Human protein tyrosine phosphatase, receptor type, N polypeptide 2

Target ID:

PTPRN2A

Aliases:

IA-2betaIARICAARPTPRP

Deposition Date:

Wednesday 27th June 2007

Families:

Phosphatases

Related Diseases:

NeurobiologySignallingMetabolism

Structure type:

apo

Download Coordinates:

2QEP

Authors:

Ugochukwu, E.; Barr, A.; Alfano, I.; Berridge, G.; Burgess-Brown, N.; Das, S.; Fedorov, O.; King, O.; Niesen, F.; Watt, S.; Savitsky, P.; Salah, E.; Pike, A.C.W.; Bunkoczi, G.; von Delft, F.; Sundstrom, M.; Edwards, A.; Arrowsmith, C.H.; Weigelt, J.; Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

2QEP

tabs

Structure Details

PTPRN and PTPRN2 form a small subfamily of receptor protein tyrosine phosphatases (PTPs) that have been reported to be catalytically inactive enzymes. The lack of enzymatic activity has been associated with a number of substitutions of conserved amino acids within the PTP domain known to be critical for PTP activity. Specifically, the conserved proline in the WPD loop is changed into a tyrosine, and the obligatory alanine located close to the catalytic cysteine is an aspartic acid residue. Due to their lack of catalytic activity a function as phosphotyrosine recognition/binding module has been suggested. High expression levels of PTPRN2 have been detected in brain, pituitary, and pancreas as well as in insulinoma, glucagoma. Furthermore, PTPRN2 expression is upregulated by ghrelin, an endogenous ligand for growth hormone (GH) secretagogue (GHS) receptor.

Interestingly, PTPRNs have been identified as the precursors of 40- and 37-kDa insulin-dependent diabetes mellitus-specific major auto-antigens. Autoantibodies to PTPRN2 appear years before the development of clinical disease and are diagnostic and predictive markers. PTPRN2 knockout mice show mild glucose intolerance and impaired glucose-stimulated insulin secretion. However, knockout of the PTPRN2 gene in NOD mice (Nonobese diabetic), the most widely studied animal model for human type 1 diabetes, fail to prevent the development of cyclophosphamide-induced diabetes.

PTPRN2 has also been implicated in the development of the nervous system and regulation of exocytosis of insulin-containing secretory granules in pancreatic beta cells. In addition, PTPRN2 may regulate PTP activity of a number of receptor type PTPs through interaction with their D1 domains.

Materials and Methods

PTPRN2

PDB:2QEP

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|11386149
Entry Clone Source:MGC
SGC Clone Accession:
Tag:NON-cleavable C-terminal His 6 tag
Host:Phage-resistant derivative of BL21(DE3)

Construct

Prelude:
Sequence:MSEEPVQSNMDISTGHMILSYMEDHLKNK NRLEKEWEALCAYQAEPNSSFVAQREENV PKNRSLAVLTYDHSRVLLKAENSHSHSDY INASPIMDHDPRNPAYIATQGPLPATVAD FWQMVWESGCVVIVMLTPLAENGVRQCYH YWPDEGSNLYHIYEVNLVSEHIWCEDFLV RSFYLKNLQTNETRTVTQFHFLSWYDRGV PSSSRSLLDFRRKVNKCYRGRSCPIIVHC SDGAGRSGTYVLIDMVLNKMAKGAKEIDI AATLEHLRDQRPGMVQTKEQFEFALTAVA EEVNAILAhhhhhh
Vector:pNIC-CH.

Growth

Medium:LB
Antibiotics:
Procedure:1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin was used to inoculate 1 litre of LB containing 50 µg/ml kanamycin. Cultures were grown at 37°C until the OD600 reached ~0.3 then the temperature was adjusted to 18°C. Expression was induced for 4 hours using 1 mM IPTG at an OD600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen.

Purification

Procedure
Column 1: Ion exchange - Nucleic acid removal. DEAE cellulose (DE52, Whatman), 10 g of resin in 2.5 x 20 cm column. The resin was hydrated in 2.5M NaCl, washed with 20 ml binding buffer prior to loading the sample.
Column 2: Ni-affinity. Ni-NTA (Qiagen), 5 ml of 50% slurry in 1.5 x 10 cm column, washed with binding buffer.
Column 3: Size Exclusion Chromatography. Superdex S200 16/60 HiLoad

Supernatant was applied by gravity flow with column 1, followed by a wash with 100 ml binding buffer. The column flow-through was collected.
The flowthrough from column 1 was loaded by gravity flow on the Ni-NTA column. The column was then washed with 100 ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5-ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM and 250 mM); fractions were collected until essentially all protein was eluted.
Eluted protein , was directly applied to a S200 16/60 HiLoad gel filtration column equilibrated in 50 mM Tris-HCl pH 7.5, 250 mM NaCl, 10 mM DTT using an AKTAxpress system

Extraction

Procedure
Frozen pellets were thawed and cells lysed using a high pressure cell disrupter. The lysate was centrifuged at 17,000 rpm for 30 minutes and the supernatant collected for purification.
Concentration:Protein was concentrated to 40 mg/ml using an Amicon 10 kDa cut-off concentrator.
Ligand
MassSpec:LC- ESI -MS TOF indicated a loss of 130 a.m.u. corresponding to the proteolytic cleavage of the initial methionine. Expected mass : 34931; Observed mass: 34799
Crystallization:Crystals were grown at 20°C in 200 nl sitting drops mixing 100 nl of 40 mg/ml protein with 100 nl of a solution containing 0.2 M Na/KPO4; 0.1 M BTProp pH 6.5; 20.0% PEG 3350; 10.0% EtGly. The crystals were cryo-protected using 25% ethylene glycol which was added to the drop 30 seconds prior to mounting and flash freezing in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 2.5 Å. X-ray source: Diffraction data were collected at the SLS beamline X10SA at a single wavelength (0.9999 Å).
Data Processing:

References

Alcalde, L., Tonacchera, M., Costagliola, S., Jaraquemada, D., Pujol-Borrell, R., and Ludgate, M. (1996). Cloning of candidate autoantigen carboxypeptidase H from a human islet library: sequence identity with human brain CPH. J Autoimmun 9, 525-528.
Morahan, G., Huang, D., Yu, W. P., Cui, L., DeAizpurua, H., and Pallen, C. J. (1998). Localization of the genes encoding the type I diabetes autoantigens, protein-tyrosine phosphatases IA2 and IAR. Mamm Genome 9, 593-594.
Vo, Y. P., Hutton, J. C., and Angleson, J. K. (2004). Recycling of the dense-core vesicle membrane protein phogrin in Min6 beta-cells. Biochem Biophys Res Commun 324, 1004-1010.
Wasserfall, C. H., and Atkinson, M. A. (2006). Autoantibody markers for the diagnosis and prediction of type 1 diabetes. Autoimmun Rev 5, 424-428.