Increased expression and secretion of r-Gsp protein, rat counterpart of complement C1s precursor, during cyclic AMP-induced differentiation in rat C6 glioma cells.
The gene, termed r-gsp, was originally isolated during identification of differentiation-associated molecules in rat C6 glial cells. Its mRNA expression was markedly increased during cAMP-induced glial cell differentiation. The deduced amino acid sequence of r-gsp was homologous to those of complement C1s precursors of hamsters and humans. In the present study, we raised anti-peptide antibody against r-Gsp protein and analyzed its change during cAMP-induced differentiation. The 90-kDa r-Gsp protein increased time-dependently and reached the maximal level ( approximately 7.6-fold increase) at 24 h in response to dibutyryl cyclic AMP (dbcAMP) and theophylline. Moreover, it was secreted into the medium and then was cleaved to form disulfide-linked fragments, one of which was 30 kDa, similar to C1s, suggesting its processing in the extracellular space. In fact, the partially purified r-Gsp from culture medium was cleaved by active human C1r to form a 30-kDa polypeptide. Moreover, secreted r-Gsp protein cleaved human C4alpha to yield C4alpha' and associated with human serum C1-esterase inhibitor, strongly suggesting that r-Gsp protein is rat C1s. However, in C6 cells overexpressing r-Gsp, their morphology and proliferation rate were similar to those in parent C6 cells. These results suggest that r-Gsp protein could not induce glial differentiation alone, and suggest that r-Gsp protein was secreted as a proenzyme and processed in culture medium. Its possible role in glial cell differentiation will be discussed.
Nakagawa
Masanori
M
Department of Neurosurgery, Gifu University School of Medicine, Tsukasamachi-40, Japan.
Nakashima
Shigeru
S
Banno
Yoshiko
Y
Yamada
Jun
J
Sawada
Motoshi
M
Yoshimura
Shin ichi
Si
Kaku
Yasuhiko
Y
Iwama
Toru
T
Shinoda
Jun
J
Sakai
Noboru
N
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D003174:Complement C1 Inactivator Proteins / Q000378:metabolism; D050718:Complement C1 Inhibitor Protein; D015923:Complement C1r / Q000378:metabolism; D003173:Complement C1s / Q000235:genetics / Q000378:metabolism*; D003181:Complement C4 / Q000378:metabolism; D016895:Culture Media, Serum-Free; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D004792:Enzyme Precursors / Q000378:metabolism; D005910:Glioma / Q000378:metabolism; D006801:Humans; D010455:Peptides / Q000276:immunology / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013806:Theophylline / Q000494:pharmacology; D014357:Trypsin / Q000235:genetics / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000906:Antibodies; D003174:Complement C1 Inactivator Proteins; D050718:Complement C1 Inhibitor Protein; D003181:Complement C4; D016895:Culture Media, Serum-Free; D004792:Enzyme Precursors; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013806:Theophylline; D000242:Cyclic AMP; D014357:Trypsin; D015923:Complement C1r; D003173:Complement C1s