Effect of glycosylation at Asn302 of pro-urokinase on its stability in culture supernatant / 中国医学科学杂志(英文版)

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the effect of glycosylation at Asn302 of pro-urokinase (pro-UK) on the stability in culture supernatant.</p><p><b>METHODS</b>Nonglycosylated 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.</p><p><b>RESULTS</b>After 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.</p><p><b>CONCLUSION</b>Higher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant.</p>