Neuroserpin: a serpin to think about

Proteinases and their inhibitors play important roles in neural development, homeostasis and disease. Neuroserpin is a member of the serine proteinase inhibitor (serpin) superfamily that is secreted from the growth cones of neurons and inhibits the enzyme tissue-type plasminogen activator (tPA). The temporal and spatial pattern of neuroserpin expression suggests a role in synaptogenesis and is most prominent in areas of the brain that participate in learning, memory and behaviour. Neuroserpin also provides neuronal protection in pathologies such as cerebral ischaemia and epilepsy by preventing excessive activity of tPA. Point mutations in neuroserpin cause aberrant conformational transitions and the formation of loop-sheet polymers that are retained within the endoplasmic reticulum of neurons, forming inclusion bodies that underlie an autosomal dominant dementia that we have called familial encephalopathy with neuroserpin inclusion bodies or FENIB. We review here the role of neuroserpin and other proteinase inhibitors in brain development, function and disease

The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity

B-type Eph receptors and ephrins induce growth cone collapse through distinct intracellular pathways

Forward and reverse signaling mediated by EphB tyrosine kinase receptors and their transmembrane ephrin-B ligands play important roles in axon pathfinding, yet little is known about the intracellular pathways involved. Here we have used growth cones from the ventral (EphB receptor-bearing) and dorsal (ephrin-B-bearing) embryonic Xenopus retina to investigate the signaling mechanisms in both forward and reverse directions. We report that unclustered, but not clustered, EphB2 ectodomains trigger fast (5-10 min) transient collapse responses in growth cones. This collapse response is mediated by low levels of intracellular cyclic GMP and requires proteasome function. In contrast, clustered, but not unclustered, ephrin-B1 ectodomains cause slow (30-60 min) growth cone collapse that depends on high cGMP levels and is insensitive to inhibition of the proteasomal pathway. Upon receptor-ligand binding, endocytosis occurs in the reverse direction (EphB2-Fc into dorsal retinal growth cones), but not the forward direction, and is also sensitive to proteasomal inhibition. Endocytosis is functionally important because blocking of EphB2 internalization inhibits growth cone collapse. Our data reveal that distinct signaling mechanisms exist for B-type Eph/ephrin-mediated growth cone guidance and suggest that endocytosis provides a fast mechanism for switching off signaling in the reverse direction. (C) 2003 Wiley Periodicals, Inc

Identification of Reissner's fiber-like glycoproteins in two species of freshwater planarians (Tricladida), by use of specific polyclonal and monoclonal antibodies

By using one polyclonal antiserum raised against bovine Reissner's fiber and seven monoclonal antibodies raised against bovine Reissner's fiber and against immunopurified bovine subcommissural organ glycoproteins, we have investigated two freshwater planarian species (Girardia tigrina, Schmidtea mediterranea) by light- and electron-microscopic immunocytochemistry. ELISA probes showed that the monoclonal antibodies recognized different, nonoverlapping, unrepeated, proteinaceous epitopes present in the same compounds of bovine Reissner's fiber. Cells immunoreactive to the polyclonal and monoclonal antibodies were found in the dorsal and ventral integument of both planarian species. Labeled cuboid epidermal cells bore cilia and displayed several types of secretory granules; they were covered by a film of immunoreactive material. Studies on adjacent thin and semithin sections revealed coexistence of label in the same regions and in the same cells when two different monoclonal antibodies were used. These results indicate that a secretory substance immunologically similar to the secretion of the vertebrate subcommissural organ is present in primitive tripoblasts such as planarians, suggesting that these secretions are ancient and well conserved in phylogeny

An antibody raised against a pathogenic serpin variant induces mutant-like behaviour in the wild-type protein

