Processing and secretion of guanylate binding protein-1 depend on inflammatory caspase activity.

Human guanylate binding protein-1 (GBP-1) belongs to the family of large GTPases. The expression of GBP-1 is inducible by inflammatory cytokines, and the protein is involved in inflammatory processes and host defence against cellular pathogens. GBP-1 is the first GTPase which was described to be secreted by eukaryotic cells. Here, we report that precipitation of GBP-1 with GMP-agarose from cell culture supernatants co-purified a 47-kD fragment of GBP-1 (p47-GBP-1) in addition to the 67-kD full-length form. MALDI-TOF sequencing revealed that p47-GBP-1 corresponds to the C-terminal helical part of GBP-1 and lacks most of the globular GTPase domain. In silico analyses of protease target sites, together with cleavage experiments in vitro and in vivo, showed that p67-GBP-1 is cleaved by the inflammatory caspases 1 and 5, leading to the formation of p47-GBP-1. Furthermore, the secretion of p47-GBP-1 was found to occur via a non-classical secretion pathway and to be dependent on caspase-1 activity but independent of inflammasome activation. Finally, we showed that p47-GBP-1 represents the predominant form of secreted GBP-1, both in cell culture supernatants and, in vivo, in the cerebrospinal fluid of patients with bacterial meningitis, indicating that it may represent the biologically active form of extracellular GBP-1. These findings confirm the involvement of caspase-1 in non-classical secretion mechanisms and open novel perspectives for the extracellular function of secreted GBP-1.

Tissue transglutaminase is not a biochemical marker for Alzheimer's disease.

Typical hallmarks of Alzheimer's disease (AD) are pathologic deposits in cortical and subcortical regions consisting of self-aggregated proteins such as amyloid-beta (Aß) or tau. Tissue transglutaminase (tTG) catalyses calcium-dependent cross-linking between proteins (transamidation) resulting in protease-resistant isopeptide bonds. Because of this ability, tTG was suspected to participate in AD pathogenesis. Aß and tau can be cross-linked by tTG in vitro. In AD neocortex, messenger RNA expression of tTG is increased. However, data on transamidation in AD specimens--activity of not only tTG but also other transglutaminases--are contradictory. The aim of our study was to investigate if tTG is involved in AD development and may be useful as biomarker for AD. We studied human brain samples for tTG concentration, tTG localization, and transamidation activity and cerebrospinal fluid (CSF) for tTG content by novel sensitive and highly specific methods. Neither tTG concentration nor transamidation was increased in AD brain homogenates. Immunohistologically, we found no colocalization of tTG in neocortex sections with tau or Aß deposits but with blood vessels. Only in rare cases, tTG was detectable in CSF samples. This could be attributed to liberation from erythrocytes. Our data contradict the view that tTG is a potential biochemical marker for AD.

Transglutaminase 2: a novel autoantigen in canine idiopathic central nervous system inflammatory diseases.

Necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis (NLE) and granulomatous meningoencephalomyelitis (GME) are common idiopathic inflammatory central nervous system (CNS) diseases with unknown etiology in dogs. We previously showed that IgG autoantibodies in the cerebrospinal fluid (CSF) of NME cases reacted to unknown brain proteins as well as to glial fibrillary acidic protein (GFAP). In the present report, we evaluated the autoantibodies against transglutaminase2 (TG2) in the canine CNS diseases. CSF samples obtained from dogs with NME (n=19), NLE (n=7), GME (n=11) and miscellaneous CNS diseases (n=12) were subjected. CSFs from 20 healthy dogs were used as controls. Indirect fluorescent antibody test on the canine cerebrum revealed astrocyte-binding IgG in the CSF of NME. After absorption of the CSF with bovine GFAP, the CSF still possessed the reactivity to astrocytes. Double-color staining showed clear colocalization of the autoantibodies and anti-human TG2 rabbit polyclonal IgG. An immunoblot assay against human recombinant TG2 revealed anti-TG2 IgG in the CSF from dogs with NME, NLE and GME. The CSF of canine idiopathic encephalitis cases, notably of NME, tended to show high ELISA OD values against human recombinant TG2 compared to healthy controls. The presence of anti-TG2 autoantibodies in the CSF may contribute to the elucidation of the etiology of canine NME, NLE and GME.

Human guanylate binding protein-1 is a secreted GTPase present in increased concentrations in the cerebrospinal fluid of patients with bacterial meningitis.

Interferon-gamma-induced GTPases are key to the protective immunity against microbial and viral pathogens. As yet, the cell interior has been regarded as the exclusive residence of these proteins. Here we show that a member of this group, human guanylate binding protein-1 (hGBP-1), is secreted from cells. Secretion occurred in the absence of a leader peptide via a nonclassical, likely ABC transporter-dependent, pathway, was independent of hGBP-1 GTPase activity and isoprenylation, and did not require additional interferon-gamma-induced factors. Interestingly, hGBP-1 was only secreted from endothelial cells but not from any of the nine different cell types tested. Clinically most important was the detection of significantly (P<0.001, Mann-Whitney U-test) increased hGBP-1 concentrations in the cerebrospinal fluid of patients with bacterial meningitis (n=32) as compared to control patients (n=74). In this first report of a secreted GTPase, we demonstrate that secreted hGBP-1 may be a useful surrogate marker for diagnosis of bacterial meningitis.

Elevated levels of the alpha subunit of GTP-binding protein Go in cerebrospinal fluid of patients with neurological disorders.

By employing a highly sensitive immunoassay method, concentration of the alpha subunit of GTP-binding protein, Go (Go alpha), recently shown to be localized mainly in nervous tissues and neuroendocrine cells, was determined in cerebrospinal fluids (CSF) of 192 patients with various neurological disorders and 50 control subjects. The results were compared with CSF levels of neuron-specific enolase (NSE) and S-100b protein (S-100b) in the same samples. Normal levels of Go alpha were 51.9 +/- 21.7 pg/ml. The levels of Go alpha, as well as NSE and S-100b, in CSF were enhanced in some patients with acute conditions, e.g., meningitis (48%), encephalitis (100%), and cerebral infarct (56%). In these disorders, cases with enhanced Go alpha levels were more frequent than those with enhanced NSE or S-100b. Three patients with encephalitis whose Go alpha levels were more than 1000 pg/ml all died; the remaining two patients with encephalitis and slightly elevated Go alpha levels had a good prognosis. Concentration of Go alpha in CSF correlated well with that of NSE but poorly with that of S-100b. However, cervical spondylosis and demyelinating diseases, CSF levels of Go alpha were generally lower than those of NSE or S-100b. These results suggest that Go alpha in CSF is a useful marker for monitoring patients with acute neuronal damage. Since these three proteins are distributed differently in the central nervous system, simultaneous determination of Go alpha, NSE, and S-100b levels in CSF might provide valuable information about the pathophysiology of neurological disorders.