How can glycoproteins act as receptors

Plant cells Figure 1b and fungal cells have a cell wall, which provides support and determines cell shape, while protists are diverse in this respect; some, such as amoebae, do not have a rigid cell wall, but many do.

All cells, however, have a cell membrane, which acts as a barrier and an interface with the external environment. The cell membrane is composed predominantly of phospholipids, arranged in a bilayer. A schematic diagram illustrating the main features of a typical animal cell membrane is shown in Figure 7.

Within the bilayer are embedded a variety of proteins and glycoproteins proteins that have sugars attached. These proteins, many of which span the membrane, play crucial roles in the interactions of the cell with its environment. Some membrane proteins act as transporters or channels that allow selective movement of ions, nutrients or other molecules into the cell.

Others, often glycoproteins, act as receptors, which respond to specific molecular changes in the extracellular environment and transduce information about the environment to the inside of the cell, thereby allowing appropriate responses to be initiated. Yet other glycoproteins act as recognition molecules, and can promote adhesion between adjacent cells in a tissue.

The cell membrane is also linked to proteins on its cytoplasmic surface intracellular proteins. These include components of the cytoskeleton, which have a structural role, maintaining the shape of the cell. Making the decision to study can be a big step, which is why you'll want a trusted University.

Take a look at all Open University courses. If you are new to University-level study, we offer two introductory routes to our qualifications. You could either choose to start with an Access module , or a module which allows you to count your previous learning towards an Open University qualification. Cells were stained with PE-coupled CD antibody. One representative out of four experiments is shown. See S10 Fig for quantitative data. B Surface expression of GluA4 blue line and GluA4short red line on stably expressing HT cells compared to the parental cell line filled curve as determined by flow cytometry.

Cells were stained with PE-coupled myc-tag antibody. Here we describe successful engineering of the NiV glycoproteins for LV pseudotyping and receptor targeting, which allowed us to rapidly generate a large series of glycoprotein variants attaching to a variety of cell surface proteins and assessing cell entry.

Our data are thus in line with those of Witting et al for G protein, and with those of Palomares et al. For both, F and G, the enhanced titers correlated well to an enhanced incorporation into LV particles, suggesting steric hindrance as likely explanation for the need for cytoplasmic tail truncations.

For identifying the most effective mutations, we relied on the G protein structure and previously identified contact residues [ 22 , 23 , 25 , 26 ]. Yet, this turned out to be challenging, since the picomolar affinity of G for ephrin-B2 is among the strongest viral envelope-receptor interactions known [ 40 , 41 ]. However, combining both double mutations ultimately diminished binding to a level below detection. Importantly, transduction via the targeted EpCAM receptor was unimpaired by these mutations.

Since ephrin-B2 is widely expressed in the organism, including microvascular endothelial cells [ 42 ], having achieved complete abrogation of LV particle attachment to ephrin-B2 is an important step towards efficient in vivo gene delivery with receptor-targeted LVs. NiV and MV enter cells by pH-independent membrane fusion at the cell membrane. EpCAM is known to be rapidly internalized upon antibody binding [ 21 , 43 ]. Bafilomycin A1 is a selective inhibitor of the V-ATPase preventing the influx of protons into endosomes [ 44 , 45 ].

Although being more unspecific, chloroquine also neutralizes the low pH in endosomes. It is well conceivable that in both settings more particles can escape the endosomes by membrane fusion and then contribute to the observed enhanced gene delivery rates. Also blocking proteolytic degradation after potential endocytosis did not restore gene transfer activity to similar levels as that of the MV-based vector particles. A Three-dimensional structures of the targeted receptors and the positions of their binding sites relative to the cell membrane.

B Molecular model for the distance effect and its implication for NiV-mediated membrane fusion. In absence of receptor binding, F is in its prefusion state with the fusion peptide light blue being covered within the globular head left.

Upon attachment of G to its cell surface receptor, conformational changes are induced resulting in the projection of the fusion peptide followed by its insertion into the cell membrane top right. If the attached binding site on the receptor is too far away from the cell membrane, the fusion peptide cannot insert and cell entry will not proceed bottom right. Model adapted from [ 2 ] and [ 50 ]. Although the 3D structure of the stalk is not available, it is assumed to be highly flexible, thus also allowing a close proximity of vector particles having attached to the Ig-like domain [ 49 ].

Taken together, we thus have experimental evidence from three different receptors that changing the distance of the attachment site relative to the plasma membrane makes a huge difference in particle entry. Receptor attachment in distances clearly beyond this results in a substantially reduced gene delivery efficiency, most likely due to inefficient or absent membrane fusion.

How can we imagine that the distance between attachment site and cellular membrane makes such a huge difference for pH-independent membrane fusion mediated by the NiV glycoproteins? Thus upon cell attachment, a rigid scaffold will be formed between viral and cellular membranes by numerous glycoprotein-receptor contacts. These trigger conformational changes in G and F, which then projects the fusion peptide on top of a long coiled-coil structure, the heptad repeat A HRA , towards the cell membrane [ 2 ].

Although the structure of the prehairpin intermediate has so far only been modeled for parainfluenza virus 5 PIV5 , we can assume a very similar distance for the NiV F protein, since structure and size of HRA and HRB adjacent to the transmembrane domain are well conserved among paramyxoviruses [ 51 , 52 ] and the recently crystallized prefusion form of NiV F exhibits an overall similar size as that of PIV5 [ 53 ].

