New insights on the structural/functional properties of recombinant human mannan-binding lectin and its variants.
Inefficient activation of complement lectin pathway in individuals with variant mannan-binding lectin (MBL) genotypes has been attributed to poor formation of higher order oligomers by MBL. But recent studies have shown the presence of large oligomers of MBL (approximately 450 kDa) in serum of individuals with variant MBL alleles. The recombinant forms of MBL (rMBL) variants except MBL/B that assemble into higher order oligomers have not yet been reported. In the present study, structural/functional properties of recombinant forms of wild type MBL (rMBL/A) and its three structural variants, rMBL/B, C, and D generated in insect cells were examined. Western blot analysis indicated covalently linked monomers to hexamers while gel filtration chromatography exhibited non-covalently linked higher order oligomers in addition to prevalent low oligomeric forms. Mannan binding determined by ELISA showed rMBL/A but not the structural variants bind to mannan. Apparent avidity of monoclonal antibody used was found to be about 18- to 52-fold weaker for rMBL structural variants than rMBL/A. Complement activation varied with maximum impairment apparent in rMBL/C followed by rMBL/B, but rMBL/D was functional to the same extent as rMBL/A. Comparison of rMBL/A to MBL purified from plasma (pMBL/A) indicated 8- and 24-fold weaker binding to mannan by BIAcore analysis and ELISA and about 5-fold lesser efficiency in activating complement. The findings provide new insights on the structural/functional properties of rMBL variants and imply that lectin pathway activation may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D.
Department of Biochemistry, University of Texas Health Science Center, 11937, US Highway 271, Tyler, TX 75708-3154, USA.
Rema
R
Rajagopalan
Veena P
VP
Salvi
Jens Chr
JC
Jensenius
Nenoo
N
Rawal
D003167:Complement Activation / Q000235:genetics / Q000276:immunology*; D003170:Complement Pathway, Alternative / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D005838:Genotype; D006801:Humans; D037601:Mannose-Binding Lectin / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D037601:Mannose-Binding Lectin; D011994:Recombinant Proteins