O–Glycosylation and Novel Processing Events During Secretion of α–Factor/GM–CSF Fusions by Saccharomyces cerevisiae

Two homologous proteins, murine (m) and human (h) granulocyte–macrophage colony–stimulating–factor (GM–CSF), were differentially glycosylated and processed during secretion by a yeast host. Secreted hGM–CSF, but not mGM–CSF was significantly O–glycosylated. Human GM–CSF was expressed mainly as a highly N–glycosylated form, while 50% of mGM–CSF was unglycosylated, or core–glycosylated. The Asn–X–Thr and Asn–X–Ser sequences of hGM–CSF were equally efficient sites for N–glycosylation, which enhanced the secretion efficiency of hGM–CSF, but not of mGM–CSF. N–terminal analysis of secreted hGM–CSF revealed a novel processing site following the pre–region of the MFα1/hGM–CSF precursor and in addition, suggested partial N–terminal degradation by the STE13–encoded dipeptidase. GM–CSFs secreted by yeast and by animal cells had identical in vitro biological activities.