Crystal structure of a cytocidal protein from lamprey and its mechanism of action in the selective killing of cancer cells

Abstract

Background In previous research, we found that lamprey immune protein (LIP) possessed cytocidal activity against tumor cells, but the mechanism of the selective recognition and killing of tumor cells by LIP was not identified. Methods Superresolution microscopy, crystallographic structural analysis, glycan chip assay, SPR experiments, FACS assays, computational studies and mass spectrometric analysis firmly establish the mode of action of LIP, which involves dual selective recognition and efficient binding. Results We determined the overall crystallographic structure of LIP at a resolution of 2.25 Å. LIP exhibits an elongated structure with dimensions of 105 Å × 30 Å × 30 Å containing an N-terminal lectin module and a C-terminal aerolysin module. Moreover, the Phe 209 -Gly 232 region is predicted to insert into the lipid bilayer to form a transmembrane β-barrel, in which the hydrophobic residues face the lipid bilayer, and the polar residues constitute the hydrophilic lumen of the pore. We found that LIP is able to kill various human cancer cells with minimal effects on normal cells. Notably, by coupling biochemical and computational studies, we propose a hypothetical mechanism that involves dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts. Furthermore, specific binding of the lectin module with biantennary bisialylated nonfucosylated N-glycan or sialyl Lewis X-containing glycan structures on GPI-APs triggers substantial conformational changes in the aerolysin module, which interacts with SM, ultimately resulting in the formation of a membrane-bound oligomer in lipid rafts. Conclusions LIP holds great potential for the application of a marine protein towards targeted cancer therapy and early diagnosis in humans. Electronic supplementary material The online version of this article (10.1186/s12964-019-0358-y) contains supplementary material, which is available to authorized users.

Molecular Evolution of Apolipoprotein Multigene Family and the Original Functional Properties of Serum Apolipoprotein (LAL2) in Lampetra japonica

“…LIP exerts a specific killing effect on certain tumor cells ( 23 , 24 ). The selective killing mechanism proposes that LIP could bind to biantennary bi-sialylated non-fucosylated N-glycan of cancer cells, such as MCF-7 and K562 cells, and not affect normal cells ( 22 ). It was speculated that the interaction between rLAL2 and LIP may also influence LIP killing activity, the MCF-7 cells sensitive to LIP are plated in 96-well plates for overnight culture, according to the experimental design.…”

“…LIP is a cytotoxic lamprey protein, which plays an important role in tumor cell survival and growth (21)(22)(23)(24). Previously, during the process of purifying the cytotoxic protein in lamprey serum, it was found that LIP and LAL2 were always present in an eluted sample ( Figure 1B).…”

“…Lamprey immune protein (LIP), a cytotoxic protein, has a jacalin-like domain and an aerolysin pore-forming domain previously identified in granulocytes of Lampetra japonica (21). We demonstrate the crystal structure of LIP and the mode of action involving dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts (22). LIP can kill a panel of human cancer cells yet has minimal effects on normal cells.…”

Lamprey Immune Protein Mediates Apoptosis of Lung Cancer Cells Via the Endoplasmic Reticulum Stress Signaling Pathway

“…It is regulated by several apoptosis-related genes and signaling pathways, including inositol-requiring transmembrane kinase/endonuclease-1 (IRE-1)/palmitate-induced splicing of X box–binding protein-1 (sXBP-1) and pancreatic ER kinase (PERK)/CCAAT/enhancer-binding protein homologous protein (CHOP) ( 7 – 9 ). Lamprey immune protein (LIP), first isolated from the lamprey, is a 313-amino acid-long protein with an N-terminal jacalin-like domain and a C-terminal aerolysin domain ( 10 , 11 ). In our previous study, LIP was shown to exert cytocidal effects on a variety of tumor cells, with minimal toxicity in normal cells ( 11 ).…”

“…Lamprey immune protein (LIP), first isolated from the lamprey, is a 313-amino acid-long protein with an N-terminal jacalin-like domain and a C-terminal aerolysin domain ( 10 , 11 ). In our previous study, LIP was shown to exert cytocidal effects on a variety of tumor cells, with minimal toxicity in normal cells ( 11 ). In addition, we explored the primary mechanism of LIP that effectively distinguishes tumor cells from normal cells, i.e., its ability to recognize biantennary non-fucosylated N-glycan or sialyl Lewis X-containing glycan structures in tumor cells ( 11 ).…”