Effect of glycosylation at Asn302 of pro-urokinase on its stability in culture supernatant.
- Author:
Bo YANG
1
;
Tian-De LI
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Substitution; Animals; Asparagine; chemistry; Base Sequence; CHO Cells; Cricetinae; Cricetulus; Culture Media; DNA Primers; genetics; Enzyme Stability; Glycosylation; Humans; In Vitro Techniques; Mutagenesis, Site-Directed; Recombinant Proteins; chemistry; genetics; metabolism; Urokinase-Type Plasminogen Activator; chemistry; genetics; metabolism
- From: Chinese Medical Sciences Journal 2006;21(2):128-130
- CountryChina
- Language:English
-
Abstract:
OBJECTIVETo investigate the effect of glycosylation at Asn302 of pro-urokinase (pro-UK) on the stability in culture supernatant.
METHODSNonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302. The pro-UK mutant and native pro-UK were transfected into dhfr(-)-CHO cells, and serum-free culture supernatant was harvested and incubated at 4 degrees C and 37 degrees C, respectively. The pro-UK activity in culture supernatant was measured by the optical density (OD) increase with time (12 hours) at 405 nm. Without thermolysin activation, the percentage of single chain pro-UK was measured.
RESULTSAfter 48 hours of incubation at 4 degrees C, the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively, and at 37 degrees C decreased 37.9% and 23.5%, respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37 degrees C. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4 degrees C and 37 degrees C.
CONCLUSIONHigher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant.
