Disease-associated mutations in human mannose-binding lectin compromise oligomerization and activity of the final protein.
Deficiency of human mannose-binding lectin (MBL) caused by mutations in the coding part of the MBL2 gene is associated with increased risk and severity of infections and autoimmunity. To study the biological consequences of MBL mutations, we expressed wild type MBL and mutated MBL in Chinese hamster ovary cells. The normal MBL cDNA (WT MBL-A) was cloned, and the three known natural and two artificial variants were expressed in Chinese hamster ovary cells. When analyzed, WT MBL-A formed covalently linked higher oligomers with a molecular mass of about 300-450 kDa, corresponding to 12-18 single chains or 4-6 structural units. By contrast, all MBL variants formed a dominant band of about 50 kDa, with increasingly weaker bands at 75, 100, and 125 kDa corresponding to two, three, four, and five chains, respectively. In contrast to WT MBL-A, variant MBL formed noncovalent oligomers containing up to six chains (two structural units). MBL variants bound ligands with a markedly reduced capacity compared with WT MBL-A. Mutations in the collagenous region of human MBL compromise assembly of higher order oligomers, resulting in reduced ligand binding capacity and thus reduced capability to activate complement.
Larsen
Flemming
F
Department of Clinical Immunology, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. flar@biobase.dk
Madsen
Hans O
HO
Sim
Robert B
RB
Koch
Claus
C
Garred
Peter
P
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D016466:CHO Cells; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D003167:Complement Activation / Q000235:genetics; D006224:Cricetinae; D004194:Disease; D014644:Genetic Variation; D006801:Humans; D046911:Macromolecular Substances; D037601:Mannose-Binding Lectin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D003062:Codon; C445619:MBL2 protein, human; D046911:Macromolecular Substances; D037601:Mannose-Binding Lectin; D011994:Recombinant Proteins