Every cell receives the required oxygen and nutrients through blood vessels for their growth and survival. As growth of an organism progresses, the need for nutrients becomes necessary, which are supplied by either existing blood vessels or newly formed ones. A new blood vessel arises from the existing blood vessels in instances to support the growth (Ex: endothelial cells sprouting to form new capillaries). Such formation of new blood vessels from the existing ones, is defined as Angiogenesis. In the case of cancer, tumour cells form their own blood vessels by deregulating the angiogeneic process. Hence, developing drugs against the deregulated angiogenesis process would be a promising treatment for cancer and other diseases. An endothelial protein, Paladin is found to be expressed both in tumour and normal vasculature. In this study we tried to find the cellular function of paladin. To study the function of protein, we used in silico tools and databases. Bioinformatics analysis reveals paladin protein is having the possibility of phosphatase activity. To verify this, site directed mutagenesis was carried out at the predicted phosphatase domains of the paladin protein. This mutated and phosphatase dead paladin protein will be used as a negative control in phosphatase assay. The results from the mutagenesis were verified by sequencing. Also, in-vitro cell signaling studies was performed in PAE/KDR cells, to analyze if Canis lupus familiaris VEGFR substrates have similar function as mouse VEGFR substrate. The result confirmed both VEGFR substrates have a similar function.
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The physiological outgrowth of new blood vessels from the existing blood vessel is defined as Angiogenesis. Multiple biological processes such as cell growth, cell proliferation, repair, survival and organ development depends on angiogenesis . This major process is facilitated by angiogeneic regulators such as vascular endothelial growth factors VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PLGF). Each growth factor works with a specific function. VEGF-A is necessary for stimulation of angiogeneic growth, remodeling and vasculogenesis  . These VEGF ligands bind to VEGF receptors VEGFR1, VEGFR2, VEGFR3. VEGFR1 and VEGFR2 are found on the endothelial cells of the blood vessels. While, VEGFR-3 is present on endothelial cells of the lymphatic vessels. VEGF-A, VEGF-B, PLGF binds to VEFGR1. VEGF-A, VEGF-C, VEGF-D binds to VEGFR2, and this receptor plays a major role in stimulation of angiogenesis process. While VEGF-C and VEGF-D binds to VEGFR3 and activates lymphangiogenesis.  
During the formation of cancerous cells, a dysregulation occurs in the process of angiogenesis, which leads to survival of tumour cells . In cancer, there is an uncontrolled production of angiogeneic factors which forms new blood vessels supplying nutrients to the tumour cells making its survival . Hence, study on anti-angiogeneic substances and their target against cancerous cells could be a hopeful treatment against cancer  . Currently two anti-angiogeneic targets VEGF-A and DLL4 (Delta like 4) are therapeutically used   . But each with their own side effects have not resulted in positive recovery for certain tumours  .
Paladin (KIAA1274) a protein expressed in vascular endothelial cells , but still the function of the paladin protein as anti-angiogeneic target remains to be determined. However, there are few reports suggests paladin to have a function in cell signaling and vascular development  . In earlier studies paladin was predicted as having tyrosine phosphatase activity. In mouse model paladin is expressed in developing and adult vasculature. In addition paladin is also found in tumour vasculature .
On the other hand, the paladin protein is also identified as inhibitor and plays a role in insulin signaling . When insulin binds to insulin receptor it phosphorylates automatically and activates IRS (insulin receptor substrate). IRS activate phosphatidylinositol-3-OH kinase (PI3K) pathway. Insulin also activates AKT signaling pathway .The metabolic process is controlled by insulin via PI3K-AKT pathway  . Overexpression of paladin in COS7 cells stimulated with insulin showed less phosphorylation of AKT at S473 and T308 at various time points. To further confirm the role of paladin in insulin induced AKT phosphorylation, C2C12 mouse myoblast cells was used. Paladin knockdown by siRNA before insulin stimulation resulted more phosphorylation of AKT and insulin receptor (IR) abundance .
