16. These agents, and the alpha 4 cytoplasmic domain, promoted increased resistance to detachment. Together the results show that VLA-4 is a versatile integrin that can mediate tethering, rolling, and firm arrest on VCAM-1.
Cell Adhesion↗
Specialized functional properties of the integrin alpha 4 cytoplasmic domain.
For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils.
Animals↗
Minimum alpha chain cytoplasmic tail sequence needed to support integrin-mediated adhesion.
Previously, we found that deletion of the integrin alpha 4 and alpha 2 subunit cytoplasmic domains, just after the conserved GFFKR motif, causes a loss of adhesive activity mediated by VLA-4 or VLA-2, respectively (Kassner, P.D., and Hemler, M. E. (1993) J. Exp. Med. 178, 649-60; Kawaguchi, S., and Hemler, M. E. (1993) J. Biol. Chem. 268, 16279-12685). Here, we show for alpha 4 and alpha 2 chains (expressed in MIP101 and Chinese hamster ovary cells) that adding only 3-4 amino acids after the GFFKR motif restores substantial adhesive activity and that 5-7 amino acids confers maximal adhesive activity (to VCAM-1, CS1 peptide, or collagen, respectively). Point mutations within the most critical 5 alpha 4 residues had no effect on alpha 4 adhesive activity, nor did exchange of the alpha 4 tail with that of alpha 2. Thus, only a short and relatively nonspecific stretch of alpha chain cytoplasmic domain amino acids may be required to achieve maximal integrin adhesive activity. Also, comprehensive divalent cation titration assays revealed (i) that deletion of alpha chain cytoplasmic domains caused a marked decrease in the efficiency of divalent cation utilization during cell adhesion assays and (ii) that cytoplasmic domain deletion effects could be either suppressed or accentuated depending on the type and amount of divalent cation and the cellular environment utilized. Notably, integrin alpha chain tail deletions did not appear to alter the intrinsic ability to interact with ligand because deletion effects were minimal in the presence of metabolic energy inhibitors and were absent during cell-free ligand binding assays.
Amino Acid Sequence↗
Adhesion receptors in lymphocyte activation.
The past several years have seen significant progress in understanding the role of T lymphocyte coreceptors in adhesion and activation. New insights have been gained in several areas: the avidity regulation of beta 1 and beta 2 integrins and their role in signal transduction; the regulation of CD8 avidity; the role of Lck in CD4 coreceptor activity; and the novel role for CD2 adhesion in the T cell antigen response.
Animals↗
Interchangeable alpha chain cytoplasmic domains play a positive role in control of cell adhesion mediated by VLA-4, a beta 1 integrin.
Integrins can exist in a range of functional states, depending on the cell type and its state of activation. Although the mechanism that controls activity is unknown, it has been suggested that for some integrins, alpha chain cytoplasmic domains may exert either a negative effect or no effect on adhesion function. To address this issue for VLA-4 (an alpha 4 beta 1 heterodimer), we constructed an alpha 4 cytoplasmic deletion mutant and chimeric alpha chains composed of the extracellular domains of alpha 4 and the cytoplasmic domains of alpha 2, alpha 4, or alpha 5. Upon stable transfection of wild-type alpha 4, VLA-4 heterodimer was obtained that mediated (a) poor adhesion to CS1 peptide, fibronectin, or vascular cell adhesion molecule 1 (VCAM-1) (in K562 cells); (b) poor adhesion to CS1 peptide but moderate adhesion to VCAM-1 (in MIP101 cells); and (c) moderate adhesion to both CS1 peptide and VCAM-1 (in PMWK cells). Chimeric alpha 4 constructs and wild-type alpha 4 yielded similar results in these cell lines. In contrast, truncation of the alpha 4 cytoplasmic domain (after the conserved GFFKR motif) caused an almost complete loss of adhesive activity in all three cell lines. Thus, several interchangeable alpha chain cytoplasmic domains play a fundamentally positive role in determining the state of constitutive activity for VLA-4. The alpha chain cytoplasmic domain is also required for agonist-stimulated adhesion, since phorbol ester stimulated the cell adhesion mediated by wild-type and chimeric alpha chains, but not by the cytoplasmic deletion mutant. The inactivity of both wild-type VLA-4 (in K562 cells), and truncated VLA-4 (in all three cell lines) was overcome by the addition of a stimulatory anti-beta 1 monoclonal antibody. Thus, the alpha cytoplasmic domain-dependent cellular mechanism controlling both constitutive and agonist-stimulated VLA-4 activity could be bypassed by external manipulation of the integrin.
Cell Adhesion↗
Distinct cellular functions mediated by different VLA integrin alpha subunit cytoplasmic domains.
