IgMBRAZOR™ in a Study on Structural Characterization of IgM
Scientists at Utrecht University and collaborators have described the use of online SEC combined with Orbitrap-based CDMS to perform real-time kinetic monitoring of pentameric IgM digestion by the protease IgMBRAZOR, a novel protease which digests specifically at a single site in the hinge region of IgM. While IgMBRAZOR was shown to efficiently digest IgM, it was observed that digestion of one of the F(ab’)2 subunits occurred more slowly than the other four. It is suggested that interactions of the J-chain within the pentameric IgM structure may influence the accessibility of the IgMBRAZOR enzyme, thus, impacting the digestion kinetics. SEC-CDMS allows the characterization of heterogeneous and large proteins, enabling unique experimental designs such as monitoring of reaction kinetics from physiological buffers.
Immunoglobulin M (IgM) plays a crucial role in adaptive immunity and under normal physiological conditions, IgM is secreted into the bloodstream as J-chain-coupled asymmetric pentamers. IgM are large, heterogeneous molecules, with 51 N-glycosylation sites and a total molecular weight of ∼950 kDa. The emerging appeal and potential of IgM as biotherapeutics has renewed the interest in studying them by MS. Their high molecular weights and extensive glycosylation are challenging, even for conventional native MS analysis, however, Charge Detection Mass Spectrometry (CDMS) has proven to be extremely powerful for high mass and/or heavily glycosylated analytes, such as IgM.
The scientists here set out to investigate enzymatic degradation of IgM and characterize kinetic properties under physiological conditions using a Orbitrap-based CDMS approach. The primary goal of this study was to monitor the processing of IgM using IgMBRAZOR. IgMBRAZOR is a unique IgM-specific protease that digests human IgM at one specific site below the CH2 domain in the heavy chain, releasing the five F(ab’)2 moieties (∼130 kDa each) from the pentameric J-coupled Fc core (MW∼ 300 kDa). The automated Size-Exclusion Chromatography (SEC)-CDMS setup enabled real-time monitoring of the IgM digestion, using IgMBRAZOR, with accurate mass identification of all intermediate digestion products formed over time.
Figure 1. Schematic overview of the IgMBRAZOR workflow.
IgMBRAZOR was shown, using SDS-PAGE, to completely and rapidly digest IgM under normal conditions, and when the protease concentration was substantially decreased, multiple bands corresponding to intermediate digestion products were detected. The masses of the F(ab’)2 and Fc regions were accurately determined by SEC-CDMS and aligned with the predicted masses. SEC-CDMS was then applied to monitor digestion intermediates following prolonged IgMBRAZOR digestion to gain insight into the mechanism of IgM digestion. Several clearly resolved intermediates were identified and correlated to IgM missing one, two, three and four F(ab’)2 arms. Removal of the first four F(ab’)2 subunits appeared to occur rapidly, however, surprisingly, removal of the final F(ab’)2 arm occurred at a rate which was more than an order of magnitude slower than the previous four.
The difference in digestion kinetics was proposed to be related to the proximity of the final F(ab’)2 subunit to the J-chain. A Cryo-EM structure of full length pentameric IgM revealed that one of the five F(ab’)2 arms was substantially more conformationally rigid than the other four, which could impede IgMBRAZOR digestion. The F(ab’)2 moieties not stabilized by the J-chain were more flexible, likely making them more accessible for IgMBRAZOR digestion. This hypothesis was validated by digesting a recombinant IgM, lacking the J-chain, where the fully processed Fc core was reached rapidly and without accumulation of partially digested intermediates. These data support the hypothesis that the presence of the J-chain influences the structure of IgM and affects its digestion kinetics, although further work may be needed to confirm the exact identity of the stalled fifth F(ab’)2 subunit.
Here, it was demonstrated that SEC-CDMS enables analysis of IgM and its proteolytic cleavage products following IgMBRAZOR digestion. By monitoring digestion kinetics of IgM, strikingly different behaviors of the five F(ab’)2 arms were revealed, with the first four cleaved off efficiently, and the final, fifth arm, more resistant to digestion. It is hypothesized that the fifth F(ab’)2 arm is distinct in IgM due to its proximity and unique interaction with the J-chain, making it less accessible to IgMBRAZOR. Native SEC-MS is well integrated into R&D pipelines of biopharmaceutical companies and, moving forward, techniques such as SEC-CDMS could be used for extended structural characterization of an expanded range of therapeutics. The online SEC-CDMS methodologies described here open new avenues into higher throughput, automated analysis of heterogeneous, high-mass protein assemblies by CDMS.
Reference
Yin et al., 2024. Not All Arms of IgM Are Equal: Following Hinge-Directed Cleavage by Online Native SEC-Orbitrap-Based CDMS. J. Am. Soc. Mass Spectrom
IgMBRAZOR™ – Digestion of IgM