A monoclonal antibody (mAb) that binds to a transient intermediate may act as a catalyst for the corresponding reaction; here we show this principle can extend on a macro molecular scale to the induction of mutant-like oligomerization in a wild-type protein. Using the common pathogenic E342K (Z) variant of α1-antitrypsin as antigen-whose native state is susceptible to the formation of a proto-oligomeric intermediate-we have produced a mAb (5E3) that increases the rate of oligomerization of the wild-type (M) variant. Employing ELISA, gel shift, thermal stability and FRET time-course experiments, we show that mAb5E3 does not bind to the native state of α1-antitrypsin, but recognizes a cryptic epitope in the vicinity of the post-helix A loop and strand 4C that is revealed upon transition to the polymerization intermediate, and which persists in the ensuing oligomer. This epitope is not shared by loop-inserted monomeric conformations. We show the increased amenity to polymerization by either the pathogenic E342K mutation or the binding of mAb5E3 occurs without affecting the energetic barrier to polymerization. As mAb5E3 also does not alter the relative stability of the monomer to intermediate, it acts in a manner similar to the E342K mutant, by facilitating the conformational interchange between these two states

Rostral floor plate (flexural organ) secretes glycoproteins immunologically similar to subcommissural organ glycoproteins in dogfish (Scyliorhinus canicula) embryos

The subcommissural organ of vertebrates secretes glycoproteins into the cerebrospinal fluid of the third cerebral ventricle. This material polymerizes in Reissner's fiber. During ontogenetic development, besides the subcommissural organ, the ependyma lining the pontine flexure constitutes an additional Reissner's fiber-secreting gland named flexural organ. We have studied the secretion of the flexural organ and the subcommissural organ in dogfish (Scyliorhinus canicula) embryos using three different antisera and the lectins concanavalin A and wheat germ agglutinin. AFRU is an antiserum against the bovine Reissner's fiber; Ab-600 is an antiserum against 600 kDa dogfish subcommissural organ glycoproteins; and APSO is an antiserum against immunoaffinity purified bovine subcommissural organ secretory glycoproteins. These three antisera immunostained the flexural organ indicating that it contains epitopes similar to those present in bovine and dogfish subcommissural organ glycoproteins. It seems highly probable that the flexural or an and the subcommissural organ of dogfish embryos secrete similar compound(s). Other ependymal regions were also immunostained with Ab-600 and APSO antisera. Then, Reissner's fiber-like glycoproteins were transiently expressed by most embryonary ependymal cells. These glycoproteins might play a role in the development of the central nervous system of vertebrates. (C) 1997 Elsevier Science B.V

Practical genetics: alpha-1-antitrypsin deficiency and the serpinopathies

Alpha-1-antitrypsin (alpha(1)-antitrypsin) is the archetypal member of the serine proteinase inhibitor or serpin superfamily. The most common severe deficiency variant is the Z allele, which results in the accumulation of mutant protein within hepatocytes. This 'protein overload' causes neonatal hepatitis, cirrhosis and hepatocellular carcinoma. The lack of circulating plasma alpha(1)-antitrypsin results in early-onset panlobular emphysema. The mechanism underlying the deficiency of Z alpha(1)-antitrypsin is due to an aberrant conformational transition within the protein and the formation of chains of polymers that tangle within the secretory pathway of hepatocytes. This mechanism also underlies the plasma deficiency of other members of the serpin superfamily to cause a class of diseases called the serpinopathies. Specifically mutant alleles of antithrombin, C1-inhibitor and alpha(1)-antichymotrypsin have been reported that favour the spontaneous formation of polymers and the retention of protein within hepatocytes. The consequent lack of plasma antithrombin, C1-inhibitor and alpha(1)-antichymotrypsin results in thrombosis, angio-oedema and emphysema, respectively. Moreover, the polymerisation of mutants of neuroserpin results in the retention of polymers within neurones to cause the inclusion body dementia, familial encephalopathy with neuroserpin inclusion bodies or FENIB. We review here the genetic and molecular basis and clinical features of alpha(1)-antitrypsin deficiency, and show how this provides a platform to understand the other serpinopathies