· cTecrem is a 73kDa membrane-bound protein
Establishment of Anti-cTecrem mAb 1F12 was assessed by Immunoprecipitation of cTecrem using KF-1 cells. Anti-cTecrem mAb trapped a 73-kDa (Fig 1) polypeptide on the membrane whereas no bands were observed in the negative control w(h)ere Anti-cTecrem mAb was not added. The theoretical molecular weight of cTecrem is 37.6 kDa. So the results suggest that the cTecrem protein expressed on KF-1 cells are highly O-glycosylated at the STP region just like the human CD46 and this could be the reason for the vast difference between the theoretical molecular weight and the result observed after the immunoprecipitation. Similar result was earlier observed in the CHO – cTecrem in a study conducted earlier in our lab.
· Production, purification and characterization of recombinant SCR 1,2 and SCR 3,4 and its polyclonal antibodies.
Primers were separately designed against the SCR 1,2 and SCR 3,4 regions of cTecrem and the recombinant proteins were made. The recombinant proteins were purified using Ni-NTA column followed by size exclusion chromatography. Clear bands were observed in 1% TAE gel at 13.5 kDa which is the estimated size of SCR 1,2 and SCR 3,4 portions separately. Western blotting was done using the Anti-His antibody to localize the protein band on the blot and then later the His Tag was removed from the proteins using Factor Xa. Fig 2
The purified protein was then immunized on to rabbit for making polyclonal antibodies. The rabbit serum was purified using HI-trap column coupled with the corresponding recombinant proteins. The ELISA and Western blot results showed that the antibodies are specific to its own antigen and are less cross reactive. Fig 3a,b
An FCM analysis was done using the carp fin epithelial cell line (KF-1) and Ginbuna crucian carp epithelial cell line (CFS), to see the expression of cTecrem on them by using Anti-SCR 1,2 pAb and Anti-SCR 3,4 pAb. The sensitivity of the polyclonal antibodies was also checked by doing FCM with a wide range of antibody concentration. Fig 4. The results showed that the antibodies can express cTecrem on KF-1 cells from a concentration as low as 0.1µg/ml to 1 µg/ml after which it enters a plateau state.
The binding of Anti-cTecrem polyclonal antibodies to the Tecrem present in Common carp and Ginbuna crucian carp was confirmed by doing a western blotting. The RBC lysates of Common carp and Ginbuna crucian carp and the cell lysates of KF-1 and CFS cells were run on SDS-PAGE and was transferred to PVDF membrane Fig 5. The membrane was later developed using both Anti-SCR 1,2 pAb and Anti-SCR 3,4 pAb. The Anti-SCR 1,2 pAb bound to Common carp RBC and KF-1 cell lysate at 58kDa,49kDa and 40 kDa and to Ginbuna crucian carp RBC and CFS cell lysate at 74.5 kDa and 56 kDa. And the Anti-SCR 3,4 pAb bound to Common carp RBC and KF-1 cell lysate at 58 kDa and 49kDa and to Ginbuna crucian carp RBC and CFS cell lysate at 74.5 kDa. The 56kDa bands present on Ginbuna crucian carp RBC and CFS lysate when developed using Anti-SCR 1,2 pAb were not seen on the membrane developed using Anti-SCR 3,4 pAb. The amino acid sequence of SCR 1 and 2 of Common carp cTecrem and Ginbuna crucian carp gTecrem-1 is 13% more identical than its SCR 3 and 4 domains 9.This could be the reason why Anti-SCR 1,2 antibody bound to gTecrem better than Anti-SCR 3,4 pAb. This data also accounts with the specificity data of Anti-SCR 1,2 pAb and Anti-SCR 3,4 pAb.
· Epitope of Anti-cTecrem mAb resides in the SCR 1,2 domains
The ELISA results showed that the epitope of Anti-cTecrem mAb resides in the SCR 1,2 domains of the gene. Fig 6.
· Activation of cTecrem results in an increase in the adhesion of KF-1 cells on to the cell culture plate
Cell ELISA results performed using the KF-1 cells and the Anti-cTecrem antibody suggested that the expression of cTecrem is directly proportional to the adherence of cells on to the ELISA plate. The A405 taken within the first two hours of seeding gave a relatively lower OD whereas the A405 taken after the cells got completely adhered to the plate gave a higher OD value Fig 7
And as per the FCM analysis done using single KF-1 cells as well as aggregated KF-1 cells, the expression of cTecrem in the aggregated cells were relatively higher compared to the single cells. This is probably because either cTecrem is directly involved in the aggregation of cells or it could be because the aggregation of cells is indirectly stimulating the cTecrem expression.Fig 8
In addition to these assays two variants of cell adhesion assays were also performed. In the first assay the Anti-cTecrem mAb, Isotype control and PBS were coated on the ELISA plate prior to the cell seeding. The number of adhered cells were counted at different time points, and the statistics showed that the number of adhered cells in the wells where the Anti-cTecrem antibodies were coated were relatively higher at the first one hour compared to its isotype and PBS controls. This result shows that the coating of cTecrem antibody has increased an early stage of KF-1 cell adhesion to the plate Fig 9.
In the second cell adhesion assay, KF-1 cells were first incubated with Anti-cTecrem mAb, Anti-mouse IgG, Anti-SCR 1,2 pAb, Anti-SCR 3,4 pAb, Anti-rabbit IgG and PBS at room temperature and then were seeded on to the plate. The number of adhered cells were counted at different time points.Fig 10 a, b In agreement with the first cell adhesion assay, number of adhered cells were significantly higher in the wells pre incubated with Anti-cTecrem antibodies. We also observed that KF-1 cells pre incubated with Anti-cTecrem mAb and Anti-SCR 1,2 pAb got adhered to the plate in a much higher number compared to the cells pre incubated with Anti-SCR 3,4 pAb. In conclusion, KF-1 cells pretreated with Anti-SCR 1,2 pAb and Anti-SCR 3,4 pAb profoundly increased the number cells which got adhered to the plate. This could be either because cTecrem is directly involved in the cell adhesion or because cTecrem is indirectly stimulating some other cell adhesion molecules.