To characterize VLA alpha subunit cytoplasmic domain functions, unaltered alpha 2 cDNA (called X2C2) and two chimeric cDNAs (called X2C5 and X2C4) were constructed with extracellular alpha 2 domains and cytoplasmic alpha 2, alpha 5, and alpha 4 domains respectively. Upon transfection into rhabdomyosarcoma (RD) cells, each construct yielded comparable expression levels, immunoprecipitation profiles, and avidity for collagen and laminin. However, while RDX2C2 and RDX2C5 transfectants mediated collagen gel contraction, RDX2C4 and a mock transfectant (RDpF) did not. Conversely, only RDX2C4 cells (but not RDX2C2 or RDX2C5) showed enhanced cell migration on collagen and laminin compared with RDpF cells. This indicates markedly differing roles for integrin alpha subunit cytoplasmic domains in post-ligand binding events. Furthermore, stable exertion of physical force (collagen gel contraction) may involve fundamentally different cellular machinery than the transient adhesion occurring during cell migration. Finally, these findings provide insight into a functional flexibility perhaps resulting from multiple integrins binding to identical ligands.
Animals↗
Functional and structural analysis of VLA-4 integrin alpha 4 subunit cleavage.
The cell surface heterodimer VLA-4 (alpha 4 beta 1), a member of the integrin family of adhesion receptors, is involved in both cell-extracellular matrix and cell-cell adhesion. Unlike any other integrin alpha subunit, the intact (150 kDa) alpha 4 subunit of VLA-4 can sometimes be cleaved into two noncovalently associated fragments (80 and 70 kDa). Using biosynthetic and mixing experiments, we found that human alpha 4 cleavage is a regulated, compartmentalized event, occurring soon after maturation of the beta 1-associated alpha 4 subunit. Cleavage of alpha 4, which is increased following T cell activation, has been suggested to correlate with altered VLA-4 functions. To address directly the functional importance of alpha 4 cleavage, we have studied VLA-4-mediated adhesion functions in cells expressing intact alpha 4 in comparison with cells expressing cleaved alpha 4. For this purpose, we first sequenced the N terminus of the endogenously produced 70-kDa alpha 4 fragment and identified the alpha 4 cleavage site between Lys557-Arg558 and Ser559. To abolish cleavage, we converted Arg558 to Leu or Lys557 to Gln by site-directed mutagenesis of the alpha 4 cDNA and then transfected both mutant and wild type alpha 4 cDNAs into VLA-4-negative K562 cells. Whereas transfection with wild type alpha 4 cDNA yielded predominantly cleaved alpha 4 subunit, the Leu558-alpha 4 yielded only intact alpha 4 subunit, and Gln557-alpha 4 yielded mostly intact alpha 4 subunit. Transfectants with the intact or the cleaved alpha 4 were equally capable of engaging in VLA-4-dependent adhesion to vascular cell adhesion molecule-1 and to the Hep II fragment of fibronectin (40 kDa) and aggregated equally well in response to anti-alpha 4 antibodies. Thus, cleavage of the alpha 4 subunit in these transfectants did not alter any of the known VLA-4-mediated adhesion functions.
Amino Acid Sequence↗
Functional role of alpha 2/beta 1 and alpha 4/beta 1 integrins in leukocyte intercellular adhesion induced through the common beta 1 subunit.
Whereas all of the integrins in the VLA protein subfamily are involved in cell-extracellular matrix interactions, only VLA-4 (through the alpha 4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein beta 1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti-beta 1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common beta 1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti-beta 1 mAb. Induction of cell aggregation by anti-beta 1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to alpha 4-mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA alpha 4/beta 1 heterodimer, intercellular adhesion induced through the VLA-beta 1 chain could be selectively inhibited by other anti-beta 1 mAb as well as by anti-alpha 4 mAb. Interestingly, anti-beta 1 mAb which induced strong aggregation of VLA-alpha 2- or VLA-alpha 4-transfected K562 cells, had minimal effect on the alpha 2- alpha 4- alpha 5+ K562 cell line. Furthermore, the beta 1-mediated induction of cell aggregation on alpha 2-K562- and alpha 4-K562-transfected cells was blocked by preincubation with either anti-alpha 2 or anti-alpha 4 mAb, respectively, as well as by other anti-beta 1 mAb. Interestingly, parental K562 cells were able to interact with both alpha 2- and alpha 4-transfected K562 cells, thus suggesting that counter-receptors for both integrins (VLA-2 and VLA-4) might exist on these cells. Together these results provide strong evidence supporting the involvement of alpha 2/beta 1 and alpha 4/beta 1 heterodimers in intercellular interactions and underline the pivotal role of the common beta 1 chain of VLA proteins in the integrin-mediated induction of cell aggregation.
Animals↗
Analyses of VLA-4 structure and function.
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Amino Acid Sequence↗
Functional roles for integrin alpha subunit cytoplasmic domains.
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Amino Acid Sequence↗
Integrin alpha 2 beta 1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells.
The capacity of cells to organize and contract collagen fibrils is fundamental to processes as diverse as embryogenesis and wound healing. We analyzed different beta 1 integrins on diploid fibroblasts for their role in modifying the tertiary structure of collagen matrices. Using monoclonal antibodies that block the interaction of integrins with their ligands, evidence was obtained that alpha 2 beta 1 integrin is required for the contraction of a type I collagen matrix. Further supporting the role of alpha 2 beta 1, cell lines expressing minimal levels of this integrin uniformly failed to contract collagen matrices. In addition, transfection of a full-length alpha 2 cDNA into one such cell line led to enhanced cell surface expression of alpha 2 beta 1 and conferred the de novo capacity to contract collagen matrices.
Antibodies, Monoclonal↗