With the above known information about paladin, we were interested in studying the actual cellular function of paladin protein. Paladin gene has 20 exons in humans and transcribed to a full length protein of 856 amino acids (aa). It has two alternative splice variants. One lacks exon 17 and transcribed to 832 aa protein and another splice variant which lacks exon 1-14 coding 237 aa. Initially in silico tools and online biological databases were used, to elucidate the role of paladin protein in humans. The result suggested paladin to have a phosphatase activity. Phosphatase activity, in general is needed to remove the activating phosphatase there by inhibiting the downstream signaling. The phosphatase activity of the paladin protein will be examined using the wild type (WT) paladin protein and the mutated paladin protein by a phosphatase assay. In this report we showed that a mutated and presumable phosphatase activity dead paladin protein is present. Site directed mutagenesis technique was used to perform mutation.
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We also verified the newly delivered Canis lupus familiaris (DOG) VEGF substrate to be a potent stimulator of the human VEGFR-2 in PAE/KDR cells. The VEGF substrate activation was analyzed by verifying the presence of phosphorylated ERK which is one of the downstream signaling molecules activated upon VEGFR-2 activation. This was performed using specific antibodies in Western Blot.
Materials and Methods:
Site Directed Mutagenesis Assay:
The phosphatase domain of paladin could be best studied upon mutating all the four predicted phosphatase domain of the paladin protein. Initially the recombinant plasmid vector pcDNATM3.1D/V5-His-Topo® (Invitrogen life technologies ) carrying wild type (WT) paladin gene was used for introducing first mutation.
All the mutations were performed by following the instructions provided by QuickChange II Site-Directed Mutagenesis kit (Agilent Technologies, San Diego CA). The principle behind this mutation process is the recombinant double stranded DNA plasmid carrying gene of interest is amplified using two synthetic primers having desired mutation. Each primer complementary to opposite strands of the vector. The plasmid is amplified by DNA polymerase without primer displacement in PCR machine. The amplified plasmid containing desired mutation with staggered nicks. The PCR product is treated with Dpn I restriction enzyme to digest the parental DNA template which is methylated. The Dpn I restriction enzyme digested PCR product is transformed to XL 10-Gold Ultracompetent cells. Transformed Ultracompetent cells are plated in LB ampicillin agar plate. The cells containing plasmid is having ampicillin gene which have capability to grown in ampicillin agar plate. The colonies are formed after incubation. Selected single clones are transformed to liquid LB ampicillin broth. After incubation time the plasmid was isolated by miniprep technique. This described method was used to introduce mutation at four different positions of paladin gene containing plasmid.
The mutagenic primers were designed using QuickChange Primer Design, a web based program at www.agilent.com/genomics/qcpd. The mutagenic primers that we used are tabulated in the table 1,
Forward Primer Sequence(5'to3')
Reverse Primer Sequence(5'to3')
Primer 1, C661
Primer 2, C312
Primer 3, C158
Primer 4, C118
The first mutation was introduced at 2262 base pair (bp) position changing the amino acid cysteine to serine at 661 position. The 10 ng (nano gram) of recombinant plasmid vector, 125 ng of oligonucleotide forward and reverse primer (primer 1 in Table 1) carrying the desired mutation, 10X reaction buffer, dNTP (deoxynucleotide triphosphate), 2.5 U/µl of Pfu Ultra HF DNA polymerase and ddH2O was added in PCR tube. The plasmid was amplified using thermo cycler. The temperature and number cycles were given in the Table 2.
The PCR product was treated with Dpn I restriction enzyme (10 U/µl) to digest the WT paladin containing plasmid (methylated). The 2 µl of digested PCR product was transformed into XL 10-Gold Ultracompetent cells. The transformation reaction was performed by incubating the eppendorf tube in the ice for 30 min than 30 sec in 42°C water bath and finally 37°C for 1 hour in shaking incubator.
The transformed ultracompetent cells were plated on LB-ampicillin agar plates. The plates were incubated at 37°C for 16 hours. The ampicillin resistant colonies were formed in the agar plate. Selected bacterial clones from the agar plates were transferred to liquid LB ampicillin broth and kept in shaking incubator for 16 hours. The ampicillin resistant bacteria cells were grown in the tubes. The plasmid was isolated by using miniprep technique described below. By sequencing the isolated plasmid (445ng), mutation at bp position 2264 of paladin gene was verified.
