Although glycosaminoglycan (GAG)–protein interactions are important in many physiological and pathological processes, the structural requirements for binding are poorly defined. Starting with GAG-binding peptide CXCL9(74-103), peptides were designed to elucidate the contribution to the GAG-binding affinity of different: (1) GAG-binding motifs (i.e., BBXB and BBBXXB); (2) amino acids in GAG-binding motifs and linker sequences; and (3) numbers of GAG-binding motifs. The affinity of eight chemically synthesized peptides for various GAGs was determined by isothermal fluorescence titration (IFT). Moreover, the binding of peptides to cellular GAGs on Chinese hamster ovary (CHO) cells was assessed using flow cytometry with and without soluble GAGs. The repetition of GAG-binding motifs in the peptides contributed to a higher affinity for heparan sulfate (HS) in the IFT measurements. Furthermore, the presence of Gln residues in both GAG-binding motifs and linker sequences increased the affinity of trimer peptides for low-molecular-weight heparin (LMWH), partially desulfated (ds)LMWH and HS, but not for hyaluronic acid. In addition, the peptides bound to cellular GAGs with differential affinity, and the addition of soluble HS or heparin reduced the binding of CXCL9(74-103) to cellular GAGs. These results indicate that the affinity and specificity of peptides for GAGs can be tuned by adapting their amino acid sequence and their number of GAG-binding motifs.
With ELISAs one detects the ensemble of immunoreactive molecules in biological samples. For biomolecules undergoing proteolysis for activation, potentiation or inhibition, other techniques are necessary to study biology. Here we develop methodology that combines immunosorbent sample preparation and nano-scale liquid chromatography—tandem mass spectrometry (nano-LC-MS/MS) for proteoform analysis (ISTAMPA) and apply this to the aglycosyl chemokine CXCL8. CXCL8, the most powerful human chemokine with neutrophil chemotactic and –activating properties, occurs in different NH2-terminal proteoforms due to its susceptibility to site-specific proteolytic modification. Specific proteoforms display up to 30-fold enhanced activity. The immunosorbent ion trap top-down mass spectrometry-based approach for proteoform analysis allows for simultaneous detection and quantification of full-length CXCL8(1-77), elongated CXCL8(-2-77) and all naturally occurring truncated CXCL8 forms in biological samples. For the first time we demonstrate site-specific proteolytic activation of CXCL8 in synovial fluids from patients with chronic joint inflammation and address the importance of sample collection and processing.
Interleukin 7 (IL-7) is a cell growth factor with a central role in normal T cell development, survival and differentiation. The lack of IL-7–IL-7 receptor(R)-mediated signaling compromises lymphoid development, whereas increased signaling activity contributes to the development of chronic inflammation, cancer and autoimmunity. Gain-of-function alterations of the IL-7R and the signaling through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) are enriched in T cell acute lymphoblastic leukemia (T-ALL) and autocrine production of IL-7 by T-ALL cells is involved in the phenotypes of leukemic initiation and oncogenic spreading. Several IL-7-associated pathologies are also characterized by increased presence of matrix metalloproteinase-9 (MMP-9), due to neutrophil degranulation and its regulated production by other cell types. Since proteases secreted by neutrophils are known to modulate the activity of many cytokines, we investigated the interactions between IL-7, MMP-9 and several other neutrophil-derived proteases. We demonstrated that MMP-9 efficiently cleaved human IL-7 in the exposed loop between the α-helices C and D and that this process is delayed by IL-7 N-linked glycosylation. Functionally, the proteolytic cleavage of IL-7 did not influence IL-7Rα binding and internalization nor the direct pro-proliferative effects of IL-7 on a T-ALL cell line (HPB-ALL) or in primary CD8+ human peripheral blood mononuclear cells. A comparable effect was observed for the neutrophil serine proteases neutrophil elastase, proteinase 3 and combinations of neutrophil proteases. Hence, glycosylation and disulfide bonding as two posttranslational modifications influence IL-7 bioavailability in the human species: glycosylation protects against proteolysis, whereas internal cysteine bridging under physiological redox state keeps the IL-7 conformations as active proteoforms. Finally, we showed that mouse IL-7 does not contain the protease-sensitive loop and, consequently, was not cleaved by MMP-9. With the latter finding we discovered differences in IL-7 biology between the human and mouse species.
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