Difference between revisions of "Part:BBa K2332312"

Revision as of 20:40, 9 October 2017

E-cadherin (Preproprotein, Mus Musculus)

This gene encodes E-cadherin, a calcium-dependent cell adhesion molecule that functions in the establishment and maintenance of epithelial cell morphology during embryongenesis and adulthood. The encoded preproprotein undergoes proteolytic processing to generate a mature protein.

Usage and Biology

Cell-cell junctions come in many forms and can be regulated by a variety of different mechanisms. The best understood and most common are the two types of cell-cell anchoring junctions which employ cadherins to link the cytoskeleton of one cell with that of its neighbour. Their primary function is to resist the external forces that pull cells apart. At the same time, however, they need to dynamic and adaptable, so that they can be altered or rearranged when tissues are remodelled or repaired or when there are changes in the forces acting on them.

Figure 1: Adherens Junction - Cadherin Mediated Cell-Cell Adhesion.

(A) Adherens junctions, in the form of adhesion belts, between epithelial cells in the small intestine. The beltlike junction encircles each of the interacting cells. Its most obvious feature is a contractile bundle of actin filaments running along the cytoplasmic surface of the junctional plasma membrane. (B) Some of the molecules that form an adherens junction. The actin filaments are joined from cell to cell by transmembrane adhesion proteins called cadherins. (Alberts B. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015)

Cadherins are a diverse family of adhesion molecules that fulfil these requirements. They are present in all multicellular animals whose genomes have been analysed. Other eukaryotes, including fungi and plants, lack cadherins, and they are also absent from bacteria and archaea. Cadherins therefore seem to be part of the essence of what it is to be an animal.

The cadherins take their name from their dependence on Ca²⁺ ions: removing Ca²⁺ from the extracellular medium causes adhesions mediated by cadherins to come apart. The first three cadherins to be discovered were named according to the main tissues in which they were found:

- E-cadherin is present on many types of epithelial cells;

- N-cadherin on nerve, muscle and lens cells;

- P-cadherin on cells in the placenta and epidermis.

All are also found in other tissues. These and other classical cadherins are closely related in sequence throughout their extracellular and intracellular domains.

Binding between cadherins is generally homophilic. This means cadherin molecules of a specific subtype on one cell bind to cadherin moleculs of the same or closely related subtype on adjacent cells. All members of the superfamily have an extracellular portion consisting of several copies of the extracellular cadherin (EC) domain. Homophilic binding occurs at the N-terminal tips of the cadherin molecules - the cadherin domains that lie furthest from the membrane. These terminal domains each form a knob and a nearby pocket, and the cadheirn molecules protruding from opposite cell membranes bind by insertion of the knob of one domain into the pocket of the other.

Figure 2: Molecular Model of E-cadherin

After processing in the late Golgi, E-cadherin contains five EC domains. The outermost EC domain forms homophilic connections with the equivalent domain of E-cadherin on the neighbouring cell. The stability of E-cadherin depends on the presence of Ca²⁺ in the extracellular space. (Alberts B. Molecular Biology of the Cell. 6th ed. Figure 19-6 New York: Garland Science; 2015)

Each cadherin domain forms a more-or-less rigid unit, joined to the next cadherin domain by a hing. Ca²⁺ ions bind to sites near each hinge and prevent it from flexing, so that the whole string of cadherin domains behaves as a rigid and slightly curved rod. When Ca²⁺ is removed, the hinges can flex, and the structure becomes floppy. At the same time, the conformation at the N-terminus is thought to change slightly, weakening the binding affinity for the matching cadherin molecule on the opposite cell.

The cadherins form homodimers in the plasma membrane of each interacting cell. The extracellular domain of one cadherin dimer binds to the extracellular domain of an identical cadherin dimer on the adjacent cell. The intracellular tails of the cadherins bind to anchor proteins that tie them to actin filaments. These anchor proteins include α-catenin, β-catenin, γ-catenin (also called plakoglobin), α-actinin, and vinculin.

UCL iGEM 2017 believes that cadherin proteins will be powerful modulators for efficient tissue engineering. We therefore investigated first the properties of one classical cadherin (E-cadherin, BBa K2332312) and then tried to make it light-responsive.

