Secretion of proteases in serglycin transfected Madin-Darby canine kidney cells.
Madin-Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules.
Zernichow
Lillian
L
Department of Nutrition, Institute of Basic Medical Sciences, University of Oslo, Norway.
Dalen
Knut T
KT
Prydz
Kristian
K
Winberg
Jan-Olof
JO
Kolset
Svein O
SO
D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004285:Dogs; D004789:Enzyme Activation / Q000187:drug effects; D005780:Gelatin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology* / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000187:drug effects / Q000378:metabolism*; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D011509:Proteoglycans; D013431:Sulfates; D033921:Vesicular Transport Proteins; C075430:serglycin; C041319:isoleucyl-prolyl-arginine-4-nitroanilide; D005780:Gelatin; D010447:Peptide Hydrolases; D013755:Tetradecanoylphorbol Acetate