Processing of natural and recombinant CXCR3-targeting chemokines and implications for biological activity.
Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated 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 an 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 CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.
Hensbergen
P J
PJ
Department of Dermatology, University of Leiden, The Netherlands. P.Hensbergen@lumc.nl
van der Raaij-Helmer
E M
EM
Dijkman
R
R
van der Schors
R C
RC
Werner-Felmayer
G
G
Boorsma
D M
DM
Scheper
R J
RJ
Willemze
R
R
Tensen
C P
CP
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002478:Cells, Cultured; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D019743:Chemokines, CXC / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002633:Chemotaxis; D006224:Cricetinae; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D015603:Keratinocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013601:T-Lymphocytes / Q000166:cytology / Q000378:metabolism; D014162:Transfection
C515202:CXCL11 protein, human; C515187:CXCR3 protein, human; D054357:Chemokine CXCL10; D054371:Chemokine CXCL11; D019743:Chemokines, CXC; C515203:Cxcl11 protein, mouse; C515188:Cxcr3 protein, mouse; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine; D011994:Recombinant Proteins; D007371:Interferon-gamma; D002118:Calcium