Functional role of glycosylation on the recombinant CD38/ADP-ribosyl cyclase in CHO cells.
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 Vmax 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.
Chidambaram
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N
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Singapore.
Chang
C F
CF
D036541:ADP-ribosyl Cyclase; D051997:ADP-ribosyl Cyclase 1; D000818:Animals; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D007150:Immunohistochemistry; D007700:Kinetics; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D009244:NAD+ Nucleosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D015703:Antigens, CD; D000943:Antigens, Differentiation; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D036541:ADP-ribosyl Cyclase; C498156:Cd38 protein, rat; D009244:NAD+ Nucleosidase; D051997:ADP-ribosyl Cyclase 1