For more information on cell-cell junctions and cadherins see Alberts B., Molecular Biology of the Cell. 6th ed., Ch.19, New York: Garland Science; 2015.

E-Cadherin Entries in the Registry

UCSF iGEM 2011 has created a BioBrick of only the extracellular domain of E-Cadherin (Mouse) BBa_K644000 but no BioBrick encoding the full E-cadherin protein has been submitted until now. BBa_K644000 also lacked detailed characterisation and the source was imprecise. Furthermore, we know now that E-cadherin requires interaction of its cytosolic domain for the production of stable cell-cell connections. (see Alberts 6th Ed. 2015, Ch. 19, p. 1040).

Experimental approach

Figure 3: Map of E-cadherin in pcDNA3

Vector Considerations

For testing this coding part we used pcDNA3 [http://www.snapgene.com/resources/plasmid_files/basic_cloning_vectors/pcDNA3/ (SnapGene File)], a standard mammalian expression plasmid, as a vector. We, thereby, created the coding device BBa_K2332313, our E-cadherin gene flanked by a CMV promoter and a bGH poly(A) tail. The well characterised strong promoter, efficient poly(A) tail and the pre-existing 5'- and 3'-UTR ensure efficient expression of E-cadherin after transfection.

There are many ways to express mammalian genes. Using a standard mammalian expression plasmid saves time and reduces the risk of low expression due to variations in 5'- and 3'- UTR.

Chassis Considerations

Choosing the correct chassis for your experiments is of equal importance to choosing the correct gene.

Since we wanted to test cell-cell aggregation induced by the E-cadherin gene, we therefore chose a mammalian cell line that naturally does not express E-cadherin and is commonly used in cadherin research, Chinese Hamster Ovary (CHO) cells. Even though they naturally lack E-cadherin expression they still maintain alpha- and beta-catenin expression, the two proteins that are essential for E-cadherin's connection to the actin cortex of the cell.

Experimental Setup

Choosi

Results

Choosi

Figure 4: Well A - Cells + Superfect + plasmid CHO cells transfected with pcDNA3 containing E-cadherin but with no Ca²⁺ show a low level of aggregation, probably due to other adhesive molecules on the plasma membrane surface.

Figure 5: Well B - Cells + Superfect + plasmid + CaCl₂

Ca²⁺ was added in the solution to CHO cells transfected with pcDNA3 containing E-cadherin. A high level of cell aggregation can be observed that significantly shifts the cell single:aggregation ration to the aggregated state (see table 1).

Figure 6: Well C - CaCl₂

Negative control well with only CaCl₂ in the solution.

Figure 7: Well D - Control cells

CHO cells treated with superfect and PBS instead of plasmid.

Figure 8: Well E - Control cells + calcium

Ca²⁺ was added in the solution to CHO cells treated with superfect and PBS instead of plasmid.

Figure 9: Well F - Untreated cells

CHO cells without any reagents added to the solution.

Diagram 1: Cell Aggregation Chart

.

Well	Contents	Single Cells	Single Cells Average	Aggregated Cells	Aggregated Cells Average	Ratio single:aggregated
A	Cells + superfect + plasmid	163, 245, 251	220	195, 242, 311	249	0.88
B	Cells + superfect + plasmid + calcium	239, 237, 213	230	251, 342, 477	356	0.65
C	CaCl2 only	/		/
D	Control cells	236, 173, 290, 185	221	157, 145, 116, 258	169	1.3
E	Control cells + calcium	187, 220, 142	183	137, 219, 136	164	1.1
F	Untreated cells	549, 385, 554	496	500, 397, 582	493	1.0

Table 1: Cell Aggregation Table Control cells = treated with superfect + PBS instead of plasmid

Usability

A functional, natural cell-cell adhesion protein like BBa_K2332312 has potential in many different fields:

Tissue engineering is dependent on the formation of lasting connections between cells. By choosing E-cadherin to form such connections you mimic the bodies natural way of adhering cells into a 3-dimensional structure. This holds the potential to one day lead to functional tissue for replacement of destroyed tissue in patients.

