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A Method For Production of Recombinant mCD1d Protein in Insect Cells.
Published on: December 10, 2007
Biochemical characterization of CD39L4
J J Mulero1, G Yeung, S T Nelken
1Functional Genomics Department, Immunology Group, Hyseq Inc., 670 Almanor Avenue, Sunnyvale, California 94086, USA.
Biochemistry
|October 21, 2000
Summary
CD39L4, an enzyme regulating nucleotide levels, was characterized. Glycosylation is not essential for its ADPase activity, and the monomeric form exhibits the highest enzymatic activity, offering insights into nucleotide regulation.
Area of Science:
- Biochemistry and Molecular Biology
- Enzymology
- Cell Biology
Background:
- Nucleotides play critical roles in physiological processes like inflammation and platelet aggregation.
- Enzymes modulating blood nucleotide levels are key regulators in these systems.
- CD39L4 is identified as a soluble E-nucleoside triphosphate dephosphohydrolase (E-NTPDase) specific for nucleotide diphosphates (NDPs).
Purpose of the Study:
- To generate and characterize recombinant CD39L4 protein expressed in mammalian and insect cells.
- To investigate the role of glycosylation in CD39L4 activity.
- To determine the active form of CD39L4 (monomer vs. dimer).
Main Methods:
- Stable mammalian (HEK 293) and insect cell lines were created for CD39L4 expression and purification.
- Glycosylation was assessed using glycosidase treatment and tunicamycin inhibition.
- Enzymatic activity was measured using ADPase assays, and protein forms (monomer/dimer) were separated by sucrose density gradient centrifugation.
Main Results:
- Recombinant CD39L4 expressed in human cells was found to be glycosylated.
- Glycosylation was not essential for the ADPase activity of CD39L4.
- CD39L4 expressed in insect cells showed different glycosylation but comparable activity; the monomeric form was determined to be the most active.
Conclusions:
- Biochemical and enzymatic characterization of CD39L4 provides a foundation for understanding its physiological roles.
- The enzyme's activity is primarily associated with its monomeric form, independent of glycosylation.
- These findings highlight CD39L4 as a significant regulator of nucleotide metabolism.
