Labeled TEMPO-Oxidized Mannan Differentiates Binding Profiles within the Collectin Families

Abstract

Establishing the rapid and accurate diagnosis of sepsis is a key component to the improvement of clinical outcomes. The ability of analytical platforms to rapidly detect pathogen-associated molecular patterns (PAMP) in blood could provide a powerful host-independent biomarker of sepsis. A novel concept was investigated based on the idea that a pre-bound and fluorescent ligand could be released from lectins in contact with high-affinity ligands (such as PAMPs). To create fluorescent ligands with precise avidity, the kinetically followed TEMPO oxidation of yeast mannan and carbodiimide coupling were used. The chemical modifications led to decreases in avidity between mannan and human collectins, such as the mannan-binding lectin (MBL) and human surfactant protein D (SP-D), but not in porcine SP-D. Despite this effect, these fluorescent derivatives were captured by human lectins using highly concentrated solutions. The resulting fluorescent beads were exposed to different solutions, and the results showed that displacements occur in contact with higher affinity ligands, proving that two-stage competition processes can occur in collectin carbohydrate recognition mechanisms. Moreover, the fluorescence loss depends on the discrepancy between the respective avidities of the recognized ligand and the fluorescent mannan. Chemically modulated fluorescent ligands associated with a diversity of collectins may lead to the creation of diagnostic tools suitable for multiplex array assays and the identification of high-avidity ligands.

Keywords: avidity; binding; fluorescent; lectin; mannan; oxidation; surfactant protein D; tempo.

Figure 1

Representation of MBL and SP-D. The chemical structures of heptose and mannose, with lectin-recognized sites in yellow.

Figure 2

Two-stage competition between PAMPs and fluorescent mannan formerly caught by lectins.

Figure 3

Formation of Mox following TEMPO-catalyzed oxidation.

Figure 4

Kinetic study of the oxidation of mannan.

Figure 5

Synthetic route to Mf.

Figure 6

Avidity determination between ligands and lectins following an ELISA-like protocol.

Figure 7

Avidity determination between oxidized mannan and fixed hMBL (A), hSP-D (B), and pSP-D (C). Lectins fixed on magnetic beads are mixed in mannan solutions for 20 min at room temperature. After three washing steps, beads are placed in a solution of hMBL-HRP for 20 min at room temperature. Avidity determination occurs after the oxidation of the TMB solution by washed beads and absorbance reading at 450 nm.

Figure 8

Avidity determination between fluorescent mannan and fixed hMBL (A), hSP-D (B), and pSP-D (C) obtained using the protocol described previously.

Figure 9

Calcium involvement in mannan recognition using EDTA-based assessments with different mannan at 250 ng/mL and MBL.

Figure 10

Avidities trends between Mannan, Mox, and human MBL, obtained using the protocol described before but with different concentrations of calcium chloride (5, 30, and 100 mM).

Figure 11

Confocal laser scanning microscopy imaging of murine macrophages in different conditions: untreated (control), after incubation with Mf 8% or Mf 24%. Macrophages were suspended in a solution of fluorescent mannan (1 mg/mL) for 30 min at 37 °C, then washed three times with buffer.

Figure 12

Flow cytometry analyses of hMBL-covered beads after 20 min in TBST Ca²⁺ buffer (Control), Mannan (100 ng/mL), and Mf 24% (300 ng/mL) solutions.

Figure 13

Cytograms of Mannan and Mf 24% bonded beads according to their fluorescence intensity.

Figure 14

Competition assays of Mf (8% and 24%) bonded to fixed hMBL (A), hSP-D (B), and pSP-D (C) versus Mannan at different concentrations (1, 10, 100 ng/mL).

Figure 15

LPS avidity array assays using fixed hMBL (A), hSP-D (B), and pSP-D (C). Lectins fixed on magnetic beads are mixed in LPS solutions for 20 min at room temperature. After three washing steps, beads are placed in a solution of hMBL-HRP for 20 min at room temperature. Avidity determination occurs after the oxidation of the TMB solution by the washed beads and absorbance reading at 450 nm.

Figure 16

Competition assays of Mf 24% bonded to fixed hMBL (A), hSP-D (B), and pSP-D (C) versus LPS at different concentrations (0.1, 1, and 10 µg/mL).