· Tecrem activation promotes carp fin epithelial cell growth as well as wound healing
Previous studies on human CD46 reports that CD46 is involved in the intestinal cell proliferation as well as wound healing through the regulation of tight junction and adherence junction (10). The proliferation of human epithelial cells is controlled by the formation and loosening up of tight junctions and adherence junctions. When the tight junctions are formed rigidly, the cell growth stops and when these junctions get deformed, the cell will initiate growth (10). As per this study, the CD46 gene has a role in the regulation of tight junction formation and hence in the cell proliferation. (10)In order to investigate if cTecrem also possess a role in the cell proliferation as well as the wound repair, a wound healing assay was performed using KF-1 cells. KF-1 cells were grown on 24 well plate until it formed a monolayer. Wounds were artificially created by scratching on the monolayer using a 200µl micropipette tip. The pipette tip was held at an angle of 300 in order to keep the scratch width limited. The monolayer was then over layered by MEM-10 with or without cTecrem antibodies and its isotype controls. The cell growth was then monitored after 12h, 24h and 48h and the percentage of wound closure was calculated Fig 11 a, b. In agreement with the wound healing assay data in human CD46, the percentage of wound closure in fish epithelial cells was also found to have accelerated upon the addition of Anti-cTecrem antibodies on to the wounds. Similar to the cell adhesion assay data, Anti-cTecrem mAb and Anti-SCR 1,2 pAb accelerated the epithelial wound healing compared to Anti-SCR 3,4 pAb.
· Hypersecretion of ZO1 protein was observed in cTecrem activated CFS cells.
There are reports on human CD46 which suggests that the CD46 gene is involved in the regulation of tight junction formation and thus in the epithelial cell proliferation. Their results showed an increased proliferation rate in the CD46 activated intestinal epithelial cells (10). In agreement to this, our cell adhesion assays and the wound healing assay also shows an increased cell adhesion as well as wound healing in the cTecrem activated carp fin epithelial cells. Tight junction proteins play a major role in the epithelial formation, development and maintenance of tissue integrity in vertebrates. (24) In order to investigate more on the cTecrem initiated cell proliferation, it is really important to study the cTecrem-TJ interaction. ZO1 or Zona Occludin1 is an important TJ protein which plays a major role in the epithelial cell proliferation. So we have done the immunohistostaining of the cTecrem activated CFS cells using Anti-ZO1 antibody. We used CFS cells here instead of KF-1 cells because the KF-1 cells were forming a spindle shape upon the addition of any fixative. This caused the distortion of the monolayer and this made it difficult to proceed the immunohistochemistry using KF-1 cells. We have already showed the similarity in the isotype 1 of the Ginbuna crucian carp Tecrem with the common carp Tecrem. Hence it is admissible to use CFS cells instead of KF-1 cells in this assay.
The results showed an increased fluorescence in the tight junctions of the cTecrem activated, fully confluent CFS cells compared to the normal CFS cells. Fig 12 a. To obtain a statistical data, the fluorescence intensity per cell was calculated for a minimum of 50 cells per experiment using ImageJ software. Graphs were plotted using the average fluorescence intensity per cell and the average florescence intensity per field. Fig 12 b. The graph was plotted by subtracting the background fluorescence in the case of Anti-SCR 12 pAb activated CFS cells, since there was a background from the direct binding of Anti-Rabbit secondary antibody with the Anti-cTecrem pAb. The calculated p values showed that the fluorescence intensity in the cTecrem activated cells are significantly higher than the normal cells. The increase in the fluorescence could be because of the hyper secretion of the ZO1 protein or its associated proteins. This hyper secretion was observed only in the cells activated with cTecrem antibodies and this shows that cTecrem has a role in the regulation of TJ.
· Compactness of the KF-1 and CFS monolayers were increased upon the activation of cTecrem.
Both KF-1 and CFS cells were grown on 6-well plates with and without the addition of cTecrem antibodies until it formed a monolayer. The area per cell was then calculated using ImageJ software. The average area of 50 cells was plotted as a graph. The average area of the cells (both KF-1 and CFS) activated with cTecrem antibodies were found to be significantly lesser than the normal cells. Fig 13 The P value between the control and the sample groups were as less as 0.001, which shows an extremely high significance. The compactness of the cell monolayer in both CFS and KF-1 have increased upon the activation of cTecrem. This resulted in the reduction of the area per cell. The cell area was calculated on the third, fourth and the seventh-day. The graphs drawn with all the three data followed the same pattern. The only exception was the fourth-day data of the CFS cells. The size of the CFS cells became bigger on the fourth-day compared to the third day and then it again became smaller on the seventh day. In all the other cases, the cell size decreased with time; which signifies the increased compactness. CFS grows at a slower rate than KF-1 cells. So when the analysis was done on the third day, the CFS wouldn’t have formed a complete monolayer like KF-1 cells. The cells might have been in the proliferation stage. This could be the reason for the increased cell size on the fourth-day in CFS cells.
The cell monolayer compactness of both KF-1 and CFS increased upon the activation of cTecrem. This pattern was observed throughout the assay. These results are in agreement with our earlier assay results as well. This signifies the role of cTecrem in the cell to cell attachment as well as in the formation of a rigid epithelial monolayer