In bioprocessing, tissue structures can be polarised and arranged for maximal surface to volume ratio. They can form separate compartments for nutrient uptake on one side and biomolecule production on a different side, thereby streamlining a production process in mammalian cells for complex molecules.

Primer Designs

PCR out Primers

5'- agcttggtacctccac -3', Ecadh_BioBrick.FwP
5'- tctagtcgtcctcgcc -3', Ecadh_BioBrick.RevP

These are the primers UCL iGEM 2017 used to clone E-cadherin out of the plasmid that Prof. Price gave us. We attached the BioBrick prefix to the 5'-end of the forward primer (+10 additional base pairs for efficient cleavage) and the BioBrick suffix to the 5'-end of the reverse primer (+10 additional base pairs for efficient cleavage). Through overhang PCR with these primers we created the BioBrick BBa_K2332312.

Sequencing Primers

We used Sanger sequencing for the sequencing of our E-cadherin gene. However, since Sanger sequencing only ensures correct results for up to around 800 bp we needed to use 2 sequencing steps ('primer walking') with two primers in each step:

Since the gene was in pcDNA3 we used the standard primers for pcDNA3.1 for the first round of sequencing:

5'- ctctggctaactagagaac -3', pcDNA3.1-FwP
5'- caaacaacagatggctggc -3', pcDNA3.1-RevP

For the second round of sequencing we designed and synthesized the following primers:

5'- tcaacacctacaacgctgc -3', E-cadh.sequ.Round2-FwP
5'- aggttctgggatgggagc -3', E-cadh.sequ.Round2-RevP

Characterisation Opportunities

This E-cadherin is not RFC25 compatible because of a single NgoMIV site at position 208. UCL iGEM 2017 suggests to use side directed mutagenesis to remove said NgoMIV cutting site and to use our primers with RFC25 overhangs to create an RFC25 compatible E-cadherin registry entry.

Sequence and Features

DNA Features

This gene was given to the UCL iGEM team 2017 by Prof. Stephen Price (UCL, not part of iGEM) after we searched for cadherin proteins suitable for our project. However, no sequence was known of the plasmid we were given and we sequenced the plasmid ourselves. Consecutive BLAST analysis of the results showed a 99% similarity with Mus musculus cadherin 1 (Cdh1), mRNA: NCBI Reference Sequence: NM_009864.3, NCBI.

Three silent mutations were added into the sequence via side directed mutagenesis in order to remove one EcoRI and two PstI sites. Afterwards we sequence confirmed the entire gene.

Protein Features

Figure 10: Protein BLAST Results from E-cadherin (Link).

Cadherin Prodomain Like - Cadherin proteins are activated through cleavage of a prosequence in the late Golgi. This prevents cadherin aggregation in the early stage of the secretory pathway. This domain corresponds to the folded region of the prosequence, and is termed the prodomain. The prodomain shows structural resemblance to the cadherin domain, but lacks all the features known to be important for cadherin-cadherin interactions.

Cadherin Repeat-Like Domain- The cadherin repeat domains occur as tandem repeats in the extracellular regions, which are thought to mediate cell-cell contact when bound to calcium. They play numerous roles in cell fate, signalling, proliferation, differentiation, and migration; members include E-, N-, P-, T-, VE-, CNR-, proto-, and FAT-family cadherin, desmocollin, and desmoglein, a large variety of domain architectures with varying repeat copy numbers. Cadherin-repeat containing proteins exist as monomers, homodimers, or heterodimers. This family also includes the cadherin-like repeats of extracellular alpha-dystroglycan.

Cadherin Cytoplasmic Region- Cadherins are vital in cell-cell adhesion during tissue differentiation. Cadherins are linked to the cytoskeleton by catenins. Catenins bind to the cytoplasmic tail of the cadherin. Cadherins cluster to form foci of homophilic binding units. A key determinant to the strength of the binding that it is mediated by cadherins is the juxtamembrane region of the cadherin. This region induces clustering and also binds to the protein p120ctn.

Functional Parameters

References

1. 2. 3. 4.