Abstract
A cDNA encoding a novel serine protease, which we designated spinesin, has been cloned from human spinal cord. The longest open reading frame was 457 amino acids. A homology search revealed that the human spinesin gene was located at chromosome 11q23 and contained 13 exons, the gene structure being similar to that of TMPRSS3 whose gene is also located on 11q23. Spinesin has a simple type II transmembrane structure, consisting of, from the N terminus, a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger receptor-like domain, and a serine protease domain. Unlike TMPRSS3, it carries no low density lipoprotein receptor domain in the stem region. The extracellular region carries five N-glycosylation sites. The sequence of the protease domain carried the essential triad His, Asp, and Ser and showed some similarity to that of TMPRSS2, hepsin, HAT, MT-SP1, TMPRSS3, and corin, sharing 45.5, 41.9, 41.3, 40.3, 39.1, and 38.5% identity, respectively. The putative mature protease domain preceded by H(6)DDDDK was produced in Escherichia coli, purified, and successfully activated by immobilized enterokinase. Its optimal pH was about 10. It cleaved synthetic substrates for trypsin, which is inhibited by p-amidinophenylmethanesulfonyl fluoride hydrochloride but not by antipain or leupeptin. Northern blot analysis against mRNA from human tissues including liver, lung, placenta, and heart demonstrated a specific expression of spinesin mRNA in the brain. Immunohistochemically, spinesin was predominantly expressed in neurons, in their axons, and at the synapses of motoneurons in the spinal cord. In addition, some oligodendrocytes were clearly stained. These results indicate that spinesin is transported to the synapses through the axons after its synthesis in the cytoplasm and may play important roles at the synapses. Further analyses are required to clarify its roles at the synapses and in oligodendrocytes.
Highlights
Serine proteases have essential functions in biological processes such as the activation of complement and blood coagulation
Nucleic Acid Sequence of Human Spinesin cDNA—PCR using degenerate primers designed from serine protease motifs, AAHC and DSGGP, amplified a 465-bp fragment from a cDNA library of human spinal cord
It apparently belongs to the TMPRSS family having an N-terminal cytoplasmic domain, a transmembrane domain, a scavenger receptor-like domain in the stem region, and a protease domain
Summary
Materials—Human tissues of the CNS for immunohistological analyses were obtained with informed consent within 12 h of death. Antibodies—Rabbit polyclonal antibodies for Western blotting and immunohistochemical analyses were raised against two KLH-conjugated peptides, KLH-CSEASAEEALLP (anti-human spinesin A) and KLH-CAGLVSHSAVRPHQG (anti-human spinesin B), and purified using protein A-Sepharose (Amersham Biosciences, Inc.) The former peptide sequence is derived from the stem region, and the latter was derived from the protease domain (see Fig. 1). A clone carrying a 465-bp fragment was found to have a novel serine protease-related sequence Based on this sequence, specific primers were synthesized for the rapid amplification of cDNA ends (RACE, Table I). Hybridization was carried out in ExpressHyb hybridization solution (CLONTECH) at 60 °C overnight, and the final wash was performed in 0.1ϫ saline/sodium phosphate/EDTA containing 0.1% SDS at room temperature for 10 min. After a wash with phosphate-buffered saline-Tween, the slides were incubated with alkaline phosphatase-labeled goat anti-rabbit IgG for 60 min at room temperature. DP-S DP-A Primer 1 Primer 2 Primer 3 Primer 4 Primer 5 Primer 6 Adaptor primer AP1 AP2
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