Sequence analysis and expression of human neuropsin cDNA and gene
Introduction
Recent studies have revealed important roles for serine proteases in the brain, e.g., plasminogen activators in the neural plasticity and neural damage caused by excitotoxins (Qian et al., 1993; Frey et al., 1996; Huang et al., 1996; Chen and Strickland, 1997). Neuropsin is a serine protease originally isolated from the mouse hippocampus (Chen et al., 1995). Biochemical analysis has revealed that neuropsin actually has proteolytic activity with trypsin-like substrate specificity (Shimizu et al., 1998). A subsequent study showed that kindling induces neuropsin mRNA expression (Okabe et al., 1996). Moreover, specific antibody against neuropsin delayed seizure progress induced by kindling (Momota et al., 1998). The amount of neuropsin mRNA is related to memory retention after a chemically induced ischemic insult (Akita et al., 1997). This protein also modifies the induction of long-term potentiation (Komai et al., 1997). Therefore, neuropsin has an important role in neural plasticity. Furthermore, neuropsin mRNA is expressed in a variety of tissues such as brain, skin and the pregnant uterus during development (Suzuki et al., 1995; Chen et al., 1998; Inoue et al., 1998). The variety of functions of this serine protease and its unique expression pattern in the mouse prompted us to analyze its structure and function and also that of a human homologue. We have cloned a cDNA from the human hippocampus and analyzed sequences of the gene for human neuropsin and its 5′ flanking region.
Section snippets
RACE of neuropsin cDNA
All oligonucleotides used for the amplifications of cDNA and DNA are listed in Table 1. According to the sequence of an EST clone (Genbank accession No. AA102333), two primers for 5′ RACE were synthesized. These were 736AS and 637AS. Two rounds of RACE reactions (nested PCR) were performed with 5 μl Marathon Ready™ cDNA of human hippocampus (Clontech, Palo Alto, CA, USA) as a template. The reaction mix and the PCR conditions were conducted according to the manufacturer's recommendations
Sequence analysis of human neuropsin cDNA
One of the human EST clones (Genbank accession No. AA102333, 539 bp) was found to have a significant homology to the sequence of the mouse neuropsin cDNA (Chen et al., 1995) during a database search with BLAST program (Altschul et al., 1990). Based on the sequence data in the database, two PCR primers were designed for 5′ RACE. Two rounds of amplifications from human hippocampus cDNA (Marathon Ready™, Clontech) resulted in a single discrete 650-bp DNA fragment. From the sequence data of the 5′
Acknowledgements
This work was supported by Grants-in-Aid from the Ministry of Education, Science, Culture and Sports of Japan. We thank Dr Y. Yamaguchi at the Department of Dermatology, Osaka University for the generous gift of primary cultures of keratinocytes.
References (27)
- et al.
Effects of oxidative stress on the expression of limbic-specific protease neuropsin and avoidance learning in mice
Brain Res.
(1997) - et al.
Basic local alignment search tool
J. Mol. Biol.
(1990) - et al.
Isolation and sequence of a rat chymotrypsin B gene
J. Biol. Chem.
(1984) - et al.
Neuronal death in the hippocampus is promoted by plasminogen-catalyzed degradation of laminin
Cell
(1997) - et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction
Anal. Biochem.
(1987) - et al.
Genomic organization and chromosomal localization of the human cathepsin G gene
J. Biol. Chem.
(1989) - et al.
Expression of neuropsin in the keratinizing epithelial tissue-immunohistochemical analysis of wild-type and nude mice
J. Invest. Dermatol.
(1998) - et al.
The physiological role of a serine protease, neuropsin on the neural plasticity in hippocampus
Neurosci. Res. Suppl.
(1997) - et al.
Two similar but nonallelic rat pancreatic trypsinogens. Nucleotide sequences of the cloned cDNAs
J. Biol. Chem.
(1982) - et al.
Kindling induces neuropsin mRNA in the mouse brain
Brain Res.
(1996)