An N-terminal truncated form of Orp150 is a cytoplasmic ligand for the anti-proliferative edible mushroom Agaricus bisporus lectin and is required for NLS-dependent nuclear protein import

Abstract

Increased cell surface expression of the oncofetal ThomsenFriedenreich antigen (TF, Galpl-3GalNAc-) is common in malignant and hyperplastic epithelia. Our previous studies haveshown that a TF antigen-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits epithelial cell proliferation (Cancer Res.1993,53:4627) in association with inhibition of nuclear localization sequence (NLS)-dependent nuclear protein import (J.Biol.Chem.l999,274:4890). The purpose of this study is to investigate how ABL produces such an unique effect on nuclear protein import. Using ABL-affinity purification, an ABL-binding 16OkDa protein was obtained from the HT29 cell cytosol extracts. N-terminal protein sequencing identified the protein as an N-terminal truncated form of the oxygen-regulated protein150 (Orp150) which lacks the ER translocation signal peptide of full length Orp150. This protein expresses sialyl-TF antigen as determined by specific sialidase/OGlyganase treatment and IectinNlrestern blotting. Introduction of an anti-Grpl50 antibody to the digitonin semi-permeabilized nuclear transport system or depletion of Orp150 inhibited the nuclear accumulation of an NLS-peptide complex. This is probably the first demonstration of an intracellular glycoprotein expressing sialyl-TF. The inhibition of nuclear accumulation of NLS-complex by anti-0rp150 antibody suggests that Orp150 is required for NLS-dependent nuclear protein import. Dietary vitamin E reduces plasma and liver markers of oxidantstress in the aluminium treated rat.Aluminium (Al) toxicity may be mediated in part via reactive oxygen species (ROS). We have investigated the effects of the antioxidant vitamin E on Al-induced effects on the liver using male Wistar albino rats. A1 was administered i.p. as aluminium lactate (10 mg/Kg body weight) 5 times a week for 4 weeks. Groups (n=5 each) were either treated with aluminium alone, or with a dietary supplement of 5, 15, and 20 mg/g, of vitamin E. Further groups received 20 mg/g vitamin E supplemented diet or normal chow without Al. Following treatment, the animals were killed and, the liver and blood removed for measurement of aluminium and markers of oxidative stress. Al treatment caused a significant increase in aluminium content of plasma (P< 0.001) and liver (P< 0.001) in all groups. Liver ROS were significantly higher (P<O.ol) in the Al-treated groups and this was significantly attenuated (P<0.05) in the vitamin E treated groups. The aluminium-induced increase in ROS was associated with a significant reduction (P<0.05) in liver reduced glutathione levels and also a reduction of hepatic catalase activity. Hence aluminium hepatotoxicity may involve oxidative stress; reduced glutathione may serve as a defence mechanism in aluminium exposure and dietary vitamin E supplementation may offer further protection.

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

“…Interestingly, HYOU1 localization was found not to be restricted to the ER. In rats, glycosylated HYOU1 was detected in mitochondria, and an N-terminally truncated form was found in the cytoplasm (98,99). Accordingly, we assume that HSP70G and its homologs may be localized to several cellular compartments as part of a multiple targeting strategy.…”

Targeting of the molecular chaperone oxygen-regulated protein 150 (ORP150) to mitochondria and its induction by cellular stress

“…5). This immunoreactive protein represents native ORP150 devoid of its glycosylated adducts as seen by other investigators (29,37).…”

“…Membranes were incubated with primary antibodies to hamster GRP170, polyclonal rabbit anti-hamster GRP170, a generous gift from Dr. John Subjeck (Roswell Park Cancer Institute, Buffalo, NY). This antibody recognizes both glycosylated (up to 170 kDa) and native ORP150 (150 kDa) and has been used for detecting mammalian ORP150 (29,37). The primary antibodies used were polyclonal rabbit anti-hamster GRP170 (1:10,000), rabbit-anticalpain 10 (1:1,000, Biogenesis), anti-␤-actin (1:1,000, Sigma), anticalnexin (1:1,000, Calbiochem), anti-HSP60 (1:1,000, Calbiochem), anti-ND6 (1:1,000, Santa Cruz), anti-OTC (1:1,000) (gift from Dr. Gary Wright, Medical University of South Carolina, Charleston, SC), anti-green fluorescent protein (GFP) (1:1,000, Calbiochem), and mouse monoclonal anti-C/enhancer-binding protein homologous protein (CHOP, 1:1,000, Alexis Biochemicals).…”

Lectin–epithelial interactions in the human colon

“…Peanut lectin has been shown to increase proliferation in colon cancer cell lines [27], in cultured human colonic mucosal biopsies [28,29] and in vivo, causing a 40% increase in rectal mucosal proliferation in individuals who ate a packet of peanuts per day for 5 days and who had increased TF expression in their colonic epithelia [25]. Conversely, the edible mushroom ABL (Agaricus bisporus lectin)/ABA (A. bisporus agglutinin), which binds not only TF but also sialylated TF, causes reversible inhibition of epithelial cell proliferation [30] as a consequence of internalization of the lectin and consequent inhibition of NLS (nuclear localization sequence)-dependent nuclear protein import ( Figure 1) [31,32]. Another TFbinding dietary lectin, jacalin, present in jackfruit, also inhibits proliferation, but via a different mechanism: activation of PHAP1 (putative HLA class II-associated protein I) with consequent release and activation of a phosphatase, PP2A (protein phosphatase 2A), that dephosphorylates and hence deactivates ERK (extracellular-signal-regulated kinase) [33].…”