Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Peptide & NMR Spectroscopy Studies of Recombinant Domain I Confirm Conformationally Correct Domain I and Non-Linear Epitope Binding to Anti-Domain I Antiphospholipid Antibodies.

Pericleous2,  Charis, Disu4,  Toluwape, Miles1,  Jennifer, Esposito1,  Diego, Garza-Garcia1,  Acely, Driscoll1,  Paul C., Latchman4,  David S.

National Institute of Medical Research, London, UK
University College London, London, United Kingdom
University College London, London, United Kingdom
University College London, London, UK

Purpose:

Recombinant Domain I (DI) is increasingly being utilised in the development of diagnostic assays and being developed as an inhibitor of pathogenicity. The critical epitope R39-R43 as well as the DI/DII interlinker region have been shown to be important in binding antiphospholipid antibodies. We undertook NMR spectroscopy studies as well as linear epitope peptide binding assays to confirm that recombinant DI was indeed conformationally correct and ascertain the potential binding abilities of different linear peptides based on the defined epitopes.

Methods:

Sera from 8 patients with APS (8/8 female; mean age 46.7±16.4; 7/8 Caucasian, 1/8 Asian; 8/8 with previous thrombosis, 4/8 with previous pregnancy morbidity) were tested in a standard direct anti-b2GPI assay and the appropriate dilution corresponding to 50% of maximal binding (MBD) was identified. Linear peptides containing region R39-R43 (PEP1, 9aa, 1kDa), DI-II interlinker (PEP2, 9aa, 1kDa), or both (PEP3, 26aa, 3kDa) were designed and synthesised (Sigma, UK). 7KDa wild type recombinant human (WT) DI was expressed in E.coli cells and folded in vitro in a cysteine/cystine-rich buffer for disulfide bond formation. Sera at a dilution that conferred 50% MBD anti-b2GPI activity were incubated (2h, RT) with increasing doses of WT DI (0, 20, 40, 60, 80 or 100mg/ml) or equimolar doses of PEP1–3, and their anti-b2GPI activity was assessed. Percentage binding was calculated based on the change in OD405nm in the presence of inhibitor. To confirm WT DI was conformationally correct, NMR spectroscopy was employed following standard triple resonance 3D NMR protocols. The Chemical Shift Index (CSI) and TALOS computer programs were used to compare the secondary structure of WT DI alone with that in whole human b2GPI based on the published 3D crystal structure.

Results:

The inhibitory ability of WT DI was statistically superior compared to all peptides, throughout the entire range of inhibitor concentrations tested (p<0.05 for 20mg/ml WT DI and equimolar PEP1–3; p<0.001 in all other cases). By calculating the mean percentage binding (±standard deviation) of all 8 sera tested, a clear positive correlation between the concentration of WT DI and level of inhibition was identified (from 75.7±29.9% with 20mg/ml WT DI to 25.0±22.0% with 100mg/ml WT DI), as opposed to PEP1–3 where binding did not fall below 91.3±30.3% (with PEP3 at the highest dose). NMR-derived parameters of WT DI were entirely consistent with the secondary structure of crystallised DI in whole b2GPI. The 3D structure of WT DI superimposed well with the b2GPI crystal structure, indicating that in solution WT DI adopts the anticipated polypeptide fold.

Conclusions:

NMR studies demonstrate that bacterially expressed recombinant DI adopts its native conformation supporting the use of this peptide in diagnostic assays and as an inhibitor of pathogenicity. Furthermore, whole DI peptide is necessary to inhibit as patient anti-DI sera failed to recognise linear epitope peptide derivatives.

To cite this abstract, please use the following information:
Pericleous, Charis, Disu, Toluwape, Miles, Jennifer, Esposito, Diego, Garza-Garcia, Acely, Driscoll, Paul C., et al; Peptide & NMR Spectroscopy Studies of Recombinant Domain I Confirm Conformationally Correct Domain I and Non-Linear Epitope Binding to Anti-Domain I Antiphospholipid Antibodies. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1358
DOI: 10.1002/art.29124

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