Interaction Properties of Human Mannan-Binding Lectin (MBL)-Associated Serine Proteases-1 and -2, MBL-Associated Protein 19, and MBL

• Published in: The Journal of immunology (1950) Vol. 166; no. 8; pp. 5068 - 5077
• Main Authors: Thielens, Nicole M, Cseh, Sandor, Thiel, Steffen, Vorup-Jensen, Thomas, Rossi, Veronique, Jensenius, Jens C, Arlaud, Gerard J
• Format: Journal Article
• Language: English
• Published: United States Am Assoc Immnol 15.04.2001
• Subjects: Amino Acid Motifs - genetics; Animals; Calcium - metabolism; Carrier Proteins - metabolism; Cations, Divalent - metabolism; Collectins; Complement C1s - genetics; Dimerization; Epidermal Growth Factor - genetics; Extracellular Matrix Proteins - genetics; Humans; Lectins - metabolism; mannan-binding lectin; Mannans - metabolism; Mannose-Binding Protein-Associated Serine Proteases; MAp19 protein; MASP-1 protein; MASP-2 protein; Peptide Fragments - biosynthesis; Peptide Fragments - genetics; Peptide Fragments - metabolism; Receptors, Complement 3b - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Serine Endopeptidases - genetics; Serine Endopeptidases - metabolism; Spodoptera - genetics; Surface Plasmon Resonance
• ISSN: 0022-1767; 1550-6606
• DOI: 10.4049/jimmunol.166.8.5068

The mannan-binding lectin (MBL) activation pathway of complement plays an important role in the innate immune defense against pathogenic microorganisms. In human serum, two MBL-associated serine proteases (MASP-1, MASP-2) and MBL-associated protein 19 (MAp19) were found to be associated with MBL. With a view to investigate the interaction properties of these proteins, human MASP-1, MASP-2, MAp19, as well as the N-terminal complement subcomponents C1r/C1s, Uegf, and bone morphogenetic protein-1-epidermal growth factor (CUB-EGF) segments of MASP-1 and MASP-2, were expressed in insect or human kidney cells, and MBL was isolated from human serum. Sedimentation velocity analysis indicated that the MASP-1 and MASP-2 CUB-EGF segments and the homologous protein MAp19 all behaved as homodimers (2.8-3.2 S) in the presence of Ca(2+). Although the latter two dimers were not dissociated by EDTA, their physical properties were affected. In contrast, the MASP-1 CUB-EGF homodimer was not sensitive to EDTA. The three proteins and full-length MASP-1 and MASP-2 showed no interaction with each other as judged by gel filtration and surface plasmon resonance spectroscopy. Using the latter technique, MASP-1, MASP-2, their CUB-EGF segments, and MAp19 were each shown to bind to immobilized MBL, with K:(D) values of 0.8 nM (MASP-2), 1.4 nM (MASP-1), 13.0 nM (MAp19 and MASP-2 CUB-EGF), and 25.7 nM (MASP-1 CUB-EGF). The binding was Ca(2+)-dependent and fully sensitive to EDTA in all cases. These data indicate that MASP-1, MASP-2, and MAp19 each associate as homodimers, and individually form Ca(2+)-dependent complexes with MBL through the CUB-EGF pair of each protein. This suggests that distinct MBL/MASP complexes may be involved in the activation or regulation of the MBL pathway.