DOI: 10.14457/tu.the.2017.1740
title: Molecular cloning and functional characterization of calreticulin from the human liver fluke, Opisthorchis viverrini

description: Opisthorchis viverrini (Ov), a human liver fluke endemic in parts of Thailand, causes opisthorchiasis and is associated with cholangiocarcinoma, a serious health problem in the country. Calreticulin (CALR) is an endoplasmic reticulum‐resident multifunctional protein in mammals that is involved in various biological functions inside and outside the cell including calcium homeostasis, chaperoning, apoptotic cell clearance, cell adhesion, as well as angiogenesis. In this study, RNA expression and protein distribution of O. viverrini calreticulin (OvCALR) were analyzed, and its intracellular and extracellular functions were investigated. A cDNA encoding OvCALR was cloned from total RNA of the adult parasite. The deduced amino acid sequence of OvCALR contains various characteristics of calreticulin including the calreticulin family signature 1 and 2, a signal peptide, an ER retention signal, three functional and structural domains (N‐, P‐, and C‐domain), three conserved cysteine residues, and three each of two tandem repeated motifs. Additionally, it has very high sequence conservation at 96.6% identity to Clonorchis sinensis calreticulin and significantly less to blood fluke calreticulin, Schistosoma mansoni (57.5%) and S. japonicum (53.6%), and mammalian calreticulin, Mus musculus (50.7%) and Homo sapiens (50.9%), respectively. OvCALR mRNA was detected by reverse transcription PCR in all analyzed developmental stages from the newly excysted juvenile to the mature parasite. Western blot analyses using mouse anti‐rOvCALR antiserum detected native protein in soluble and insoluble crude parasite extracts as well as in the excretory/secretory product of the adult parasite. Moreover, rOvCALR was recognized by Ov‐infected hamster sera starting eight weeks postinfection. Immunohistochemical detection of OvCALR in adult O. viverrini revealed its presence in several tissues and cell types, including tegumental cell bodies, cecal epithelium, testes, ovary, prostate gland, Mehlis’ gland, vitelline cells, eggs, and lining of seminal vesicle. OvCALR was not detected in liver tissue of Ov‐infected hamster. Recombinant OvCALR demonstrated in vitro protection of citrate synthase from thermal‐induced aggregation. Recombinant OvCALR C‐domain showed a mobility shift in native‐PAGE in the presence of calcium. Recombinant OvCALR was shown to bind to human C1q in a dose‐dependent manner and to suppress C1q‐mediated hemolysis of human sensitized red blood cells. In addition, it also was shown to inhibit proliferation, migration, and sprouting of human endothelial cells in vitro. The observed effects were similar to those observed in rMmCALR. However, transacetylase activity of rOvCALR as previously reported for Haemonchus contortus calreticulin could not be observed. In conclusion, OvCALR has ER‐functions including chaperoning and calcium binding that are comparable to human calreticulin. Moreover, OvCALR is a released protein from the parasite with potential to modulate the host immune system and cholangiocarcinoma development from its anti‐angiogenesis activity and C1q‐mediated interference of the host immune system. OvCALR is an interesting molecule that might be a target for opisthorchiasis control and diagnosis.