Processing of natural and recombinant CXCR3-targeting chemokines and implications for biological activity

Chemokines comprise a class of peptides with chemotactic activity towards l eukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, posttr anslational modifications have been shown to affect the effectiveness of ch emokines. Although in several studies, natural CXCR3-targeting chemokines have been i solated, detailed information about the proteins and their possible modific ations is lacking. Using a combination of liquid chromatography and mass sp ectrometry we studied the protein profile of CXCR3-targeting chemokines exp ressed by interferon-gamma -stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more det ail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the g enerally accepted sequence. In addition we identified a C-terminally trunca ted CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to a n N-terminally truncated human CXCL11, lacking the first two amino acids FP indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL1 1 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expr essing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein leve l is important to characterize the exact profile of these chemotactic pepti des as modifications can severely alter their biological activity.