The second mutation at 1215 bp was introduced in the plasmid carrying first mutation. Upon confirmation of the second mutation by sequencing, the plasmid was introduced with third mutation at 754 bp. Finally mutation on position 634 bp was introduced to the plasmid carrying the three mutations. All the four successive mutations were carried out in a way similar to mutation at position 2262 bp of WT paladin gene containing plasmid.
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The plasmid isolation was performed by instructions provided by the QIAprep Spin Miniprep Kit (Qiagen). The liquid LB broth tubes were removed from shaking incubator after 16 hours incubation. Plasmids were isolated using the QIAprep Spin Miniprep Kit. Briefly, bacteria was harvested by centrifugation at 4000 rpm for 10 min. The bacterial cells were settled down in the pellet after centrifugation. The pellet from the bacterial cells was resuspended in 250µl of buffer P1 and transferred to microcentrifuge tube. The mixtures were treated with 250µl of buffer P2 (Lysis buffer) and mixed by inverting the tubes 4-6 times. This made cell debris and chromosomal DNA to precipitate after that 350µl of buffer N3 (neutralization buffer) was added. The tubes were centrifuged for 10 min at 13000 rpm and the supernatant alone was added to spin column by pipetting. After centrifugation the plasmid were bound to the membrane and washed with 0.75 ml of ethanol containing buffer PE and kept in centrifuge for 60 sec. The flow-through was discarded and kept it in centrifuge for 1 min to remove residual wash buffer. QIAprep column was placed it in 1.5ml microcentrifuge tube and 50µl of buffer EB (elution buffer) was added. Plasmid DNA was eluted by centrifuge the tube for 1 min. The amount of DNA present in the tubes was measured by using spectrophotometer.
Western Blot Analysis:
The protein samples were prepared by addition of 4X sample buffer and 10X reducing agent. The prepared samples were incubated at 70°C for 10 min to denature the proteins. The appropriate amounts of samples were then loaded in 4-12% gradient Bris-Tris acrylamide gel. The proteins were separated according to the molecular weight by running the gel at 170V for 1 hour in the presence of mops buffer (1X-running buffer).
After 1 hour the gel was removed from the cassette, and the proteins were transferred into Hybond nitrocellulose membrane (GE healthcare) in the presence of transfer buffer (1X) by electrophoresis (30v for 2 hours). The membrane was then washed with 1X TBS-0.1%Tween for 3x10 min. Unspecific binding to the membrane is blocked using 5% of milk in 1X TBS-0.1%Tween for 1 hour. The membrane was incubated with suitable primary antibody 1:1000 dilution and for overnight at 4°C. Next day the membrane was washed with 1X TBS-0.1%Tween for 3x10 min. The secondary antibody conjugated with horseradish peroxidase was added to the washed membrane at 1:5000 dilution and incubated at room temperature for 1 hour. The membrane was later washed with 1X TBS-0.1%Tween solution for 3x10 min. The proteins were detected using ECL-Plus kit (GE Healthcare).
Mops SDS running buffer, sample buffer, antioxidant, sample reducing agent, sample buffer, transfer buffer were purchased from Invitrogen (NUPAGE). Anti-VEGFR-2 (TYR 1175), anti total VEGF, anti phosphorylated ERK (T202/Y204) antibodies were purchased from Cell Signaling Technology Inc (Beverly MA). The secondary antibodies anti mouse IgG, HRP linked and anti rabbit IgG HRP linked were purchased from GE Healthcare.
In silico analysis of Paladin Protein:
To elucidate the function of the human paladin protein, we used in silico tools and online biological databases. According to bioinformatics databases, amino acid (aa) region C(X)5R is predicted to be the signature motif of the active site of phosphatase. The paladin protein sequence carries four such phosphatase signature motif regions. The regions are aa 118-124, 158-164, 312-318, 661-667 [Figure 1].
In addition to this SCOP Super Family and InterPro databases shows similar predictions. SCOP Super Family database shows paladin protein carries two PTP (Protein Tyrosine Phosphatase) regions at the position of aa 247-319, 619-677.
