| Summary: | Leukosialin is a major cell-surface sialoglycoprotein of unknown function expressed on all leukocytes except B cells. Two clones for mouse leukosialin were isolated from a genomic library and sequencing revealed one of these to be a non-functional pseudogene, whereas the other contained an uninterrupted open reading frame that encoded leukosialin. The possibility that leukosialin might be involved in cell migration pathways was tested by expressing the antigen on B cells in transgenic mice but these cells did not show any aberrant migratory behaviour. The heavy glycosylation of the leukosialin extracellular domain, with one O-linked glycan per 3 amino acids, suggests an extended structure and this has now been confirmed by electron microscopy. The antigenicity of this domain was studied using nine monoclonal antibodies (mAbs) whose binding is differentially affected by the cell type on which leukosialin is expressed and by the removal of sialic acid. Seven of these mAbs reacted clearly with the extracellular domain expressed in an unglycosylated form in E.coli and epitope mapping demonstrated that these epitopes correspond to short protein sequences. It thus appears that linear protein epitopes are recognized and these can be modified in the native structure by glycosylation. MAbs to human leukosialin are potent at inducing cell clustering and this was shown to hold also for mAbs to rat leukosialin. Monomeric Fab fragments were inactive, demonstrating that cross-linking of leukosialin molecules is required for cell clustering. The cytoplasmic domain of rat leukosialin was expressed in E.coli and shown to have an elongated structure and to be amenable to phosphorylation by protein kinase C. CD2 is an important T cell adhesion molecule, interacting with LFA-3 on other cells through its NH2-terminal domain. This domain is also of interest as it has been predicted to have an immunoglobulin type fold despite the absence of the normally conserved disulphide bond. The expression of rat CD2 domain 1 in E.coli and the three-dimensional structure of the domain, determined by NMR spectroscopy, are reported.
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