Biochemical and structural characterization of a recombinant fibrinogen-related lectin from Penaeus monodon

Abstract

Fibrinogen-related lectins are carbohydrate-binding proteins of the innate immune system that recognize glycan structures on microbial surfaces. These innate immune lectins are crucial for invertebrates as they do not rely on adaptive immunity for pathogen clearance. Here, we characterize a recombinant fibrinogen-related lectin PmFREP from the black tiger shrimp Penaeus monodon expressed in the Trichoplusia ni insect cell. Electron microscopy and cross-linking experiments revealed that PmFREP is a disulfide-linked dimer of pentamers distinct from other fibrinogen-related lectins. The full-length protein binds N-acetyl sugars in a Ca2+ ion-independent manner. PmFREP recognized and agglutinated Pseudomonas aeruginosa. Weak binding was detected with other bacteria, including Vibrio parahaemolyticus, but no agglutination activity was observed. The biologically active PmFREP will not only be a crucial tool to elucidate the innate immune signaling in P. monodon and other economically important species, but will also aid in detection and prevention of shrimp bacterial infectious diseases.

Biochemical and ligand binding properties of recombinant Xenopus laevis cortical granule lectin-1

“…After the establishment of the signal peptide cleavage site, His 6 XCGL-1 and His 6 XCGL-1 CRD constructs were made for insect cell expression. XEEL signal peptide was used as it is known to be an efficient secretion signal [ 14 , 31 , 32 ]. Ni-NTA affinity chromatography is a superior approach due to higher binding capacity compared to in-house-generated lactose resin.…”

The role of pattern recognition receptors in crustacean innate immunity

“…In P. vannamei , 10 transcripts of FREP were isolated with conserved FBG domains at the C‐terminus that were shown to agglutinate V. harveyi in the presence of Ca 2+ ions facilitating its clearance in vivo 213,214 . A FREP with lectin specificity has also been characterized from P. monodon binding to N‐acetyl sugars in a Ca 2+ ‐dependent manner with strong agglutination and weak binding properties against Pseudomonas aeruginosa and V. parahaemolyticus , respectively 215 . The antibacterial immunity of P. japonicus requires the participation of FREP2 as impairing its activity led to a reduction in the phagocytosis function of haemocytes 216 .…”

“…213,214 A FREP with lectin specificity has also been characterized from P. monodon binding to N-acetyl sugars in a Ca 2+ -dependent manner with strong agglutination and weak binding properties against Pseudomonas aeruginosa and V. parahaemolyticus, respectively. 215 The antibacterial immunity of P. japonicus requires the participation of FREP2 as impairing its activity led to a reduction in the phagocytosis function of haemocytes. 216 The ficolin from P. monodon with FBG domain also showed positive results from the bacterial binding assay showing agglutination properties against Gram-negative and Grampositive bacteria in the presence of Ca 2+ ions and effectively clearing V. harveyi from the host system.…”