InterPro database predicted one PTP catalytic domain motif at 262-384 aa position of paladin protein.
Figure1: The amino acid sequence of human paladin retrieved from www.ncbi.nlm.nih.gov. The highlighted amino acid regions are the phosphatase signature motif of the paladin protein. Mutation was carried out by replacing Serine for Cysteine (red).
To examine the phosphatase activity of paladin protein in vitro, point mutations were introduced at four individual phosphatase signature motifs by site directed mutagenesis. The nucleotide positions that we introduced mutations were described in Table 2. These mutations changing the amino acid cysteine to serine at the position mentioned in the Table3. All the four successive mutations were verified by sequencing the plasmid [Fig 2] [Fig 3] [Fig 4] [Fig 5].
Table 1: The four (661, 312, 158, 118) positions of the paladin protein contributing to the phosphatase domain. Mutation was introduced in the nucleotide positions 2262, 1215, 754, 634. The 2265, 1215 nucleotides were mutated from Thymidine to Adenine. The 754, 634 nucleotides were mutated from Guanine to cytosine.
Verification of Canis lupus familiaris VEGF:
To analyze the effect of Canis lupus familiaris (DOG) VEGF substrate on cell signaling, we stimulated VEGF receptor-2 in PAE /KDR cell line by using mouse and two different batch of dog VEGF substrate. The activated VEGFR-2 was traced by probing against Anti-P-VEGFR-2 (TYR 1175) while p44, p42 ERK, Total VEGF, β actin were traced by probing against anti phosphorylated ERK (T202/Y204), anti total VEGF, anti β actin respectively. The western blot result showed both mouse (A) and dog batch 2 (C) VEGF substrate function in a similar way. While dog batch 1 (B) slightly differs from mouse VEGF at 15 min time point of pERK [Fig 6].
The online biological database www.ensembl.org was used for finding the information about paladin gene. Superfamily database http://supfam.cs.bris.ac.uk/SUPERFAMILY/ was used to find the phosphatase domains of paladin protein. By using http://smart.embl-heidelberg.de, we collect information about highly conserved active site of phosphatases.
Paladin plays a role in insulin signaling pathway. The over expression of paladin in insulin stimulated cells inhibits AKT phosphorylation which is necessary for metabolic process controlled by insulin. But the phosphatase activity of paladin protein was not examined . Therefore we would like to investigate the actual molecular function of paladin.
Figure 2: Figure 3:
Figure 4: Figure 5:
Figure 2, 3, 4, 5 are the sequencing results of the paladin gene by using specific primer to sequence the specific mutated position. Fig 2 and 3 confirms the mutation at base pair position 2262 and 1215, the wild type paladin gene has T at the yellow colour highlighted position followed by GC which codes for cysteine in 661 and 313 aa position by site directed mutagenesis we mutated T with A followed by GC codes for serine at that position. Fig 4 and 5 confirms the mutation at base pair position 754 and 634. As in fig 2 and 3 the yellow highlighted position is mutated from TGT to TCT and it codes serine instead of cysteine at the aa position 158 and 118.
Figure 6, Stimulation of Mouse and two batches of dog VEGF in PAE/KDR cells. PAE/KDR cells were serum starved prior to treatment with mouse VEGF (A), dog B1 VEGF (B), dog B2 VEGF (C) for times indicated and analyzed for VEGFR-2 (TYR 1175), p42, p44 ERK, Total VEGF and β actin. Total VEGF and β actin were included as loading control.
The four predicted phosphatase domains were successfully mutated by QuickChange II Site Directed Mutagenesis Kit. In the future we will transfect the mutated and WT paladin to eukaryotic cells. The protein can be isolated using the V5 epitope and used in phosphatase assays.
In the phosphatase assay radioactive labeled phosphorylated peptides are mixed with target protein under optimized conditions. After certain incubation time the amount of free (non-peptide bound) phosphate groups can be measured. The mutated paladin also will be used in cell signaling experiments as a negative control in comparison to WT paladin. To this is an important step to be able to found out if the effect of paladin is due to a phosphatase activity or if paladin mediates its function in another way.