Molecular basis of sugar recognition by collectin-K1 and the effects of mutations associated with 3MC syndrome

Abstract

BackgroundCollectin-K1 (CL-K1, or CL-11) is a multifunctional Ca2+-dependent lectin with roles in innate immunity, apoptosis and embryogenesis. It binds to carbohydrates on pathogens to activate the lectin pathway of complement and together with its associated serine protease MASP-3 serves as a guidance cue for neural crest development. High serum levels are associated with disseminated intravascular coagulation, where spontaneous clotting can lead to multiple organ failure. Autosomal mutations in the CL-K1 or MASP-3 genes cause a developmental disorder called 3MC (Carnevale, Mingarelli, Malpuech and Michels) syndrome, characterised by facial, genital, renal and limb abnormalities. One of these mutations (Gly204Ser in the CL-K1 gene) is associated with undetectable levels of protein in the serum of affected individuals.ResultsIn this study, we show that CL-K1 primarily targets a subset of high-mannose oligosaccharides present on both self- and non-self structures, and provide the structural basis for its ligand specificity. We also demonstrate that three disease-associated mutations prevent secretion of CL-K1 from mammalian cells, accounting for the protein deficiency observed in patients. Interestingly, none of the mutations prevent folding or oligomerization of recombinant fragments containing the mutations in vitro. Instead, they prevent Ca2+ binding by the carbohydrate-recognition domains of CL-K1. We propose that failure to bind Ca2+ during biosynthesis leads to structural defects that prevent secretion of CL-K1, thus providing a molecular explanation of the genetic disorder.ConclusionsWe have established the sugar specificity of CL-K1 and demonstrated that it targets high-mannose oligosaccharides on self- and non-self structures via an extended binding site which recognises the terminal two mannose residues of the carbohydrate ligand. We have also shown that mutations associated with a rare developmental disorder called 3MC syndrome prevent the secretion of CL-K1, probably as a result of structural defects caused by disruption of Ca2+ binding during biosynthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0136-2) contains supplementary material, which is available to authorized users.

l‐Fucose prevention of renal ischaemia/reperfusion injury in Mice

“…Previous studies have shown that it binds to MASPs with high affinities, 31 similar to those of MBL-MASP and ficolin-MASP complexes. 26 This ELISA establishes l-fucose blocks rCL-11 ( Figure 2) at a concentration that approximates the amount of fucose present in the kidney at the induction of I/R injury. 26 This ELISA establishes l-fucose blocks rCL-11 ( Figure 2) at a concentration that approximates the amount of fucose present in the kidney at the induction of I/R injury.…”

“…The rCL-11 (3 µg/mL) 26 and monosaccharide inhibitor (l-fucose [Sigma, Gillingham, Dorset, UK], d-mannose [Sigma Gillingham, Dorset, UK], or dgalactose [Sigma, Gillingham, Dorset, UK]), diluent alone, or 10 mM ethylenediaminetetraacetic acid (EDTA) solution were mixed in 1:1 ratio prior to incubation overnight at 4°C. Plates were blocked for 2 hours at room temperature (RT) with tris buffered saline (TBS)/Tw/Ca + 0.1% bovine serum albumin (BSA).…”

“…Human rCL-11 (0.9 µg/mL) 26 was added to CL-11 -/mouse serum 30 minutes prior to addition to PTECs alongside l-fucose, d-galactose or culture media (without FCS). A, Confirmation of K-fucose kit (megazyme) specificity, showing detection of increasing concentrations of l-fucose, d-mannose and d-galactose compared with manufacturer's l-fucose standard.…”

A journey through the lectin pathway of complement—MBL and beyond

“…On the other end of the spectra, lack of secreted CL‐11 was found to be responsible for the development of the 3MC (Malpuech, Michels, Mingarelli, and Carnevale) syndrome due to the missing CL‐11‐guided migration of neural crest cells . COLEC11 mutations were shown to interfere with the binding to calcium and carbohydrates, probably resulting in misfolded CL‐11 peptides that are targeted for degradation within the cell …”

Collectin-11 Is an Important Modulator of Retinal Pigment Epithelial Cell Phagocytosis and Cytokine Production

“…Previous studies have shown that CL-11 preferentially binds to L -fucose, D -mannose, and highmannose glycans, among other possible monosaccharide ligands [4,13,23] . We therefore performed lectin staining in mouse eye tissue, using 2 fluorescein-labeled lectins: GNL (for detecting structures containing terminal [α-1,3] mannosyl residues) and LCA (for detecting branched fucose/α-linked mannose residues).…”