Molecular and biochemical characterization of a serine proteinase predominantly expressed in the medulla oblongata and cerebellar white matter of mouse brain.
A full-length cDNA clone of a serine proteinase, mouse brain serine proteinase (mBSP), was isolated from a mouse brain cDNA library. mBSP, which has been recently reported to be expressed in the hair follicles of nude mice, is most similar (88% identical) in sequence to rat myelencephalon-specific protease. The mBSP mRNA was steadily expressed in the brain of adult mice with a transient expression in the early fetal stage during development. The genomic structure of the mouse gene for mBSP was determined. The gene, which is mapped to chromosome 7B4-B5, is about 7.4 kilobases in size and contains 7 exons. Interestingly, the 5'-untranslated region of the mBSP gene was interrupted by two introns. In situ hybridization analyses revealed that mBSP is expressed in the white matter of the cerebellum, medulla oblongata, and capsula interna and capsula interna pars retrolenticularis of mouse brain. Further, mBSP was immunolocalized to the neuroglial cells in the white matter of the cerebellum. Recombinant mBSP was produced in the bacterial expression system and activated by lysyl endopeptidase digestion, and the activated enzyme was purified for characterization. The enzyme showed amidolytic activities preferentially cleaving Arg-X bonds when 4-methylcoumaryl-7-amide-containing peptide substrates were used. Typical serine proteinase inhibitors, such as diisopropyl fluorophosphates, phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, aprotinin, leupeptin, antipain, and benzamidine, strongly inhibited the enzyme activity. The recombinant mBSP effectively hydrolyzed fibronectin and gelatin, but not laminin, collagens I and IV, or elastin. These results suggest that mBSP plays an important role in association with the function of the adult mouse brain.
Matsui
H
H
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
Kimura
A
A
Yamashiki
N
N
Moriyama
A
A
Kaya
M
M
Yoshida
I
I
Takagi
N
N
Takahashi
T
T
D000818:Animals; D001483:Base Sequence; D002531:Cerebellum / Q000201:enzymology*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D008526:Medulla Oblongata / Q000201:enzymology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D011994:Recombinant Proteins; D012697:Serine Endopeptidases