Abstract
Current evidence suggests that CD38, a lymphocyte differentiation antigen, has been found to be functionally indistinguishable from the native form when it was expressed as a soluble and non-glycosylated protein in yeast. Studies were conducted to evaluate the functional role of glycosylation on the membrane-bound CD38 in mammalian cells. The stable transformants of CHO cells were established with pXJ41–CD38 construct and the recombinant CD38 was detected at the surface of CHO cells using a polyclonal antibody to rat CD38 by immunocytochemistry. The recombinant protein displaying ADP-ribosyl cyclase activity was purified from CHO cells, which appeared as 42 and 46 kDa bands on immunoblot under non-reducing and reducing conditions, respectively. The recombinant CD38 was then subjected to deglycosylation with N-glycosidase F that resulted in a 33 kDa band on immunoblot under reducing condition. Further the partial deglycosylation of the recombinant CD38, performed at various time intervals, resulted in a series of bands (33–46 kDa) on immunoblot. Kinetic analysis indicated that deglycosylation of the recombinant CD38 showed a considerable decrease in V max and an increase in K m of ADP-ribosyl cyclase activity. These observations clearly suggest that glycosylation plays an important role to maintain the enzymatic activity and substrate affinity of CD38/ADP-ribosyl cyclase for mediating the signalling events in mammalian cells.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
More From: International Journal of Biochemistry and Cell Biology
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.