Prion infections cause lethal neurodegeneration. This process requires the cellular prion protein (PrP(C); ref. 1), which contains a globular domain hinged to a long amino-proximal flexible tail. Here we describe rapid neurotoxicity in mice and cerebellar organotypic cultured slices exposed to ligands targeting the 1 and 3 helices of the PrP(C) globular domain. Ligands included seven distinct monoclonal antibodies, monovalent Fab1 fragments and recombinant single-chain variable fragment miniantibodies. Similar to prion infections, the toxicity of globular domain ligands required neuronal PrP(C), was exacerbated by PrP(C) overexpression, was associated with calpain activation and was antagonized by calpain inhibitors. Neurodegeneration was accompanied by a burst of reactive oxygen species, and was suppressed by antioxidants. Furthermore, genetic ablation of the superoxide-producing enzyme NOX2 (also known as CYBB) protected mice from globular domain ligand toxicity. We also found that neurotoxicity was prevented by deletions of the octapeptide repeats within the flexible tail. These deletions did not appreciably compromise globular domain antibody binding, suggesting that the flexible tail is required to transmit toxic signals that originate from the globular domain and trigger oxidative stress and calpain activation. Supporting this view, various octapeptide ligands were not only innocuous to both cerebellar organotypic cultured slices and mice, but also prevented the toxicity of globular domain ligands while not interfering with their binding. We conclude that PrP(C) consists of two functionally distinct modules, with the globular domain and the flexible tail exerting regulatory and executive functions, respectively. Octapeptide ligands also prolonged the life of mice expressing the toxic PrP(C) mutant, PrP(Δ94-134), indicating that the flexible tail mediates toxicity in two distinct PrP(C)-related conditions. Flexible tail-mediated toxicity may conceivably play a role in further prion pathologies, such as familial Creutzfeldt-Jakob disease in humans bearing supernumerary octapeptides. (Fig. 1b). None of three high-affinity antibodies to the octapeptide repeats (OR, residues 50-90 embedded within the FT) were neurotoxic (Fig. 1b). Antibodies POM3 and D13, which bind the "charged cluster-2" 11 (CC2, residues 95-110), were innocuous at 67 nM but neurotoxic at 200 nM (Fig. 1b). None of the tested antibodies were toxic to Prnp o/o COCS ( Supplementary Fig. 2a). The identity of the targeted epitopes appeared to be a better predictor of PrP C antibody toxicity than their affinity to PrP C , suggesting that neurotoxicity resulted from the interaction of antibodies with specific PrP C domains (Supplementary Table 2).The mechanisms of neurotoxicity were further explored using POM1, a highly toxic antibody targeting the GD. Wild-type (wt) and tga20 COCS lost most granule cells (CGC) within 28 and 14 days post-exposure (dpe) to POM1, respectively (Fig. 2a-c). Controls included POM1-treated Prnp o/o COCS 12 , t...

