CD66 monoclonal antibodies recognize a phosphotyrosine-containing protein bearing a carcinoembryonic antigen cross-reacting antigen on the surface of human neutrophils

K. M. Skubitz, T. P. Ducker, S. A. Goueli

Research output: Contribution to journalArticlepeer-review

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Abstract

The CD66 Ag is a neutrophil-specific 'activation Ag' in that it is detected in low density on resting cells but its surface expression is up- regulated by stimulation (with the chemotactic peptide FMLP, the calcium ionophore A23187, and 12-O-tetradeconoyl-phorbol-13-acetate). Phosphorylation is an important mechanism of regulation of protein function. Although most studies of protein phosphorylation have focused on intracellular reactions, recent studies have provided evidence for the existence of ectoprotein kinase activity on the surface of several types of cells including human neutrophils. The role of ectoprotein kinase activity in cell function is unknown and little is known about the endogenous substrates of this enzyme system. The identification and characterization of physiologic substrates of ectoprotein kinase activity should aid the understanding of the role of this enzyme activity in cell function. Immunoprecipitation and subsequent gel electrophoresis of proteins from neutrophils labeled with [γ-32P]ATP revealed that CD66 mAb specifically recognize a ~180-kDa phosphoprotein on the surface of human neutrophils. This protein was one of the major endogenous substrates for human neutrophil ectoprotein kinase activity. Phosphoamino acid analysis of the 180-kDa protein revealed that it contained predominantly phosphotyrosine. Preclearing studies demonstrated that this protein was also recognized by CD15 mAb, and by polyclonal anticarcinoembryonic Ag antiserum. In addition, the CD66 mAb reacted with purified carcinoembryonic Ag, biliary glycoprotein, and 'nonspecific cross- reacting Ag.' Thus, the neutrophil protein recognized by CD66 mAb appears to be a ~180-kDa form of the classical 'nonspecific cross-reacting Ag' on human neutrophils.

Original languageEnglish (US)
Pages (from-to)852-860
Number of pages9
JournalJournal of Immunology
Volume148
Issue number3
StatePublished - 1992

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