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Ficolins and mannose-binding lectins (MBLs) are the first components of the lectin branch of the complement system. They comprise N-terminal collagen-like domains and C-terminal pathogen-recognition domains (fibrinogen-like domains in ficolins and C-type carbohydrate-recognition domains in MBLs), which target surface-exposed N-acetyl groups or mannose-like sugars on microbial cell walls. Binding leads to activation of MBL-associated serine protease-2 (MASP-2) to initiate complement activation and pathogen neutralization. Recent studies have shown that MASP-2 binds to a short segment of the collagen-like domain of MBL. However, the interaction between ficolins and MASP-2 is relatively poorly understood. In this study, we show that the MASP-2 binding site on rat ficolin-A is also located within the collagen-like domain and encompasses a conserved motif that is present in both MBLs and ficolins. Characterization of this motif using site-directed mutagenesis reveals that a lysine residue in the X position of the Gly-X-Y collagen repeat, Lys(56) in ficolin-A, which is present in all ficolins and MBLs known to activate complement, is essential for MASP-2 binding. Adjacent residues also make important contributions to binding as well as to MASP activation probably by stabilizing the local collagen helix. Equivalent binding sites and comparable activation kinetics of MASP-2 suggest that complement activation by ficolins and MBLs proceeds by analogous mechanisms.

Integrin a2β1 is a major cellular receptor for collagens, but the molecular basis of its function is unknown. The α2 subunit contains a von Willebrand factor A-domain (I-domain) in its N-terminal region, and it has been demonstrated recently that this domain binds specifically to collagen I. This interaction requires divalent cations (e.g., Mg2+) and native collagen conformation, as does binding of the parent integrin to collagen. The α2 A-domain therefore has a number of functional similarities to the parent integrin, α2β1. However, while sequence specificity has been demonstrated for the parent integrin, no such observations have been made for the A-domain. In particular, it is not known whether the A-domain is responsible for sequence-specific recognition of collagens or whether it binds to the generic collagenous motif. To investigate the ligand specificity of the α2 A-domain, its binding to a range of collagenous ligands has been studied, with cation dependence, collagen triple-helicity, and inhibition by function blocking antibodies as criteria for specificity. Binding of the parent integrin was examined for comparison. The α2 A-domain was found to bind specifically to collagens l, 11, IV and XI. The complement component Clq has a collagenous domain but this was unable to support specific binding of α2 A domain or α2β1. Furthermore, synthetic triple-helical collagenous peptides failed to act as specific ligands. In conclusion, the α2 A-domain binds specifically to a range of extracellular matrix collagens, but it is not a receptor for all collagenous triple helices. By inference, these findings indicate the existence of an integrin-specific sequence motif within collagenous ligands recognised by the α2 A-domain. [ABSTRACT FROM AUTHOR]

A mouse macrophage λgtl1 cDNA library was screened using a genomic DNA clone coding for the C-chain gene of human Clq. Approximately 600000 recombinant phage plaques were hybridized with peroxidase-labeled human C-chain probe and detected by enhanced chemiluminescence. Five positive clones were obtained. The size of the full-length cDNA is 1019 bp. The sequence identity of the nucleotide sequence with human Clq C chain is 79%, the identity of the deduced amino acid sequences is 73%. The mouse Clq C chain exhibits the same structural features as the human C chain, e.g. conservation of the cysteine residues. Like the mouse A chain, the mouse C chain has an RGD sequence that may be recognized by receptors of the integrin family. No RGD sequences have been found in any of the human Clq chains. The size of the C-chain mRNA (1.2 kb) and its tissue distribution (macrophages being the cell type with the highest mRNA concentration) are identical to the mRNA of the mouse A and B chains. Alignment of human and mouse Clq A, B and C chains exhibits two blocks of highly conserved residues within the C-terminal globular regions. Three other proteins, collagen type VIII and type X and precerebellin share this similarity with Clq, indicating the structural and probably functional importance of these regions within the non-collagenous domains of the molecules. [ABSTRACT FROM AUTHOR]

Collectins are a family of innate immune proteins that contain fibrillar collagen-like regions and globular carbohydrate recognition domains (CRDs). The CRDs of these proteins recognize various microbial surface-specific carbohydrate patterns, particularly hexoses. We hypothesized that collectins, such as pulmonary surfactant proteins (SPs) SP-A and SP-D and serum protein mannose-binding lectin, could recognize nucleic acids, pentose-based anionic phosphate polymers. Here we show that collectins bind DNA from a variety of origins, including bacteria, mice, and synthetic oligonucleotides. Pentoses, such as arabinose, ribose, and deoxyribose, inhibit the interaction between SP-D and mannan, one of the well-studied hexose ligands for SP-D, and biologically relevant D-forms of the pentoses are better competitors than the L-forms. In addition, DNA and RNA polymer-related compounds, such as nucleotide diphosphates and triphosphates, also inhibit the carbohydrate binding ability of SP-D, or ∼60 kDa trimeric recombinant fragments of SP-D that are composed of the α-helical coiled-coil neck region and three CRDs (SP-D(n/ CRD)) or SP-D(n/CRD) with eight GXY repeats (SPD(GXY)8(n/CRD)). Direct binding and competition studies suggest that collectins bind nucleic acid via their CRDs as well as by their collagen-like regions, and that SP-D binds DNA more effectively than do SP-A and mannose-binding lectin at physiological salt conditions. Furthermore, the SP-D(GXY)8(n/CRD) fragments co-localize with DNA, and the protein competes the interaction between propidium iodide, a DNA-binding dye, and apoptotic cells. In conclusion, we show that collectins are a new class of proteins that bind free DNA and the DNA present on apoptotic cells by both their globular CRDs and collagen-like regions. Collectins may therefore play an important role in decreasing the inflammation caused by DNA in lungs and other tissues. [ABSTRACT FROM AUTHOR]