Purpose: A tumor-derived proteolysis-inducing factor (PIF) is suggested to be a potent catabolic factor in skeletal muscle of mice and humans.We aimed to establish the clinical significance of PIF in cancer patients and to elucidate its structural features. Experimental Design: PIF was detected in human urine using a monoclonal antibody (mAb) and related to clinicaloutcomes. PIFimmunoaffinity-purifiedusing the mAbwaspurified/separated using reverse-phase high-performanceliquid chromatography and two-dimensional electrophoresis.Tenhuman cancer cell lines were tested for expression of mRNA encoding PIF core peptide. Results: PIF immunoreactivity was present in 160 of 262 patients with advanced cancers of the lung, esophagus/stomach, and other organs. In a Kaplan-Meier survival analysis of 181 lung cancer patients, PIF was unrelated to survival; PIF status was also unrelated to skeletal muscle loss confirmed by computed tomography imaging. PIF was seen in 16 of 24 patients with chronic heart failure and thus is not exclusive to malignant disease. In-gel digestion and mass spectrometric analysis of immunoaffinity purified PIF from cancer patients consistently identified human albumin and immunoglobulins.We showed nonspecific binding of purified albumin and immunoglobulins to the anti-PIF mAb, which is thus not a useful tool for PIF detection or purification in humans. Finally, the human PIF core peptide was detected in human cancer cell lines using reverse transcription-PCR and nucleotide sequencing; however, none of the amplified products had a site for the glycosylation critical to the proteolysis-inducing activity of murine PIF. Conclusions: A putative human homologue of murine PIF and its role in human cancer cachexia cannot be verified.A proteolysis-inducing glycoprotein [proteolysis-inducing factor (PIF)] of tumor origin mediates muscle wasting in mice bearing the MAC16 adenocarcinoma (1). PIF elicits intense skeletal muscle catabolism in muscle cells or animals (1 -4). Purified PIF has a mass of f24,000 Da and consists of a short polypeptide containing both N-linked (f10 kDa) and O-linked (f6 kDa) sulfated oligosaccharides (5). Preliminary evidence (6, 7) suggested that an identical factor is associated with weight loss in cancer patients (6 -10). Additionally, PIF was absent in cancer patients without weight loss or weight-losing patients with benign disease (7). These results suggested discovery of a critical factor underlying human cancer cachexia, but attempts at further confirmation suggested that PIF was not necessarily associated with clinical outcomes (i.e., survival and weight loss) in cancer patient populations (11,12).Regulation of expression of the human PIF core peptide, the sites at which glycosylation occurs to form the functional glycoprotein, and the structure of the oligosaccharides (5, 7), which confer the proteolysis-inducing activity to this unusual molecule, remain unresolved. A peptide sequence (7) and two patents describing the human cachexia-associated protein (HCAP) gene enc...

Prion diseases are neurodegenerative diseases characterized by the conversion of the cellular prion protein PrP(c) into a pathogenic isoform PrP(sc). Passive immunization with antiprion monoclonal antibodies can arrest the progression of prion diseases. Here, the crystal structure of the Fab fragment of an antiprion monoclonal antibody, POM1, in complex with human prion protein (huPrP(c)) has been determined to 2.4 Å resolution. The prion epitope of POM1 is in close proximity to the epitope recognized by the purportedly therapeutic antibody fragment ICSM18 Fab in complex with huPrP(c). POM1 Fab forms a 1:1 complex with huPrP(c) and the measured K(d) of 4.5 × 10(-7) M reveals moderately strong binding between them. Structural comparisons have been made among three prion-antibody complexes: POM1 Fab-huPrP(c), ICSM18 Fab-huPrP(c) and VRQ14 Fab-ovPrP(c). The prion epitopes recognized by ICSM18 Fab and VRQ14 Fab are adjacent to a prion glycosylation site, indicating possible steric hindrance and/or an altered binding mode to the glycosylated prion protein in vivo. However, both of the glycosylation sites on huPrP(c) are positioned away from the POM1 Fab binding epitope; thus, the binding mode observed in this crystal structure and the binding affinity measured for this antibody are most likely to be the same as those for the native prion protein in vivo.

The proteolysis-inducing factor is a putative mediator of cancer-associated weight loss. The goal of this study was to examine for the first time: (i) its prevalence in patients with metastatic gastric/esophageal cancer; and (ii) whether it possibly correlated with weight loss and anorexia and whether it predicted tumor response and patient survival. This study recruited 41 patients as part of a phase II therapeutic, chemotherapy protocol for patients with metastatic gastric/esophageal cancer. Patient eligibility criteria were designed to select a group of patients who would tolerate treatment with the drugs capecitabine and oxaliplatin. Urine for assaying the proteolysis-inducing factor was obtained at registration and then 6 weeks later. Patients completed the FACT-E questionnaire every 6 weeks and had their weights checked at the same interval. Patients were followed prospectively for tumor response and patient survival. Twenty-three (56%) patients had the proteolysis-inducing factor in their urine at registration, and 18 (64%) had it at 6 weeks. There was no statistically significant correlation between the presence of the proteolysis-inducing factor and weight loss or between its presence and anorexia. Moreover, there was no evidence that the presence of the proteolysis-inducing factor in urine was able to predict tumor response or patient survival. The proteolysis-inducing factor in urine does not appear to be tied to weight loss, anorexia, tumor response, or patient survival in the clinical setting of metastatic gastric/esophageal cancer.

