Potential Involvement of the South American Lungfish Intelectin-2 in Innate-Associated Immune Modulation

Abstract

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 β-sheets: an anti-parallel β-sheet, a β-hairpin, and a disordered β-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.

Keywords: carbohydrate-binding protein; innate immunity; lectin; molecular docking; molecular modeling.

Figure 1

Venn diagram of Lepidosiren paradoxa lectins identified at 21 days post-amputation and 2 and 7 days post-skin injury through transcriptomes.

Figure 2

Gene ontology of the identified lectins at (A) 2 days post-skin injury (dpi), (B) 7 dpi, and (C) 21 days post-amputation (dpa). Colors are as follows: biological processes (red), cellular component (blue), molecular function (green).

Figure 3

Multiple alignment of the amino acid sequence of Lepidosiren paradoxa intelectin-2-B with O. mykiss (UniProt Accession P0DMV4), M. musculus (UniProt Accession O88310), H. sapiens (UniProt Accession Q8WWU7), and X. laevis (UniProt Accession Q5PPM0) intelectins. Colors are as follows: cysteine residues (yellow), carbohydrate-binding site (blue), calcium-binding site and carbohydrate binding site (red), identical residues (grey), partially conserved amino acid residues (light grey). Symbols are as follows: helices (ŋ), α-strands (α), β-turns (TT).

Figure 4

Model quality assessment and validation. (A) Superposition of the generated model and the template (PDB ID: 6USC). (B) Ramachandran plot by PROCHECK. (C) Verify3D graphic result and (D) ERRAT graphic result.

Figure 5

(A) Overall three-dimensional structure of LpITLN2-B. (B) Ribbon diagram of LpITLN2-Bstructure (β-strands, β1–11). Colors are as follows: (A) Chain A is colored in light pink, Chain B is in Green, Chain C is colored in lime green; (B) β-sheets are in red, α-helix in cyan, and loops are in pink.

Figure 6

(A) 3D interaction of Ca²⁺ ion with binding site residues. (B) 2D representation, with the distance of hydrogen bonds expressed in Å.

Figure 7

(A) Carbohydrate-binding site of LpITLN2-B forming a complex with maltose (cyan sticks). Yellow dashes represent polar contacts. (B) 2D representation of hydrogen bonds and hydrophobic interactions around maltose.

Figure 8

(A) Carbohydrate-binding site of LpITLN2-B with complex to poly(I:C) in yellow sticks. Yellow dashes represent polar contacts. (B) 2D representation of hydrogen bonds and hydrophobic interactions around poly(I:C).

Figure 9

(A) Carbohydrate-binding site of LpITLN2-B forming a complex with LPS, represented by pink sticks. Yellow dashes represent polar contacts. (B) 2D representation of hydrogen bonds and hydrophobic interactions around LPS.