The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases.
Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 +/- 170 Da (MBL-I) and 304,899 +/- 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 +/- 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable K(D) values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.
Teillet
Florence
F
Laboratoires d'Enzymologie Moléculaire, Institut de Biologie Structurale Jean-Pierre Ebel (Commissariat à l'Energie Atomique-Centre National de la Recherche Scientifique-Université Joseph Fourier), Grenoble, France.
Dublet
Bernard
B
Andrieu
Jean-Pierre
JP
Gaboriaud
Christine
C
Arlaud
Gérard J
GJ
Thielens
Nicole M
NM
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism*; D002850:Chromatography, Gel; D004220:Disulfides / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008358:Mannose / Q000378:metabolism*; D037601:Mannose-Binding Lectin / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D037241:Mannose-Binding Lectins; D050606:Mannose-Binding Protein-Associated Serine Proteases; D011485:Protein Binding / Q000276:immunology; D025941:Protein Interaction Mapping; D012697:Serine Endopeptidases / Q000378:metabolism*; D012709:Serum Albumin / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020349:Surface Plasmon Resonance
D004220:Disulfides; C445619:MBL2 protein, human; D037601:Mannose-Binding Lectin; D037241:Mannose-Binding Lectins; C050080:N-acetylglucosamine-bovine serum albumin conjugate; D012709:Serum Albumin; C023840:mannose-bovine serum albumin conjugate; D012710:Serum Albumin, Bovine; C084067:MASP1 protein, human; C105296:MASP2 protein, human; D050606:Mannose-Binding Protein-Associated Serine Proteases; D012697:Serine Endopeptidases; D008358:Mannose; D000117:Acetylglucosamine