Expression of the Major Vault Protein (MVP) and Cellular Vault Particles in Fish

Abstract

Cellular vaults are ubiquitous 13 mega Da multi-subunit ribonuceloprotein particles that may have a role in nucleocytoplasmic transport. Seventy percent of the vault's mass consists of a 100 kDa protein, the major vault protein (MVP). In humans, a drug resistance-associated protein, originally identified as lung resistance protein in metastatic lung cancer, was ultimately shown to be the previously described MVP. In this study, a partial MVP sequence was cloned from channel catfish. Recombinant MVP (rMVP) was used to generate a monoclonal antibody that recognizes full length protein in distantly related fish species, as well as mice. MVP is expressed in fish spleen, liver, anterior kidney, renal kidney, and gills, with a consistent expression in epithelial cells, macrophages, or endothelium at the interface of the tissue and environment or vasculature. We show that vaults are distributed throughout cells of fish lymphoid cells, with nuclear and plasma membrane aggregations in some cells. Protein expression studies were extended to liver neoplastic lesions in Atlantic killifish collected in situ at the Atlantic Wood USA-EPA superfund site on the southern branch of the Elizabeth River, VA. MVP is highly expressed in these lesions, with intense staining at the nuclear membrane, similar to what is known about MVP expression in human liver neoplasia. Additionally, MVP mRNA expression was quantified in channel catfish ovarian cell line following treatment with different classes of pharmacological agents. Notably, mRNA expression is induced by ethidium bromide, which damages DNA. Anat

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“…Five days later, mice were euthanized by slow lethal CO 2 asphyxiation, followed by bilateral pneumothorax according to an approved Clemson University IACUC Animal Use Protocol, blood collected by cardio-puncture, and spleens harvested to isolate plasma cells. Blood was clotted overnight at 4 • C, then centrifuged for 20 min at 10,000× g to collect overlying serum as the crude source of polyclonal anti-sera if needed and stored at −20 • C. Procedures for fusion of spleen cells with Sp2/0-Ag14 myelomas have been described elsewhere [31,32]. Primary hybridomas were typically grown in Dulbecco's Modified Eagle Medium (Cellgro) supplemented with heat inactivated fetal bovine serum (FBS, Gibco), 20 mM HEPES, 10 mM L-glutamine, 100 µg/mL penicillin, 100 µg/mL streptomycin, 110 µg/mL sodium pyruvate, 1% non-essential amino acids (100× stock), 4.5 g/L glucose, 10 µg/mL gentamycin and 5 µg/mL nystatin.…”

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“…Perhaps one of the most interesting finds regarding etoposide resistance is that the R428W mutation in the P53 gene produces an aberrant protein that transcriptionally activates the tyrosyl-DNA phosphodiesterase 2 ( TDP2 ) DNA repair gene, causing resistance to etoposide 56 . Efflux pump-induced resistance of etoposide is modulated by the ABCB1 , major vault protein ( MVP ), and ABCG2 57 , 58 , 59 genes. Capicua transcriptional repressor 3 ( CIC-3 ), a member of the CLC family, is an important component for drug sequestration as it regulates vesicular acidity; over-expression appears to correlate with etoposide resistance, possibly due to accelerated break-down of the compound through upregulating P-glycoprotein expression 60 .…”

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“…Recently, immunohistochemical staining for MVP in rainbow trout showed high levels of MVP protein in the epithelium of gills and intestine, macrophages of the head kidney and secretory cells of the renal kidney (Margiotta et al, 2017).…”

“…Proteomic analysis of zebrafish liver tissue exposed to tunicamycin might provide evidence for the possible synergism between MVP and Krt8/18 in the protection against liver diseases. Given the expression of MVP in fish liver, implication of mouse and human Krt8 and Krt18 in liver diseases, (Margiotta et al, 2017) and the frequent involvement of ER stress in liver diseases (review in (Malhi and Kaufman, 2011)), it is possible that MVP plays a role in ER stress response in the liver.…”

“…Recently, MVP was shown to be expressed in macrophages, epithelial and endothelial cells of the kidneys, gills, spleen and liver in rainbow trout. MVP expression was also detected in fish lymphoid cells (Margiotta et al, 2017).…”