The Protein Tyrosine Phosphorylation Biology Essay

INTRODUCTION

The protein tyrosine phosphorylation is important in various physiological

functions of the eukaryotic cells, being involved in many functions such as cell

proliferation, survival, migration and differentiation. The Protein Tyrosine Kinases

(PTKs) and Protein Tyrosine Phosphatases (PTPs) play opposite roles in tight

regulation of protein phosphorylation in the cells (1). Receptor Protein Tyrosine

Phosphatases (RPTPs) belong to classical protein tyrosine phosphatases and show

strong expression in central and peripheral nervous system. Many RPTPs are

involved in axogenesis, synaptogenesis, target contact and plasticity (2, 3). Several

in vivo roles of RPTPs in neural development and function have been demonstrated

(1, 3). Although numerous effectors of the downstream RPTP signaling are known,

the information about the ligands of RPTPs is limited (4).

The mouse Ptprr gene encodes PTPBR7, a receptor-type isoform. PTPBR7 is

the longest of the four isoforms, containing 656 amino acids (5). Ptprr belongs to

the R7 subgroup of RPTPs composed of a short extracellular region, a transmembrane

region, a cytosolic region containing the Kinase Interacting Motif (KIM), and a

phosphatase domain (6). PTPBR7 is expressed during early embryogenesis in

spinal ganglia and Purkinje cells. After birth, the expression of PTPBR7 occurs in

all regions of the brain, whereas it is decreased in maturing Purkinje cells (7).

PTPBR7 forms homo-oligomers located at the cell surface, and shows reduced

activity in comparison with the cytosolic isoforms that are monomeric (8). All the

Ptprr isoforms contain Kinase Interacting Motif (KIM), and are known to bind and

inactivate Mitogen-Activated Protein Kinases (MAPKs) (5). Ptprr knockout mice

performed poorly in various locomotive tests, but they show no brain malformation

(9). Impairment in cerebellar calcium ion homeostasis is also known to cause

ataxia in animal models, similar to Ptprr knockout mice (10). Highly myelinated

areas in the brain have been recently proved to be sites for PTPBR7 ligands (11).

Identified PTPBR7 ligand candidates suggest/indicate the involvement of this

receptor in cell-cell adhesion complex during cerebellar development and calcium

ion regulated events that are important in neuronal development and plasticity (11).

MATERIAL AND METHODS

CLONING OF PTPBR7 CONSTRUCTS

The gene coding for the full PTPBR7 extracellular region cloned into pHLsec

mammalian expression vector was a kind gift from Dr Hendriks (11). The pHLsec

vector contains a signal sequence for secretion of the protein fused with a Cterminal

6X His-tag. The shorter constructs of PTPBR7 extracellular region were

cloned into pHLsec vector at AgeI and KpnI restriction sites. The plasmid

containing full-length extracellular region of PTPBR7 was used as template and the

following primers were used: BR7S1 forward, 5’-

GAAACCGGTAGTTGGAAGCCGGTGTTCATTTATGACC-3’; BR7S2 forward,

5’- GAAACCGGTAGCCTGGACATCGCACAAGAGGC-3’; BR7S3 forward, 5’-

GAAACCGGTCATAACTACCACTCCCCTTCCGAAAG-3’; BR7S reverse, 5’-

CTTGGTACCCTGCCCTTGTAAAACTTTTTCTCAAGGGG-3’ for the PCR

amplification. The PCR product and the pHLsec vector were restriction digested

with AgeI and KpnI enzymes and ligated to obtain desired constructs. The pHLsec

vector with RPTPμ coding gene inserted between AgeI and KpnI restriction sites

was a kind gift from Dr. Aricescu (12).

