EXPRESSION AND FUNCTIONAL STUDIES OF CD157/BST-1

Abstract

In this work, we have studied the expression and transcriptional regulation of CD157 and evaluated its functions. We first sought to raise polyclonal Ab against CD157 that would facilitate characterization and functional studies of CD157. A recombinant 103-amino acid 6xHis-CD157 polypeptide (~14 kDa) was expressed in bacteria, which was purified using Ni-NTA/agarose beads and injected into rabbits for producing Ab (Chapter 1). Purified lgG from immunized rabbit serum recognized the 6xHis-CD157 immunogen and a yeast- expressed 38.5-kDa CD157. We further affinity-purified the CD157-specific Ab from total lgG samples by a novel method using a Ni-NTA/agarose-based column on which the 6xHis CD157 antigen was immobilized. This method was able to capture almost all of the CD157- specific Ab and the purified Ab displayed a very high specificity. In Chapter 2, we describe the expression of a recombinant CD157 (rCD157) having structural and functional integrity in yeast anticipating that it would facilitate studies aimed at determining the relationship between its enzymatic activity and cellular function. The yeast-expressed 6xHis-rCD157 was purified to homogeneity using a Ni-NTA/agarose column and appeared as a 38.5-kDa protein on SDS-PAGE gels and on immunoblots. Treatments with N-glycosidase demonstrated that two of the four potential N-glycosylation sites were recognized in yeast. The rCD157 displayed the ADP-ribosyl cyclase activity. CD157 is expressed on the mature mycloid cells but not on the immature precursors in the bone marrow. Further CD38, a homologous gene to CD157, has been found to be expressed in the promyeloid HL-60 and/or U937 cell lines when treated with the monocyte and granulocyte differentiation inducing 1?,25-Dihydroxyvitamin D3 (VD3) and all-trans- retinoic acid (ATRA), respectively (Chapter 3). Hence, we have evaluated whether CD157 is expressed or upregulated in HL-60 and/or U937 cells when induced to differentiate into mature phenotypes with special emphasis on the VD3- and ATRA-induced differentiation (Chapter 3). The monocytic (VD3, TNF-?. and arabinosylcytosine (ara-C)) and granulocytic (ATRA and dimethylsulfoxide) lineage inducers upregulated CD157 while the macrophage lineage-inducing phorbol-12-myristate-13-acetate (PMA) induced its down-regulation. The time-kinetics of VD3-induced antigen expression demonstrated a quick (within 6 hours) induction of CD38 but a delayed (after 24 hours) initiation of CD157 and CD11b (differentiation marker). VD3-induced expression of CD157 was irreversible while those of CD38 and CD11b were reversible upon withdrawal of VD3 from culture medium. Expression of CD157 in VD3-treated HL-60 cells appeared to be dependent on the cAMP- mediated mechanism. TNF-? and ara-C seemed to mediate CD157 expression in HL-60 cells via VD3-independent mechanisms. The upregulated CD157 in VD3-treated HL-60 cells was able to activate the Ab-ligation dependent tyrosine phosphorylation signal. In Chapter 4, we investigated the detailed kinetics, behavior and cell-type specificity of CD157-stimulated p130 phosphorylation. CD157-mediated p130 phosphorylation was ligand-independent in rCD157-expressing CHO, MCA102 and COS-7 cells but ligand- dependent in VD3-differentiated HL-60 monocytes (mHL-60) having enhanced CD157 expression. This p130 phosphorylation was activated at 0-4°C in MCA102, COS-7 and mHL-60 cells but was temperature-insensitive in CHO cells. CHO/CD157 cell clones had ~22-28% slower rates of proliferation than that of a CHO/mock clone while the MCA102 cell proliferation remained unaffected by CD157 expression.