Molecular diversity of calpastatin in human erythroid cells.
According to differences in mobility on SDS-polyacrylamide gel electrophoresis, calpastatins (inhibitor proteins of the calcium-dependent proteinase calpain) are classified into the tissue type (100-120 kDa) and the erythrocyte type (70 kDa), which lacks the amino-terminal domains (domains L and 1). We investigated the molecular diversity of calpastatin in human hematopoietic cells by Western-blot analysis and by the reverse-transcription-polymerase-chain reaction method. While the mononuclear and polymorphonuclear cells in peripheral blood showed the tissue type (110 and 114 kDa), a cell line of erythroid cells (JK-1) showed both the tissue type (110 kDa) and the erythrocyte type (70 kDa) at approximately equal ratios. When the lysate of JK-1 cells was incubated in the presence of ATP, the 110-kDa form was degraded much faster than the 70-kDa form. In human erythrocytes, the 110-kDa form was identified as the tissue type by an antibody recognizing domain L, and this form was also present in addition to the predominant 70-kDA form. JK-1 cells, as well as nucleated cells in peripheral blood, contained calpastatin mRNA with exon-3-deleted. Glioblastoma and fibroblast cell lines expressed the nondeleted calpastatin mRNA in addition to the deletion type, and they showed bands corresponding to 117 kDa as well as 110 and 114 kDa. The 117-kDa band was detectable by an anti-exon 3 peptide antibody. These results suggest that diversity among the tissue type calpastatins is caused by both alternative splicing and post-translational processing whereas the apparent conversion from the tissue type to the erythrocyte type is caused by proteolytic processing.
Institute for Virus Research, Faculty of Medicine, Kyoto University, Japan.
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E
Takano
T
T
Nosaka
W J
WJ
Lee
K
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Nakamura
T
T
Takahashi
M
M
Funaki
H
H
Okada
M
M
Hatanaka
M
M
Maki
D000255:Adenosine Triphosphate / Q000494:pharmacology; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000097:blood* / Q000235:genetics; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005091:Exons; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D005910:Glioma / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D006801:Humans; D015636:Magnesium Chloride / Q000494:pharmacology; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000097:blood; D014407:Tumor Cells, Cultured
D002135:Calcium-Binding Proteins; D012333:RNA, Messenger; D015636:Magnesium Chloride; C033668:calpastatin; D000255:Adenosine Triphosphate