Charles F.B. Holmes

Mannose 6-Phosphate/Insulin-like Growth Factor II Receptor Is a Death Receptor for Granzyme B during Cytotoxic T Cell–Induced Apoptosis

The serine proteinase granzyme B is crucial for the rapid induction of target cell apoptosis by cytotoxic T cells. Granzyme B was recently demonstrated to enter cells in a perforin-independent manner, thus predicting the existence of a cell surface receptor(s). We now present evidence that this receptor is the cation-independent mannose 6-phosphate/insulin-like growth factor receptor (CI-MPR). Inhibition of the granzyme B-CI-MPR interaction prevented granzyme B cell surface binding, uptake, and the induction of apoptosis. Significantly, expression of the CI-MPR was essential for cytotoxic T cell-mediated apoptosis of target cells in vitro and for the rejection of allogeneic cells in vivo. These results suggest a novel target for immunotherapy and a potential mechanism used by tumors for immune evasion.

Motuporin, A Potent Protein Phosphatase Inhibitor Isolated from the Papua New Guinea Sponge Theonella swinhoei Gray

Abstract Motuporin ( 1 ), a cyclic pentapeptide that is a potent protein phosphatase-1 inhibitor and cytotoxin, has been isolated from the marine sponge Theonella swinhoei collected in Papua New Guinea. The structure of motuporin was elucidated by spectroscopic analysis and chemical degradation.

Chemical and biological evidence links microcystins to salmon ‘netpen liver disease’

Evidence is presented that links microcystins to a severe liver disease that occurs in Atlantic salmon that are netpen-reared in coastal British Columbia. Liquid chromatography-linked protein phosphatase bioassay analysis of extracts of liver tissue taken from Atlantic salmon afflicted with netpen liver disease showed the presence of an inhibitor of protein phosphatase that was chromatographically indistinguishable from microcystin-LR. Analysis of liver tissue from healthy control fish showed a complete absence of microcystin-LR. Intraperitoneal injection of microcystin-LR into healthy Atlantic salmon re-created the pathologic changes of netpen liver disease, including diffuse necrosis and hepatic megalocytosis.

Bioaccumulation and clearance of microcystins from salt water mussels, mytilus edulis, and in vivo evidence for covalently bound microcystins in mussel tissues

Over a period of 3 days saltwater mussels, Mytilus edulis, were fed a cyanobacteria, Microcystis aeruginosa, that contained a high concentration of microcystins. The mussels were killed on a periodic basis over the course of 2 months. Mussels were also collected at two sites were high levels of microcystins in tissues had been noted. A strategy based on the chemically unique nature of the C20 beta-amino acid, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6- dienoic acid (Adda), portion of the microcystins was used in conjunction with a protein phosphatase (PPase) assay to analyse for both covalently bound microcystins and free microcystins in the mussel tissues. The mussel PPase assay results were compared with the Lemieux oxidation gas chromatography-mass spectrometry (GCMS) analysis. Less than 0.1% of the total microcystin burden in the mussel tissue was found to be extractable with MeOH. Thus, direct evidence was provided for the existence of covalently bound microcystins in mussel tissues in vivo. The mussels rapidly cleared the covalently bound microcystins when transferred to untreated seawater. Within 4 days the total microcystin burden dropped from a high of 336.9 (+/- 45.8) micrograms/g wet tissue to 11.3 (+/- 2.6) micrograms/g. After 4 days postexposure until completion of the experiment the total levels remained below the detection limits of the GCMS method. The levels of free microcystins, extracted with MeOH and detected by the PPase assay, fell from 204 ng/g wet tissue to a residual 14 ng/g over a 53 day postexposure period. Presumably the bound microcystin present in the mussel tissue exists as a covalent complex with the PP-1 and PP-2A enzymes. We conclude that in any shellfish monitoring program it is the total tissue microcystin burden that needs to be considered.

