Genetic variation and bacteria binding functions of mouse mannan-binding lectins and ficolins
Mannan-binding lectin (MBL) and ficolin are collagenous lectins that are involved in innate resistance to microbial pathogens. The objectives of this thesis were to determine whether genetic variation in collagenous lectins exists among different strains of laboratory mice, and, to investigate bacterial binding properties of murine MBL-A and MBL-C. MBL-A and MBL-C were eluted with N-acetylglucosamine (GlcNAc) from intact bacteria incubated in mouse plasma and identified in non-reducing SDS-PAGE using Western blot analysis and monoclonal antibodies. GIGNAc eluates of plasma incubated with mannan-sepharose beads, 'Klebsiella oxytoca, Pseudomonas aeruginosa' and ' Staphylococcus aureus' contained similar bands (mainly ~50 kDa) that were immunoreactive with MBL-C antibody. By comparison, immunoreactive MBL-A (a ladder of ~175 kDa and larger bands) was identified in GlcNAc eluates from mannan-sepharose beads, 'S. aureus' and ' K. oxytoca' but not 'P. aeruginosa.' SNPs in expressed collagenous lectin genes 'Mbl1, Mbl2, Fcna, ' and 'Fcnb' were compared in ten strains of mice designated high priority Group A strains by the Mouse Phenome Project (129S1/SvImJ, A/J, BALB/cByJ, C3H/HeJ, C57BL/6J, DBA/2J, FVB/NJ, SJL/J, CAST/EiJ and SPRET/EiJ). Sequence comparisons identified a total of 15 SNPs in 'Mbl1' in two strains, 27 SNPs in 'Mbl2' in five strains, and 19 and 15 SNPs in 'Fcna' and 'Fcnb,' respectively, in two strains. Two non-synonymous SNPs were identified in the collagen-like domain of mouse 'Fcnb' that are similar to the coding polymorphisms in the collagen-like domain of human 'MBL2.' Several non-synonymous SNPs identified in 'Mbl1' and 'Mbl2' occurred in the carbohydrate-recognition domains (CRDs) and some resulted in altered residues close to known ligand binding sites. The miscoding SNPs found in the CRD regions of mouse 'Mbl1, Mbi2, Fcna,' and 'Fcnb ' might be associated with strain differences in glycan binding avidity and disposition of microbial or host ligands. Further, the mutations in the collagen-like domain of 'Fcnb' may alter the structure of mature ficolin-B leading to functional deficiencies. Results of these studies demonstrate that mouse 'Mbl1, Mbl2, Fcna,' and 'Fcnb' are polymorphic and suggest that mouse MBL-A and MBL-C differ in their lectin activity for the surfaces of bacteria that are pathogens of mice.