Our studies are directed toward understanding the structure and function of human interferon alphas and their receptors. The objective of these studies is to delineate the rationale for the existence of this family of structurally-related proteins and to understand the mechanism by which they elicit their pleiotropic biological activities. To date, 22 components of human IFN-a derived from Sendai virus-induced human lymphoblastoid cells (Namalwa) were isolated by sequential monoclonal antibody affinity chromatography using four different monoclonal antibodies, ultrafil-tration and reverse-phase HPLC. Many biological properties of these components have been examined. One component of interest, IFN-alpha component o, exhibits high antiproliferative activity on Daudi and AU937 cells, but has low affinity for the IFN-a2b binding site on both cell lines. Cross-linking experiments using Daudi cells revealed a 134-KD complex for component o. Partial amino acid sequence of IFN-a component o revealed that it is indistinguishable from IFNA21. In order to learn the relationship between component o and IFNA21, we cloned and expressed the IFNA21 gene in a pQE-30 expression system. The expressed protein was purified by Ni-NTA affinity chromatography and 4F2 monoclonal antibody affinity chromatography. Analysis of IFNA21 protein by SDS-PAGE revealed only one major band and like IFN-alpha component o, IFNA21 has a very high antiproliferative activity on Daudi cells; the concentration for 50% inhibition of cell growth is 0.01ng/ml. In addition, binding experiments using 125I-IFN-alpha2b suggested that IFNA21, like IFN-alpha component o, competed poorly for IFN-alpha2b binding sites on Daudi and AU937 cells. Cross-linking experiments using Daudi cells and 125I-IFNA21 revealed a 125KD complex with IFNA21 on SDS- PAGE. Chemical characterization of these IFN-a components has been a major program in our laboratory eg. N-terminal amino acid sequences. Currently, we are continuing our studies of the carbohydrate structure of the IFN-a components and have identified 3 major glycosylated components and 11 components with low levels of glycosylation. Employing the techniques of monosaccharide analysis by high pH anion exchange chromatography with pulsed amperometric detection and oligosaccharide mapping using the same technology, we have learned that IFN-alpha components pre-a, a, and b are complex glycoforms that have a combination of N- and O-linkages. In addition, pre-a has a combination of mono-, bi- , and trisialylatrd structures, while components a and b have a combination of mono-, and bisialylated structures. Glycolytic enzyme treatment showned a change in the apparent molecular weight of these components as detected by SDS-PAGE, but no detectable change in their antiviral (MDBK and WISH cells) and antiproliferative (Daudi cells) activities.
