Characterization of Recombinant β2GPI Using Hydrogen-Deuterium Exchange and X-Ray Crystallography
Authors: Alluri Ravi Kumar , Arce Nicholas , Klenotic Philip , Su Chih Chia , Kundu Suman , Yu Edward , Li Renhao , McCrae Keith R 
Published: November 2019

Introduction:
Antiphospholipid syndrome (APS) is an autoimmune disorder caused by "antiphospholipid" antibodies (aPL) directed against β2-glycoprotein I (β2GPI). However, there remains significant inconsistency in measurements of anti-β2GPI antibodies, possibly due to different sources of β2GPI. To address this issue and others, we have developed a system in which recombinant β2GPI (rβ2GPI) is expressed in mammalian (HEK293) cells using a lentiviral construct with a CMV promoter, in which the signal peptide of β2GPI has been mutated. In previous work, we demonstrated that patient-derived anti-β2GPI antibodies recognize rβ2GPI similarly to purified plasma β2GPI. Plasma Β2GPI has been crystallized and found to be in a "fishhook" or "J" conformation, however the protein was isolated under harsh conditions that included perchloric acid precipitation. Moreover, electron microscopy and other approaches have suggested that β2GPI may adopt several conformations in addition to the fishhook, including circular and other intermediate forms.
Methods:
cDNA encoding full length APOH (the gene encoding β2GPI), was cloned into pLenti CMV Puro DEST. Lentivirus was produced using the Lentiviral Gateway Expression kit and stable cell lines developed by transducing HEK293 cells with lentivirus encoding APOH. Cell lines were grown in suspension, and supernatants containing secreted full length β2GPI were purified using a hitrap-heparin column.
Anti-β2GPI antibody binding to rβ2GPI was determined using a standard β2GPI ELISA. For hydrogen-deuterium exchange (HDX) mass spectrometry, proteins were in 10 mM phosphate buffer, 99.9% D2O in an autosampler at 20 °C. After 10-10,000 seconds of incubation, the reaction was quenched. Sequences of peptic fragments were identified and mass assignment for each peptide without deuterium exchange was checked manually. The average of relative fractional deuterium uptake for each amide proton residue was calculated using HDsite equations. Crystallization of plasma derived and recombinant β2GPI-WT was performed using a hanging-drop vapor diffusion method at 4°C in 150 mM NaCl and 50 mM Tris-HCl pH 7.3.
Results:
After transduction with lentiviral β2GPI constructs, HEK-293 cells efficiently secreted rβ2GPI-WT, site-directed mutants and several domains constructs. Hydrogen-deuterium exchange (HDX) analysis of plasma derived and rβ2GPI-WT revealed that both have very similar surface deuterium exchange rates and heat map profiles (Figure 1A, B). In both plasma derived and rβ2GPI, limited digestion of domains D-I, II and III by pepsin was observed, consistent with other sushi domain containing proteins. However, solid coverage of domain IV and V was obtained and the heat maps generated for both plasma derived and rβ2GPI-WT were very similar, suggesting similar structure and conformation. Moreover, although crystallization of plasma derived and rβ2GPI-WT revealed differences in crystal size and appearance, X-ray structural analysis to a resolution of 3.1 Å (Figure 1C, D) demonstrated that both plasma derived and rβ2GPI had the same structure and conformation under the conditions employed.
Conclusion:
These studies demonstrate that recombinant β2GPI produced in mammalian cells following lentiviral transduction has a similar structure and conformation as plasma-derived β2GPI. This methodology may be used for additional mutagenesis studies to better define the underlying structural features regulating the conformation of β2GPI as well as its interactions with other proteins, diagnostic utility and functions.

Disclosures
McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.