Any distance beyond that would not allow insertion of the fusion peptide into the cell membrane. While most of the paramyxoviruses use sialic acid as receptor and can thus choose between many attachment sites exposed at various distances from the cell membrane, Henipa- and morbilliviruses using protein receptors must have adapted to receptors bringing them so close to the cell membrane that the distance between both viral and cellular membrane can be covered by their F protein.

Supporting this model, a study analyzing a panel of chimeric CDCD4 proteins to function as MV receptors demonstrated that putting the MV binding domains of CD46 on top of the complete CD4 molecule four extracellular Ig domains strongly reduced membrane fusion [ 54 ].

Thus, LV particles pseudotyped with MV glycoproteins bind to cells via very few or even single receptor contacts, which leaves them more flexibility to take a position within an optimal distance to the cell surface for membrane fusion.

This may well help MV-LVs to better compensate when being bound to a membrane distal domain of a receptor. For NiV-LVs, in contrast, this may not be as easily possible since they form many receptor contacts resulting in a much more rigid complex between virus particle and target cell. Moreover, the henipavirus G proteins are unique among all paramyxoviruses, including MV, in forming covalently linked tetramers dimers-of-dimers [ 56 ]. This could further contribute to a more rigid receptor-attachment protein complex for NiV than for MV, which in turn results in higher sensitivity towards membrane-distal receptor attachment.

In summary, the data presented in this manuscript imply that for the engineering of cell-type-specific LVs, binding domains should be used bringing the particles within a close distance to the cell membrane. Titers of these vectors are substantially enhanced compared to vectors pseudotyped with engineered MV glycoproteins. The reasons for this could be allocated to an increased number of particles released from packaging cells which is most likely due to the intrinsic budding capability of the NiV glycoproteins [ 57 ].

Second, the particles are more active in delivering the packaged gene which is likely the consequence of the substantially higher glycoprotein density of NiV-LV particles compared to MV-LVs. Since NiV-LVs can be produced at titers exceeding 10 6 t. Each mutation was generated by amplification of two fragments carrying the designated mutation with homologous regions at the mutation site. These fragments were fused and amplified by a flanking primer pair. Following transduction, cells were cultivated in the same medium without OKT3 antibody and CD28 antibody.

Twenty-four hours before transfection, 2. For initial experiments Fig 1B and 1D 1. Following optimization of G to F ratios, 0. The amounts of packaging plasmid and transfer vector remained unchanged. G kindly provided by Didier Trono, Lausanne, Switzerland , At day two post transfection, cell supernatants containing the vector particles were passed through a 0. The pellet was resuspended in phosphate-buffered saline PBS. For titration, cells were transduced with at least four serial dilutions of vector particles.

After 72 h, the percentage of GFP-positive cells was determined by flow cytometry and the transducing units per milliliter t. Percentages of GFP-positive cells were determined by flow cytometry at the indicated days post transduction.

For surface expression experiments of Nipah virus G constructs, HEKT cells were transfected with the corresponding expression plasmid. Data were analyzed using FCS Express version 4. Samples were analyzed by flow cytometry. After 72 h, the percentage of GFP-positive cells was determined by flow cytometry. To determine the photon intensities of bands corresponding to the glycoproteins and to p24, areas corresponding to the respective bands were manually defined using the Living Image 4.

Background activities were identified by the negative control samples and subtracted from the glycoprotein and p24 signals. Buffy-oats obtained from anonymous blood donors were purchased from the German blood donation center.

Abstract Receptor-targeted lentiviral vectors LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Author Summary Pseudotyping of lentiviral vectors LVs with glycoproteins from other enveloped viruses has not only often been revealing in mechanistic studies of particle assembly and entry, but is also of practical importance for gene delivery.

Introduction Cell entry as first step in the viral replication cycle is initiated by the attachment of virus particles to distinct cell surface proteins. Results Setting up the system Previous studies showed that LVs can be pseudotyped with the NiV glycoproteins resulting in vector stocks with high titers [ 17 — 19 ]. Download: PPT. Fig 1. Fig 2. Mutation of the NiV glycoprotein to ablate natural receptor recognition.

Fig 3. Expanding the system to additional target receptors Next, we asked if other surface proteins can be targeted by engineered NiV glycoproteins as well. Thus, both surfaces of the plasma membrane are hydrophilic. In contrast, the interior of the membrane, between its two surfaces, is a hydrophobic or nonpolar region because of the fatty acid tails. This region has no attraction for water or other polar molecules. Proteins make up the second major chemical component of plasma membranes.

Integral proteins are embedded in the plasma membrane and may span all or part of the membrane. Integral proteins may serve as channels or pumps to move materials into or out of the cell.

Peripheral proteins are found on the exterior or interior surfaces of membranes, attached either to integral proteins or to phospholipid molecules. Carbohydrates are the third major component of plasma membranes. They are always found on the exterior surface of cells and are bound either to proteins forming glycoproteins or to lipids forming glycolipids. These carbohydrate chains may consist of 2—60 monosaccharide units and may be either straight or branched.

Along with peripheral proteins, carbohydrates form specialized sites on the cell surface that allow cells to recognize each other. How Viruses Infect Specific OrgansSpecific glycoprotein molecules exposed on the surface of the cell membranes of host cells are exploited by many viruses to infect specific organs. For example, HIV is able to penetrate the plasma membranes of specific kinds of white blood cells called T-helper cells and monocytes, as well as some cells of the central nervous system.

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Cite this Article Format. Helmenstine, Anne Marie, Ph. What Is a Peptide? Definition and Examples. Polysaccharide Definition and Functions. Ribosomes - The Protein Builders of a Cell. Fat Definition and Examples Chemistry. The Structure and Function of a Cell Wall.