Structure of the mouse gene for the serine protease inhibitor neuroserpin (PI12)
Introduction
Extracellular proteases are known to play an important role in neural development and function. During the formation of the nervous system, proteases regulate cell migration and axon growth (Pittman and Williams, 1989; Gurwitz and Cunningham, 1988). In the mature nervous system they may be involved in processes linked to neuronal plasticity, such as the reorganization of synaptic connections (for a review see Nguyen and Lichtman, 1996). The action of serine proteases is controlled by serine protease inhibitors, e.g. those belonging to the serpin family. Up to now, two serpins were shown to be expressed in the nervous system: protease nexin-1/glia-derived nexin (PN-1/GDN) (Gloor et al., 1986) and neuroserpin (Osterwalder et al., 1996). PN-1 has been shown to promote neurite outgrowth (Gloor et al., 1986), to rescue neurons from cell death (Houenou et al., 1995), and to prevent synapse elimination at the neuromuscular junction by inhibiting thrombin (Liu et al., 1994).
Neuroserpin, originally termed axonin-2, was identified as an axonally secreted protein from chicken dorsal root ganglion cultures (Stoeckli et al., 1989). It was purified from chicken vitreous fluid and subsequently cloned using a PCR-based strategy (Osterwalder et al., 1996). The amino acid sequence, as deduced from a full-length cDNA, qualified neuroserpin as a member of the serpin family. The expression of neuroserpin mRNA is restricted to neurons, as shown by in situ hybridization (Osterwalder et al., 1996). The onset of neuroserpin expression was shown to coincide with the stages of neurogenesis occurring after the axons have reached their targets. In some regions of the brain, including the cerebral cortex, the hippocampus, and the amygdala, neuroserpin mRNA could be detected in the adult (Krueger et al., 1997). The neuroserpin protein, a glycoprotein of 394 amino acids with a molecular weight of approximately 55 kDa, was produced using a eukaryotic expression system (Osterwalder et al., 1998). In vitro results revealed that neuroserpin inhibits tissue-type plasminogen activator (tPA), and to a lesser extent urokinase-type plasminogen activator (uPA) and plasmin, but not thrombin (Osterwalder et al., 1998). Because neuroserpin and tPA do not only interact but are also co-expressed in the same brain regions (Thewke and Seeds, 1996; Krueger et al., 1997), it is possible that neuroserpin has a function as a regulator of tPA activity in the developing and adult nervous system.
We have recently cloned the cDNA of neuroserpin of the mouse (Krueger et al., 1997). Murine neuroserpin exhibits an amino acid sequence identity of 75% and 85%, with its chicken and human homologues, respectively (Osterwalder et al., 1996; Schrimpf et al., 1997), and has an identity of 42% and 39%, respectively, with its closest relatives, PN-1 and plasminogen activator inhibitor-1 (PAI-1). To understand the biological function and the molecular mechanisms contributing to the specific neuronal expression of neuroserpin, we cloned the mouse neuroserpin gene and determined its transcription initiation site. In this report, we describe the structural organization of the mouse neuroserpin gene and give the first account of its basic promoter region.
Section snippets
Isolation of genomic clones encoding mouse neuroserpin
A mouse genomic LambdaGEM-11 library with inserts derived from partially Sau3A1-digested 129SvEv AB-1 embryonic stem cell DNA (kindly provided by Dr Ulrike Mueller, Institute of Molecular Biology I, University of Zurich) was used for the isolation of the neuroserpin gene. A total of 6×106 phages in three different rounds of screening were plated using the E. coli strain LE 392 as a host at a density of 250 000 pfu/500 cm2. Filters were hybridized with the following radioactively labelled probes (
Isolation of genomic clones containing the mouse neuroserpin gene
Approximately 6×106 phage clones of a mouse genomic LambdaGEM-11 library were screened with several radioactively labelled probes derived from either cDNA or genomic DNA of mouse neuroserpin. Thirteen independent clones were identified in the primary screen and plaque-purified to homogeneity by several rescreening rounds. The DNA isolated from the phage clones was analysed by restriction enzyme digestion and Southern blotting. Two groups of overlapping phages were identified. The first group
Discussion
The gene encoding the axonally secreted serine protease inhibitor neuroserpin is present in a single copy in the haploid genome of the mouse. It spans over more than 55 kb and is subdivided into nine exons and eight introns. Both Northern blot analysis and the determination of the transcription initiation site suggest that the expression of the mouse gene is controlled by a single promoter. Nevertheless, the use of an alternative exon 1 during embryogenesis cannot be excluded. The genes of human
Acknowledgements
We thank Dr. A. Shakhov for help with primer extension and Dr. O. Georgiev and J. Kinter for helpful discussion. This work was supported by the Hartmann Müller-Stiftung, the Betty und David Koetser-Stiftung für Hirnforschung and the Stiftung für Wissenschaftliche Forschung an der Universität Zürich.
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