Adsorption of microcystin-LR by activated carbon and removal in full scale water treatment

Abstract Removal of microcystin toxins from drinking water was evaluated at two full scale treatment plants that employed coagulation-sedimentation, dual media filtration and chlorination combined with either granular activated carbon filtration or powdered activated carbon. The influence of natural organic matter on the adsorption of the cyanobacterial toxin, microcystin-LR, by activatedcarbon was also evaluated in laboratory studies over a range of toxin concentrations similar to those typically observed in raw water at these plants. The sensitive protein phosphatase inhibition bioassay was used to quantify microcystin. Conventional treatment processes combined with activated carbon generally removed more than 80% of microcystin from raw water, but a residual concentration of 0.1-0.5 μg equivalents of microcystin-LR per liter was observed considering both (GAC and PAC) treatment facilities. Most values of residual microcystin-LR were at the low end of this range, but the upper end approaches the guidance level being considered by Health Canada for these toxins in drinking water

Regulation of matrix metalloproteinase-2 (MMP-2) activity by phosphorylation.

The regulation of matrix metalloprotein‐ases (MMP) has been studied extensively due to the fundamental roles these zinc‐endopeptidases play in diverse physiological and pathological processes. However, phosphorylation has not previously been considered as a potential modulator of MMP activity. The ubiquitously expressed MMP‐2 contains 29 potential phosphorylation sites. Mass spectrometryreveals that at least five of these sites are phosphorylated in hrMMP‐2 expressed in mammalian cells. Treatment of HT1080 cells with an activator of protein kinase C results in a change in MMP‐2 immunoreactivity on 2D immuno‐blots consistent with phosphorylation, and purified MMP‐2 is phosphorylated by protein kinase C in vitro. Furthermore, MMP‐2 from HT1080 cell‐conditioned medium is immunoreactive with antibodies directed against phosphothreonine and phosphoserine, which suggests that it is phosphorylated. Analysis of MMP‐2 activity by zymography, gelatin dequenching assays, and measurement of kinetic parameters shows that the phosphorylation status of MMP‐2 significantly affects its enzymatic properties. Consistent with this, dephos‐phorylation of MMP‐2 immunoprecipitated from HT1080 conditioned medium with alkaline phospha‐tase significantly increases its activity. We conclude that MMP‐2 is modulated by phosphorylation on multiple sites and that protein kinase C may be a regulator of this protease in vivo.—Sariahmetoglu, M., Crawford, B. D., Leon, H., Sawicka, J., Li, L., Ballermann, B. J., Holmes, C., Berthiaume, L. G., Holt, A., Sawicki, G., Schulz, R. Regulation of matrix metalloproteinase‐2 activity by phosphorylation. FASEB J. 21, 2486–2495 (2007)

A Molecular Basis for Different Interactions of Marine Toxins with Protein Phosphatase-1 MOLECULAR MODELS FOR BOUND MOTUPORIN, MICROCYSTINS, OKADAIC ACID, AND CALYCULIN A

The hepatotoxic cyclic heptapeptide microcystins and cyclic pentapeptide nodularins are powerful liver tumor promoters and potent inhibitors of the catalytic subunits of protein phosphatase-1 and −2A (PP-1c and PP-2Ac). In marked contrast to microcystins, which interact covalently with PP-1 and PP-2A, the nodularins do not bind covalently to PP-1 and PP-2A and may additionally possess unique carcinogenic properties. The conformation of microcystin-LR has been determined in solution and bound to PP-1c. We show here that the free NMR solution structures of two distinct microcystin structural congeners (microcystin-LR and -LL) are remarkably similar to the bound crystal structure of microcystin-LR. We have exploited this finding by using Metropolis Monte Carlo modeling to dock the solution structures of microcystin-LL and the marine toxin motuporin (nodularin-V) onto the crystal structure of PP-1c. Both of these toxins occupy a position similar to that of microcystin-LR when bound to PP-1c. However, although there are relatively minor differences in the structural orientation of microcystin-LL compared with microcystin-LR, there is a striking difference in the position of the N-methyldehydrobutyrine residue in motuporin relative to the comparable N-methyldehydroalanine residue in microcystin-LR. We propose that this difference in orientation provides a molecular explanation for why nodularins are incapable of forming a covalent linkage with PP-1c. Furthermore, the predicted position of N-methyldehydrobutyrine in motuporin is at the surface of the PP-1c-toxin complex, which may thus facilitate chemical interaction with a further macromolecule(s) possibly relating to its carcinogenic properties. PP-1c and PP-2Ac are also targets for other marine toxins such as okadaic acid and calyculin A. It was therefore of interest to use Metropolis Monte Carlo modeling to dock the known free crystal structures of okadaic acid and calyculin A to the crystal structure of PP-1c. These experiments predict that both okadaic acid and calyculin A are strikingly similar to microcystins and motuporin in their tertiary structure and relative PP-1c binding position.

