Background

Our laboratory, supervised by Dr Chang Chan Fong, is mainly involved in signal transduction work which will include basic molecular biology techniques as well as using standard biochemical techniques tools. Bioinformatics and biocomputing will play an integral part in our project work if implemented in our laboratory. We can rely on online databases and biocomputing tools for experimental design as well as data analysis. One area of research is in the signaling pathway of the ectoenzyme, CD38. In my Honour's Project, I have characterized and expressed the CD38-GFP protein in mammalian cells.    

What is CD38?

CD38 is a type II transmembrane glycoprotein of 300 amino acids expressed in many vertebrate cells. The deduced amino acid sequences of CD38 have shown that the polypeptide consists of three domains, a short NH₂-terminal cytoplasmic tail, a single transmembrane region, and a large extracellular COOH-terminal catalytic domain. It is a bifunctional ectoenzyme that catalyzes both the synthesis of cyclic ADP-ribose (cADPR) from NAD⁺ and the degradation of cADPR to ADP-ribose by means of its ADP-ribosyl cyclase and cADPR-hydrolase activities, respectively.

Roles of CD38 and cADPR

ADP- ribosyl cyclase and CD38 are multi-functional enzymes involved in Ca²⁺ signaling. Both can cyclize NAD⁺ and its guanine analog, NGD⁺, to produce cyclic ADP-ribose and cyclic GDP-ribose, respectively. Both enzymes can also catalyze the exchange of nicotinamide group NADP⁺ with nicotinic acid (NA) at acidic pH, producing another potent activator of Ca²⁺ mobilization, nicotinic acid adenine dinucleotide phosphate (NAADP). The Ca²⁺ release mechanism activated by NAADP is totally independent of cADPR and inositol triphosphate (IP₃).

CD38 plays a role in the signaling system of insulin secretion. Glucose induces an increase in intracellular Ca²⁺ concentration in pancreatic b-cells of the islets of Langerhans to secrete insulin. Recently, CD38 has been demonstrated to be a catalytically active, unidirectional transmembrane reporter of cADPR, which then reaches its receptor–operated intracellular calcium stores. Moreover, CD38 was reported to undergo a selective and extensive internalization through non clathrin-coated endocytotic vesicles upon incubating CD38⁺ cells with either NAD⁺ or thiol compounds: these endocytotic vesicles can convert cytosolic NAD⁺ into cADPR.

cADPR is a universal second messenger that releases calcium from intracellular stores (Clapper et al.,1987).In cells, Ca²⁺ -induced- Ca²⁺ -release (CICR) mechanism requires calmodulin and Ca²⁺ . Ca²⁺ release is also induced by the CICR modulators, ryanodine and caffeine.Another well-known mechanism for mobilizing Ca²⁺ is mediated by inositol  1,4,5-trisphosphate (IP₃). Binding of external ligands to surface receptors can activate phospholipase C and the production of IP₃, which, upon binding to a specific receptor (IP₃-sensitive Ca²⁺ channel) on the endoplasmic reticulum, activates Ca²⁺ release.

Approach used in studying CD38

The conventional methodology used in the study of CD38 has always been immunostaining. GFP was chosen because its fluorescence is an intrinsic property of the protein, which does not require additional gene products. Besides, time and effort can be saved in raising antibodies to localize CD38 in live cells. This model system enables one to monitor the trafficking and internalization of receptors in living cells in real time under confocal microscopy.

Other research interests in the laboratory

The other research interest in our laboratory is on BST-1 also known as CD157. cADP-ribose acts as an agent potent in mobilizing Ca²⁺ from ryanodine-sensitive, Ins(1,4,5)P₃-independent intracellular stores. cADP-ribose is thought to be an endogenous modulator of ryanodine receptors. Biological functions of cADP-ribose have been studied in sea-urchin eggs, as well as in a variety of mammalian cell systems. Convincing evidence is accumulating that cADP-ribose is involved in cardiac muscle contraction, glucose-induced release of insulin in endocrine pancreas and contraction of intestinal longitudinal muscle. In mammalian cells, cADP-ribose is synthesized by the membrane-bound enzymes CD38 and BST-1 /BP-3 , which is now renamed CD157 . Human BST-1/CD157, which was identified in a bone marrow stromal cell line, consists of 290 amino acids. The amino acid sequence corresponding to BST-1 cDNA contains an N-terminal signal sequence and a short C-terminal hydrophobic region, suggesting that BST-1 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein. Thus BST-1 and CD38 are bifunctional ecto-enzymes, having both ADP-ribosyl cyclase and cADP-ribose hydrolase activities. However, both products, cADP-ribose and ADP-ribose, do not appear to cross the plasma membrane themselves. How cADP-ribose, which is presumably produced by extracellularly facing ADP-ribosyl cyclase, enters into the cell interior is currently under investigation.