Elsevier

Gene

Volume 213, Issues 1–2, 15 June 1998, Pages 9-16
Gene

Sequence analysis and expression of human neuropsin cDNA and gene

https://doi.org/10.1016/S0378-1119(98)00232-7Get rights and content

Abstract

Neuropsin is a serine protease which is thought to function in a variety of tissues including the brain and skin. This protease has been shown to have important roles in neural plasticity in mice. Here we have cloned a cDNA and analyzed the gene for human neuropsin by polymerase chain reaction-based strategies. The cDNA had 72% identity to mouse neuropsin. The deduced amino acid sequence showed 72% identity to mouse neuropsin. Key amino acid residues for the enzyme activity and all cysteine residues were conserved between human and mouse neuropsin. The gene for human neuropsin had six exons and five introns, and the gene organization is similar to trypsin-type serine proteases. The mRNA was expressed in primary cultures of keratinocytes.

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.

Cited by (0)

View full text