Analysis of physiological functions of CEA family cell adhesion molecules expressed on neutrophils

点击翻译按钮获取中文摘要

For clarifying physiological functions of carcinoembryonic antigen (CEA)-related cell adhesion molecules (CD66, NCAs) in human neutrophils, the following studies were carried out : 1.Recombinant proteins of BGP (CD66a) , CGM6 (CD66b) and NCA-50 (CD66c) were prepared in silkworm larvae. Transfer vectors were constructed with pBK283 and cDNAs of the CD66 antigens that were modified to encode secretory forms of the proteins. The vectors were introduced to the baculovirus DNA by co-transfection of BmN4 cells. Silkworm larvae were infected with the virus, and recombinant proteins secreted into hemolymph, at the concentrations of 0.2-1.3 mg/ml, were purified by affinity chromatography with anti-NCA-50 antibody (Ab) or cation exchange chromatography. The purified proteins were coated on plates and examined for cell adhesion activity against CHO cells expressing CEA family proteins. The recombinant proteins exhibited cell adhesion activity against the cells with the same specificities to those … More observed between the CHO transfectants. Deglycosylated form of the antigens also showed the same adhesion activity, indicating that the adherence is dependent on the peptide portion of the antigens. By using the BGP protein, monoclonal Abs specific to BGP were obtained. These recombinant antigens and monoclonal Abs will be valuable for investigating the functions of CD66 antigens in adhesion and activation of neutrophils. 2.The colon tumor cell line HT29 was cloned in glucose-deficient media so that the cells develop a monolayr with polarity similar to normal colonic epithelia. The adherence of neutrophils to the monolayr was not inhibited but was augmented by anti-CD66, implying that CD66 antigens are not directly involved in the adherence. This monolayr will be useful to study the role of CD66 antigens in adherence and migration of neutrophils on mucosal epithelial cells. 3.In vitro phosphorylation experiments using immunoprecipitates from neutrophil lysates revealed that an about 55-kDa phosphorylated protein is associated with CD66b (CGM6) , indicating that CD66b may be linked with intracellular signal transduction pathways. Less