Cloning of a complementary DNA encoding the unique dendritic cell antigen Ki-M9.

Abstract

Human sinus-lining cells (SLC) of the lymph node sinuses most probably represent accessory cells for the primary humoral immune response and have been shown to express a unique antigen recognized by the monoclonal antibody Ki-M9. To characterize this SLC-specific antigen further, a spleen cDNA library established in the expression vector lambda gt-11 was searched immunochemically for clones expressing the Ki-M9 antigen. Two recombinant phage clones revealed a cDNA with an open reading frame of 1666 bp encoding a 68-kDa protein. When fused with an expression vector that codes for the bacterial maltose-binding protein (MBP), the purified MBP-Ki-M9 fusion protein could be clearly detected by Western blot analysis. Furthermore, in situ hybridization with Ki-M9 cDNA as a probe confirmed the SLC-specific expression of the cloned cDNA. Additionally, Ki-M9 mRNA transcripts were detected in follicular dendritic reticulum cells of the secondary germinal centers, in a few cells of the perifollicular zone of the spleen, in some sinusoidal cells of liver, and in thymic reticular cells. A sequence database research revealed a strong homology to a murine cDNA. By applying non-radioactive in situ hybridization on mouse tissue, strong expression in SLC of the lymph node and metallophilic cells of the spleen in mouse tissue could be seen indicating that the Ki-M9 cDNA is highly conserved in the two species. Further computer analysis of the deduced amino acid sequence of the Ki-M9 antigen showed a large number of potential glycosylation sites and a PEST motive, which are characteristic for rapidly degraded membrane bound proteins and represent prerequisites for the function of this cell system in initiating the humoral immune response.