Effects of N‐Glycosylation Deletions on Cypridina Luciferase Activity

Abstract 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.