Horseshoe crab acetyl group-recognizing lectins involved in innate immunity are structurally related to fibrinogen

Abstract

We have characterized and cloned newly isolated lectins from hemolymph plasma of the horseshoe crab Tachypleus tridentatus, which we named tachylectins 5A and 5B (TLs-5). TLs-5 agglutinated all types of human erythrocytes and Gram-positive and Gram-negative bacteria. TLs-5 specifically recognize acetyl group-containing substances including noncarbohydrates; the acetyl group is required and is sufficient for recognition. TLs-5 enhanced the antimicrobial activity of a horseshoe crab-derived big defensin. cDNA sequences of TLs-5 indicated that they consist of a short N-terminal Cys-containing segment and a C-terminal fibrinogen-like domain with the highest sequence identity (51%) to that of mammalian ficolins. TLs-5, however, lack the collagenous domain found in a kind of "bouquet arrangement" of ficolins and collectins. Electron microscopy revealed that TLs-5 form two- to four-bladed propeller structures. The horseshoe crab is equipped with a unique functional homologue of vertebrate fibrinogen, coagulogen, as the target protein of the clotting cascade. Our observations clearly show that the horseshoe crab has fibrinogen-related molecules in hemolymph plasma and that they function as nonself-recognizing lectins. An ancestor of fibrinogen may have functioned as a nonself-recognizing protein.

Figure 1

Purification of TLs-5 by using the N-acetyl group-immobilized resin. (A) Hemolymph plasma (500 ml) was applied to the resin (5 ml), equilibrated with 20 mM Tris⋅HCl (pH 8.0) containing 10 mM CaCl₂ and washed extensively with the same buffer containing 0.5 M NaCl. Two protein peaks with hemagglutinating activity were obtained by stepwise elution of 25 mM GlcNAc and 250 mM GlcNAc in the equilibration buffer. No proteins were further eluted by extensive washing with the buffer containing 6 M guanidine⋅HCl. The fractions indicated by bars were pooled and dialyzed against 20 mM Tris·HCl (pH 8.0) containing 0.15 M NaCl and 5 mM CaCl₂. (B) SDS/PAGE of purified TLs-5 under nonreducing (lanes 1 and 2) and reducing (lanes 3 and 4) conditions.

Figure 2

A synergistic effect of TLs-5 on antimicrobial activity of big defensin. (A) Enterococcus hirae; (B) Staphylococcus aureus 209P; (C) Escherichia coli B; (D) Escherichia coli K12. The bar graph represents the means ± SE of the three measurements. Big defensin (2.5 μg/ml) was mixed with bacteria in the presence or absence of TLs-5 (1 μg/ml). Bar 1, no big defensin and no TLs-5; bar 2, TL-5A; bar 3, TL-5B; bar 4, big defensin; bar 5, big defensin + TL-5A; bar 6, big defensin + TL-5B.

Figure 3

Tissue-specific expression patterns of TLs-5. (A) The membrane was probed with polyclonal antibody against TL-5A. (B) The membrane was probed with polyclonal antibody against TL-5B. The anti-TL-5B antibody cross-reacts with TL-5A. Lane 1, hemocytes; lane 2, hepatopancreas; lane 3, heart; lane 4, stomach; lane 5, intestine; lane 6, nervous tissue; lane 7, skeletal muscle; lane 8, purified TL-5A; lane 9, purified TL-5B.

Figure 4

Alignment of amino acid sequences of TLs-5, human l-ficolin/P35 (HFCL), and the C-terminal domains of human fibrinogen β- (HFBE) and γ- (HFGA) chains. In the nucleotide sequence, the N-terminal amino acid of TL-5B was aspartic acid; nevertheless, asparagine was identified by amino acid sequence analysis. This discrepancy cannot be clearly explained, but there may be several genes coding isoproteins for TL-5B. The bold letters represent residues identical to TLs-5.

Figure 5

Schematic diagrams of the inter- and intrachain disulfide bonds of TLs-5. Arrows indicate cysteine residues that are possibly involved in interchain disulfide linkages. The disulfide bonds are predicted according to those in human fibrinogen (35).

Figure 6

Genomic Southern blot analyses of TLs-5. (A) For TL-5A: lane 1, BamHI; lane 2, HindIII; lane 3, XhoI. (B) For TL-5B: lane 1, BamHI; lane 2, XhoI; lane 3, KpnI.

Figure 7

The apparent molecular masses of TLs-5 in solution. (A) Gel filtration for TL-5A. (B) Gel filtration for TL-5B. Gel filtration was carried out on a Bio-Silect TM SEC400–5 column (Bio-Rad Laboratories), equilibrated with 20 mM Tris⋅HCl (pH 7.5) containing 0.15 M NaCl and 5 mM CaCl₂, by using a FPLC system (Amersham Pharmacia). Proteins eluted were monitored at 280 nm.

Figure 8

Electron micrographs of negatively stained TLs-5. (A) A hexameric structure of TL-5A. (B) An octameric structure of TL-5A. (C) A tetrameric structure of TL-5B. (D) Schematic models of the oligomeric structures of TLs-5. Electron microscopy was performed by using the JEM200EX electron microscope (JEOL, Tokyo) by negative staining with 0.5% uranyl acetate. The length measurements of the protein molecules were made on micrographs printed at ×360,000 magnification.