CELL CULTURE AND TRANSIENT TRANSFECTION

HEK293T cells and N-acetylglucosaminyl transferase I-negative 293S GnTI¯

cells (13), unable to synthesize complex N-glycans, a kind gift from Dr. Aricescu,

were used for the expression of recombinant PTPBR7 constructs. HEK293T and

293S cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM high

glucose, Sigma) supplemented with L-glutamine, non-essential amino acids

(Gibco) and 10% fetal calf serum (FCS, Sigma). The cells were maintained in

standard flask in a humidified incubator at 37ºC and 5% CO2. Small-scale

transfections were carried out in 6-well plates, while the large-scale cultures for

protein productions were performed using expanded-surface polystyrene roller

bottles (2125 cm2, Greiner Bio-One). Cells were transfected when the confluence

of the adherent cells reached about 90%. PTPBR7 constructs purified with

Endotoxin-Free Plasmid Mega Kit (Qiagen) were used for transfection with PEI

(1:1.5 v:v). After reaching 90% confluence, the cells were transferred into a lower

(2%) serum containing media, and the transfection mixture containing DNA-PEI

complex was added. HEK293T and 293S cells were grown for 2-3 days and 4-5

days, respectively, for protein expression (12).

PTPBR7 DETECTION BY WESTERN BLOTTING

Small aliquots of conditioned media 2-3 days post transfection were

separated by SDS-PAGE and then transferred to Immobilon P membrane

(Millipore) for 1 h at 75 mA, using a semidry transfer system. The membrane was

treated with blocking agent (5% low fat skim milk (Fluka)) for 1 h at room

temperature with blocking buffer in a 50 mM Tris, pH 8.0, 150 mM NaCl buffer).

Then, the membrane was incubated with PentaHis monoclonal (against 6XHis tag)

primary antibody (1:1000 dilution, Qiagen) for 1 h at room temperature, followed

by incubation with the goat anti-mouse IgG peroxidase-conjugated secondary

antibody (1:2000, Sigma) for 1 h at room temperature. The blots were developed

using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific)

and exposed to Amersham Hyperfilm ECL (GE Healthcare).

PTPBR7 PROTEIN PURIFICATION

The conditioned media were collected 3 days post transfection in case of

HEK293T cells and 5 days in case of 293S cells. Conditioned media were filtered

through a 0.2 μm membrane (Express filter, Millipore) and diluted three folds

using PBS at pH 8.0 adjusted with Tris buffer. Immobilized metal affinity

chromatography (IMAC) purification is performed using nickel-coated chelating

Sepharose (GE Healthcare). Beads equilibrated with Phosphate Buffer Saline

(PBS) were added to the filtered, diluted conditioned media and incubated for

about 1 hour on a shaker incubator at 16ºC for the affinity binding of the Histagged

protein. The beads were separated by filtration and the bound protein was

eluted using 10 mM Tris-HCl buffer containing 150 mM sodium chloride and 300

mM imidazole pH 8.0. The elute profile was analyzed by SDS-PAGE. The

fractions containing the fused PTPBR7 were pooled and the protein was further

purified by size exclusion chromatography using Superdex 75 10/300GL (GE

Healthcare). The purity of the protein obtained was determined by SDS-PAGE.

N-TERMINAL SEQUENCING

Protein samples containing PTPBR7 were separated on 12% SDS-PAGE and

then transferred to Immobilon P membrane at 75 mA for 1 h using transfer buffer

that contained 10 mM CAPS, 10% methanol, pH 11. The membrane was washed

with water and stained with Coomassie Blue, and the stain excess was removed.

The bands corresponding to PTPBR7 were excised from the membrane and sent to

N-terminal sequencing (Proteomics facility, University of Leeds).

DEGLYCOSYLATION OF PTPBR7 USING PNGASE F

Purified protein samples were denatured in the presence of glycoprotein

denaturing buffer at 100ºC for 10 minutes, and further incubated with reaction

buffer, NP40 and PNGase (NEB) at 37ºC for 1 hour. The samples were further

analyzed by western blotting using PentaHis monoclonal (against 6XHis tag)

antibody.

RESULTS AND DISCUSSION

The full extracellular region of PTPBR7 is about 24 kDa and the construct

cloned into pHLsec obtained from Dr Hendriks was transfected into HEK293T

cells. Large-scale transfection in roller bottles using 0.5 mg of endotoxin free

purified plasmid DNA/bottle was used in complex with polyethylenimine (PEI).

