Effects of N-Glycosylation Deletions on Cypridina Luciferase Activity

Cypridina luciferase (Cluc), a secreted luminescent protein identified from Cypridina noctiluca, has two N-glycosylation sites. In this study, we evaluated the effects of N-glycosylation on Cluc properties by creating site-directed mutagenic modifications at the consensus sequence for N-glycosylation (Asn-X-Ser/Thr). Eight variants consisting of four single- and double-residue mutants each were characterized. The producibility and relative specific activity were apparently reduced in mutant Cluc although the thermostability and secretion efficiency were not affected. These results suggested that N-glycosylation modifications and the proper amino acid sequence of the N-glycan binding sites of Cluc are required for the complete protein folding to form a stable catalytic center, for the proper conformation of substrate-protein interaction residues, or for both and that defects in the glycosylation modification are not related to secretion process and stability of the protein.