The adsorption of microcystin-LR by natural clay particles.

The microcystin cyanobacterial hepatotoxins represent an increasingly severe global health hazard. Since microcystins are found world wide in drinking water reservoirs concern about the impact on human health has prompted investigations into remedial water treatment methods. This preliminary study investigates the scavenging from water of microcystin-LR by fine-grained particles known to have a high concentration of the clay minerals kaolinite and montmorillonite. The results show that more than 81% of microcystin-LR can be removed from water by clay material. Thus, microcystin-LR is indeed scavenged from water bodies by fine-grained particles and that this property may offer an effective method of stripping these toxins from drinking water supplies.

14C-Labeled microcystin-LR administered to Atlantic salmon via intraperitoneal injection provides in vivo evidence for covalent binding of microcystin-LR in salmon livers

The tissue distribution and clearance of radiolabeled microcystin-LR administered to Atlantic salmon via i.p. injection has been re-examined using uniformly 14C-labeled toxin. Significant differences were found to exist between these results and those obtained when fish received an i.p. injection of tritium-labeled dihydromicrocystin-LR. In addition, MeOH liver extracts were assayed by both phosphatase assay and 14C counts and the results compared with the total levels of incorporation determined by digestion and subsequent 14C counting of the same live tissues. An attempt to investigate the metabolism and to document the putative products was also undertaken. It was found that microcystin-LR was extensively metabolized to compounds that are more polar than the parent compound.

Quantitation of the microcystin hepatotoxins in water at environmentally relevant concentrations with the protein phosphatase bioassay.

Toxic cyanobacteria (blue-green algae) blooms have been documented in lakes and drinking water sources in Europe, China, Australia, western Canada, and the American Midwest (1). A primary cause of cyanobacterial toxicity has been attributed to the microcystin class of cyclic heptapeptides. The microcystin toxins are very potent hepatotoxins causing death to a variety of animals, and they have shown evidence of being potent tumor promoters (1-3). The overall toxicity of a bloom can be uncertain because of variations in toxin concentration over a short time and spatially within a water body experiencing a bloom (4). Several incidents of human illness have been attributed to the presence of cyanobacterial toxins in drinking water in Australia, Africa, and the United States (5-8). Water treatment studies conducted at laboratory and pilot plant scale have concluded that activated carbon and ozone are capable of removing microcystins from drinking water below the detection limit of the mouse bioassay and high performance liquid chromatography using UV absorbance detection (HPLC/UV) (9-14). Conventional water treatment practices (coagulation/sedimentation, filtration, and chlorination) have been found to be ineffective at removing the toxins (9-11, 14). Because of the lack of a sensitive analytical technique, the water treatment studies have been conducted at concentrations higher than would likely be encountered at treatment facilities. Microcystin-LR is one of the most common cyanobacterial hepatotoxins, but there are a t least 40 structural variations ( I ) . Microcystin-LR has been shown to be a potent inhibitor of the catalytic subunits of the serine/ threonine protein phosphatases PP-1 and PP-2A (termed PP-lc and PP-2Ac) (15-18), and this probably underlies its toxicity to animals (I 7,18). Microcystin-LR inhibited either PP-lc or PP-2Ac at concentrations of -0.1 nM when assays were performed at phosphatase concentrations of 0.2 munit/mL (16). There are currently two methods for the quantitation of microcystin toxins in water a t low levels; the protein phosphatase (PP) bioassay (19) and an enzyme-linked immunosorbent assay (ELISA) for quantitative analysis of microcystins (20). This paper presents an application of the protein phosphatase (PP) bioassay (19) for the