2012•Doctoral thesis, UCL (University College London).Requires access
Complement-carbohydrate interactions : studies of mannose binding lectin and complement factor H
Ami Miller
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Abstract
The complement system is a fundamental component of innate immunity that
orchestrates complex immunological and inflammatory processes. Complement
comprises over 30 proteins that eliminates invading microorganisms while
maintaining host cell integrity. Protein-carbohydrate interactions play critical roles in
both the activation and regulation of complement.
Mannose binding lectin (MBL) activates the lectin pathway of complement
via the recognition of sugar arrays on pathogenic surfaces. X-ray scattering and AUC
combined with constrained modelling were used to identify a bent structure for the
MBL monomer in terms of crystal structures for its carbohydrate-recognition domain
and its triple helical region. Near-planar solution structures were determined for the
MBL dimer, trimer and tetramer. These solution structures clarified how MBL binds
to pathogenic surfaces and provides a template for the binding and autoactivation of
the MASP protease to initiate the lectin pathway of complement activation.
Factor H (FH) with 20 short complement regulator (SCR) domains regulates
the alternative pathway of complement by facilitating the breakdown of the central
component C3b. FH binds to heparan sulphate (HS) and to heparin (a HS analogue)
on host cell surfaces where it regulates C3b activity and protects these cells from
complement attack. A Tyr402His polymorphism in SCR-7 is a major risk factor for
the development of age-related macular degeneration (AMD), and is involved in
heparin binding. Both the Tyr402 and His402 allotypes of the SCR-6/8 fragment of
FH were cloned and expressed in an E. coli system. X-ray scattering, analytical
ultracentrifugation and surface plasmon resonance were used to characterise the
interactions of FH Tyr402 and His402 with heparin and HS. These polyanions induce
the strong self-association of FH SCR-6/8, the extent of which was found to be
dependent on the length of the polyanion and the presence of the His402 allotype.
The formation of large FH-heparin aggregates may provide a molecular explanation
for the link between the Tyr402His polymorphism and the initial formation of drusen
deposits in AMD.