1 Evidence indicates that imidazoline I 2 binding sites (I 2 BSs) are present on monoamine oxidase (MAO) and on soluble (plasma) semicarbazide-sensitive amine oxidase enzymes. The binding site on MAO has been described as a modulatory site, although no effects on activity are thought to have been observed as a result of ligands binding to these sites. 2 We examined the effects in vitro of several imidazoline binding site ligands on activities of bovine plasma amine oxidase (BPAO) and porcine kidney diamine oxidase (PKDAO) in a spectrophotometric protocol. 3 While both enzymes were inhibited at high concentrations of all ligands, clonidine, cirazoline and oxymetazoline were seen, at lower concentrations, to increase activity of BPAO versus benzylamine, but not of PKDAO versus putrescine. This effect was substrate dependent, with mixed or biphasic inhibition of spermidine, methylamine, p-tyramine and b-phenylethylamine oxidation observed at cirazoline concentrations that increased benzylamine oxidation. Abbreviations: a, factor by which K M is altered by imidazoline drug; AM, amiloride; b, factor by which V max is altered by imidazoline drug; 2-BFI, 2-(2-benzofuranyl)-2-imidazoline; BPAO, bovine plasma amine oxidase; BZ, benzylamine; CIR, cirazoline; D, imidazoline drug binding to any site on BPAO; E, enzyme; E max , maximum degree to which measured parameter can be changed by drug of interest; g, factor by which affinity of enzyme for substrate is reduced by a mixed inhibitor; H, imidazoline drug binding to high-affinity site on BPAO; I, imidazoline drug binding to (very low-affinity) inhibitory site on BPAO; IBS, imidazoline binding site; IDZ, idazoxan; I 1 R, imidazoline type-1 receptor; k p , rate constant for enzymatic formation of product; K H , dissociation constant for ligand H from high-affinity site on BPAO; K L , dissociation constant for ligand L from low-affinity site on BPAO; K S , dissociation constant for substrate S from active site of BPAO; L, imidazoline drug binding to low-affinity site on BPAO; MA, methylamine; MOX, moxonidine; P, product (of an enzyme reaction

Prion diseases are neurodegenerative diseases that are characterized by the conversion of the cellular prion protein PrP c to the pathogenic isoform PrP sc . Several antibodies are known to interact with the cellular prion protein and to inhibit this transition. An antibody Fab fragment, Fab POM1, was produced that recognizes a structural motif of the C-terminal domain of mouse prion protein. To study the mechanism by which Fab POM1 recognizes and binds the prion molecule, the complex between Fab POM1 and the C-terminal domain of mouse prion (residues 120-232) was prepared and crystallized. Crystals of this binary complex belonged to the monoclinic space group C2, with unit-cell parameters a = 83.68, b = 106.9, c = 76.25 Å , = 95.6 .

A comparative study was undertaken to characterize the oligosaccharides released by endo-P-Nacetylglucosaminidase H (endo H) from the membrane glycoproteins of rat hepatocytes and three different Morris hepatoma cell lines HTC and MH,C,). It is shown that the membrane glycoproteins of hepatocytes and hepatoma cells contain markedly different quantities and forms of high-mannose-type carbohydrate chains.After radiolabelling of the cells with D-[Z3H]mannose, in the absence and presence of 1 mM 1 5 dideoxy-I ,5-imino-~-mannitol (1-deoxymannojirimycin), high-mannose-type oligosaccharides were released from delipidated membrane glycoproteins by enzymic digestion with endo H. The carbohydrate chains were converted to their corresponding oligosaccharide alditols by reduction with sodium borohydride, then further analysed by HPLC using an APS-2 Hypersil column.In the absence of 1-deoxymannojirimycin, up to 10% of the radiolabelled oligosaccharides were released by endo H-treatment of the membrane glycoprotein fraction from rat hepatocytes. In contrast, the quantity of radiolabelled high-mannose-type carbohydrate chains released by endo Htreatment from tumour-cell membrane glycoproteins of hepatoma cell lines NA-MH 7777 (313 %), MH,C,-MH 7795 (37,2%) and HTC-MH 7288c (48%) was increased up to fivefold. The formation of higher-mannosylated structures after oligosaccharide analysis was observed in all hepatoma cell lines, with Man,GlcNAc, as the major component, whereas in hepatocytes Man,GlcNAc, was the predominant high-mannose-type structure.In contrast, in the presence of the Golgi a-D-mannosidase I inhibitor, 1 -deoxymannojirimycin, no significant differences were observed between the distribution of high-mannose-type oligosaccharides in the membrane glycoproteins of hepatocytes and hepatoma cells. However, in the presence of this inhibitor, the proportion of radiolabelled glycans sensitive to deglycosylation by endo H was greatly increased (>85%) in all the cell lines investigated, the predominant structures being Man,_,-GlcNAc,,. This study shows that an increased content of high-mannose-type sugar chains is a general characteristic of membrane-bound glycoproteins for malignant transformed hepatocytes.The carbohydrate moieties of cell-surface glycoconjugates are thought to be important in cell-cell interaction and recognition [l -51. Structural analysis of the carbohydrate chains for the membrane glycoprotein y-glutamyltranspeptidase, from various organs and species, has shown that the structure of asparagine-linked carbohydrate chains organ specific as well as species specific [6]. It has been proposed that the altered glycosylation of cell-surface carbohydrates for Dedication. Dedicated to Professor Hans Kroger on the occasion of his 65th birthday.Abbreviation. Endo H, endo-P-N-acetylglucosaminidase H; Mega-10, decanoyl-N-methyl-glucamide; DMEM, Dulbecco's modification of Eagle's medium ; I-deoxymannojirimycin, 1,Sdideoxy-1,5-imino-~-mannitol; the subscript OH is used to indicate NaBH,-reduced oligosaccharides : ...

