Native top-down mass spectrometry reveals role of glycans in protein oligomers
Many proteins contain patterns of sugar molecules (glycans) and are
made of several aggregated subunits. This glycosylation and
oligomerization has a decisive influence on protein function and must be
considered in biopharmaceutical development. In the journal Angewandte Chemie,
a British team has introduced an approach based on native top-down mass
spectroscopy (MS) that can be used to analyze the interplay between
glycosylation and oligomerization in various therapeutic hormones and
cytokines.
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In conventional MS, molecules break into fragments, but native MS
makes it possible to examine folded protein oligomers bound to glycans.
In the top-down method, the oligomers are subsequently separated by
means of a gas-phase dissociation and measured. In a final step,
gas-phase fragmentation is used to split off and analyze the glycans.
For a given protein complex, it is thus possible to determine the
proportions of monomers and oligomers present—whether in an organism or
in a batch of medication—and which different glycosylation patterns
occur and in what amounts.
Carol V. Robinson and Di Wu at the University of Oxford (UK)
concentrated on glycans that are located at the interface between two
subunits and can play an important role in oligomerization. They
compared the results of their measurements with a theoretical model that
was computed based on the dissociated subunits. This made it possible
to draw conclusions about the stabilization effect of the glycans.
One of the therapeutic glycoproteins they studied was interferon-β
(IFN-β), an anti-inflammatory cytokine that is used to treat conditions
such as multiple sclerosis. IFN-β1a forms an asymmetrical homodimer. The
MS analyses showed that most of the primary forms of both the monomer
and dimer are glycosylated. However, for this cytokine, dimerization is
independent of glycosylation status.
Things are different for tumor necrosis factor-α (TNF-α), an
inflammatory cytokine. Antibody-based biopharmaceuticals that neutralize
TNF-α are used in cases of autoimmune disease, such as rheumatoid
arthritis, Crohn’s disease, and psoriasis. TNF-α is a homotrimer with
one glycan at the interface of each subunit. The MS analyses, and
experiments with a small molecule that disrupts the trimerization,
indicated that the glycans significantly stabilize the TNF-α trimer.
Robinson and Wu also studied the follicle-stimulating hormone (FSH,
follitropin), a heterodimer made of α and β subunits. Drugs based on
follitropin α are used for fertility treatments. The team discovered an
unusual distribution of glycans on the α subunit. One of these glycans
interacts extensively with the β subunit and is clearly involved in
regulation of the dimerization.
The knowledge gained through native top-down MS experiments could
help to tailor glycans in therapeutic proteins to improve their
stability and effectiveness.
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About the Author
Prof. Carol Robinson
is the director of the Kavli Institute in Oxford and has a long-term
interest in the application of mass spectrometry to understanding
protein structure and interactions.
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