The conditioned medium was collected 3 days post transfection as the protein is

secreted into the medium. The filtered medium was diluted with PBS and

incubated with nickel-coated chelating Sepharose on a shaker incubator. The beads

with bound His-tagged protein were separated, washed with 10 mM Tris-Hcl buffer

containing 150 mM sodium chloride, 5 mM imidazole, pH 8.0, and the protein was

eluted with 10 mM Tris-HCl buffer containing 150 mM sodium chloride and 300

mM imidazole pH 8.0. The protein was analyzed on 12% SDS-PAGE. A second

purification step was performed by size exclusion chromatography using Superdex

75 10/300GL column equilibrated with 10 mM Hepes pH 7.5 buffer containing

150 mM sodium chloride. The elution fractions were analyzed on 12% SDS5

PAGE. The obtained PTPBR7 protein was pure, but presented multiple fragments

of lower molecular weight in addition to the full-length protein of 24 kDa (Fig. 1

A). This could be due to the presence of different levels glycosylation of PTPBR7,

but the bioinformatics analysis using NetNGlyc server predicted only one N-linked

glycosylation site. Deglycosylation using PNGase F, which reduces the

heterogeneity in the protein caused by differential gycosylation did not reduce the

number of protein fragments (Fig. 1 B) and most of the protein fragments presented

lower size than the theoretical MW of ~24 KDa in the absence of glycosylation.

This suggested an N-terminal proteolytic cleavage of the secreted protein, ruling

out a C-terminal cleavage that would have affected the binding of this C-terminus

6X His-tag fused protein to IMAC matrix. To confirm this, the N-terminal

sequencing of several low mass protein fragments was performed. The purified

proteins were separated on 12% SDS-PAGE, and electroblotted on to an Imobilon

P (Millipore) membrane. The membrane was then stained with Coomassie Blue

and the corresponding protein bands were excised from the membrane and

analyzed by N-terminal sequencing. The results clearly showed three N-terminal

tryptic-like cleavage sites where the protein was cleaved (Fig. 2).

In order to obtain a stable extracellular region of PTPBR7, three shorter

constructs were obtained, coding for proteins with N-termini corresponding to each

of the identified proteolytic sites. The DNA fragments unique restriction sites AgeI

and KpnI in the pHLsec vector allowed the insertion of the shorter constructs of

ecto region of PTPBR7, named PTPBR7 S1 (SWKPVF), PTPBR7 S2 (SLDIAQ),

and PTPBR7 S3 (HNYHSP). PTPBR7 S2 and S3 constructs were used for largescale

protein production. HEK293S cells were used, which allows homogeneous

glycosylation of the recombinant protein. The shorter PTPBR7 protein produced by

293S cells was more stable and less prone to proteolytic cleavage (data not shown).

Therefore, the expression of PTPBR7 S2 and S3 constructs was performed in 293S

cells. The proteins obtained were purified by affinity chromatography (IMAC)

followed by a final step of purification by size exclusion chromatography as

described earlier. The proteins thus obtained were stable with less proteolytic

degradation as compared to the full ecto construct, the shortest construct being the

most stable as expected (Fig. 3).

In conclusion, the large-scale expression of the ecto domain of PTPBR7 was

obtained in mammalian cells. The full-length extracellular protein of PTPBR7 was

unstable during expression and purification processes, due to proteolytic cleavage

at multiple sites in the N-terminal region. This was confirmed by protein

sequencing of the N-terminal protein fragments obtained after purification. cDNAs

coding for N-terminal shorter constructs starting at the proteolytic cleavage sites

were cloned, expressed and purified as the full-length ecto BR7. The proteins thus

purified were resistant to proteolysis as compared to the full-length ecto region, the

shortest construct being the most stable. Consequently, by using this mammalian

expression system, we were able to obtain stable pure ecto domain of PTPBR7.

This protein can be used for further structural and functional in vitro studies.