Lipscombe, Richard John; (1995) Mannose binding protein deficiency: Immunochemistry and mutation analysis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mannose Binding Protein (MBP) is a serum lectin believed to play a role in innate immunity by specifically recognising foreign carbohydrate structures, and evolutionary studies have shown that MBP is an ancient molecule found in species as diverse as shark and man. Previous work has linked human MBP deficiency with a common opsonisation defect that predisposes to frequent infections during infancy, and associated low protein levels with a point mutation in codon 54 of the MBP gene. The investigations described here extend our knowledge of MBP deficiency from its original discovery in Caucasians into other population groups using both functional and quantitative ELISA procedures. MBP deficiency was found in the Chinese and was associated with the codon 54 mutation, whereas in Gambians (West Africa) a new point mutation was discovered in codon 57. Both mutations are predicted to disrupt the structure of the collagenous region of MBP. The frequencies of the structural MBP gene mutations were investigated in these populations by PCR followed by restriction enzyme digestion, oligospecific hybridisation, or direct sequencing, and compared with serum protein concentrations for each group. This showed conclusively that the Eurasian and African mutations account for most of the observed low MBP levels. The geographical distribution of the mutations was further investigated by studies on a range of populations including Xhosa (South Africa), San Bushmen (Namibia), and inhabitants from Papua New Guinea and Vanuatu (South West Pacific). A physicochemical evaluation of the effects of the mutations on circulating MBP was also undertaken. Protein forms within sera were separated on the basis of charge and size by electrophoresis, gel filtration and sucrose density centrifugation, and analysed by immunoblotting. This suggested that the major serum forms of wild type MBP are dimers, trimers and tetramers of three-chain subunits, whilst the mutant phenotypes are predominantly of lower molecular mass.

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