The amino acid sequence of GTP :AMP phosphotransferase (AK3) from beef-heart mitochondria has been determined, except for one segment of about 33 residues in the middle of the polypeptide chain. The established sequence has been unambiguously aligned to the sequence of cytosolic ATP :AMP phosphotransferase (AK1) from pig muscle, allowing for six insertions and deletions. With 30 % of all aligned residues being identical, the homology between AK3 and AKI is well established. As derived from the known three-dimensional structure of AKI, the missing segment is localized at a small surface area of the molecule, far apart from the active center. The pattern of conserved residues demonstrates that earlier views on substrate binding have to be modified. The observation of three different consecutive N-termini indicates enzyme processing.The enzyme GTP: AMP phosphotransferase (AK3, Mr = 26000) catalyzes the reaction MgGTP + AMP + MgGDP + ADP. The enzyme AK3 is located in the inner matrix of the mitochondria [I -31 and is involved in the reaction sequence of substrate level phosphorylation [l]. Purification procedures, physical, chemical, kinetic and inhibition studies of AK3 from beef-heart mitochondria [2 -41 have emphasized similarities with the adenylate kinases from mammalian cytosol [3] and mitochondria [5], AKI and AK2 respectively, as well as from yeast cytosol [6]. All these enzymes are water-soluble, globular, low-M, proteins relatively stable in a wide range of p H and are purified by using similar purification protocols.The catalytic centers of adenylate kinases carry two binding sites for the nucleotides. This is borne out by the strong binding of adenosine(5')pentaphospho(5')adenosine by AK1 [7] and AK2 [S] and guanosine(5')pentaphospho(5')adenosine by AK3 [3]. Moreover, kinetic, spectrophotometric and equilibrium dialysis binding studies [9] (and citations therein) demonstrate two binding sites. One site, the AMP site, of AKI, AK2 and AK3 is very specific for AMP. Rigorous specificity does not apply to the other site. The GTP-binding site of AK3 can accommodate both guanosine and inosine nucleotides but does not accommodate ATP, CTP or UTP [4]; the ATP-binding site of AK1 and AK2 can accommodate GTP, CTP, ITP and UTP 161.The amino acid sequence and the three-dimensional structure of AK1 from pig muscle have been elucidated [lo-121. Sequences of other species have been determined completely [13] or partially [14, 151. The active-center region of AKI was established by binding studies of substrate analogs in crystals [16]. The enzyme molecule is to some extent deformed in these crystals as evidenced by the break up of crystals on binding substrates. Further X-ray analyses of other crystal forms and/or of other adenylate kinases from different species are necessary for elucidating the catalytic mechanism. The analysis of AK3 crystals is in progress [17]. In this paper we report most of the amino acid sequence of AK3 and its comparison with the sequence of AK1 and modify earlier views on substrate binding pos...