Isolation of the CXC chemokines ENA‐78, GROα and GROγ from tumor cells and leukocytes reveals NH<sub>2</sub>‐terminal heterogeneity

Wuyts, Anja; Govaerts, Cindy; Struyf, Sofie; Lenaerts, Jean‐Pierre; Put, Willy; Conings, René; Proost, Paul; Damme, Jo Van

doi:10.1046/j.1432-1327.1999.00166.x

Cited by 80 publications

(81 citation statements)

References 40 publications

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“…Considering that these residues are buried in the GAG-bound form, GAG-bound hCXCL1 is much less susceptible to proteolysis, indicating that GAG-binding also plays a direct role in evading microbial infection. Endogenously pro-duced CXCL1 has also been shown to be heterogeneous because of the N-terminal cleavage, and the isoforms have differential neutrophil activity (44). Our observation that the N-terminal residues are also involved in GAG binding suggests that GAG binding also indirectly influences receptor activation by regulating accessibility to proteases.…”

Section: Discussionmentioning

confidence: 80%

CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine

Sepuru

Rajarathnam

2016

Journal of Biological Chemistry

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In humans, the chemokine CXCL1/MGSA (hCXCL1) plays fundamental and diverse roles in pathophysiology, from microbial killing to cancer progression, by orchestrating the directed migration of immune and non-immune cells. Cellular trafficking is highly regulated and requires concentration gradients that are achieved by interactions with sulfated glycosaminoglycans (GAGs). However, very little is known regarding the structural basis underlying hCXCL1-GAG interactions. We addressed this by characterizing the binding of GAG heparin oligosaccharides to hCXCL1 using NMR spectroscopy. Binding experiments under conditions at which hCXCL1 exists as monomers and dimers indicate that the dimer is the high-affinity GAG ligand. NMR experiments and modeling studies indicate that lysine and arginine residues mediate binding and that they are located in two non-overlapping domains. One domain, consisting of N-loop and C-helical residues (defined as ␣-domain) has also been identified previously as the GAG-binding domain for the related chemokine CXCL8/IL-8. The second domain, consisting of residues from the N terminus, 40s turn, and third ␤-strand (defined as ␤-domain) is novel. Eliminating ␤-domain binding by mutagenesis does not perturb ␣-domain binding, indicating two independent GAG-binding sites. It is known that N-loop and N-terminal residues mediate receptor activation, and we show that these residues are also involved in extensive GAG interactions. We also show that the GAG-bound hCXCL1 completely occlude receptor binding. We conclude that hCXCL1-GAG interactions provide stringent control over regulating chemokine levels and receptor accessibility and activation, and that chemotactic gradients mediate cellular trafficking to the target site.Chemokines, a large family of small soluble proteins, are highly versatile and play fundamental roles in diverse functions, from combating infection and initiating tissue repair to regulating metabolism and organ development (1, 2). Common to these various functions is the directed movement of various cell types to distal and remote locations. Cellular trafficking must be highly coordinated to elicit the required biological function, and its dysregulation could be detrimental, resulting in disease. There is now increasing evidence that the ability of a chemokine to reversibly exist as monomers and dimers and binding glycosaminoglycans is coupled and plays important roles in mediating these functions (3, 4).Glycosaminoglycans (GAGs) 2 such as heparan sulfate (HS) are highly sulfated polysaccharides. They are expressed ubiquitously by many cell types, are anchored to the cell surface by covalent attachment to membrane proteins, and form non-covalent complexes with proteins in the extracellular matrix (5-7). Animal models and cellular studies have established that GAG interactions dictate chemokine concentration gradients and that these gradients orchestrate cellular trafficking (8 -10). GAGs are acidic, and chemokines are basic or contain clusters of basic residues, indicating that electr...

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Section: Discussionmentioning

confidence: 80%

CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine

Sepuru

Rajarathnam

2016

Journal of Biological Chemistry

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“…This can be explained by the fact that large volumes (liters) of conditioned medium were used as start material for purification of minute quantities of different natural GCP-2 isoforms. In addition to GCP-2, IL-8, and GRO, ENA-78, which is most homologous to GCP-2 (77% identical amino acids), was isolated in small amounts from the supernatant of these cytokine-stimulated osteosarcoma cells (Proost et al, 1993;Wuyts et al, 1999). The availability of various natural NH 2 terminally truncated forms of GCP-2 allowed confirmation that all of these were equally well recognized by our specific and sensitive GCP-2 ELISA.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, monocytes were purified from peripheral blood as previously described (Wuyts et al, 1999) and were stimulated with LPS (Escherichia coli 0.111.B4; Difco, Detroit, Michigan; 2 g/ml) and conA (Calbiochem, La Jolla, California; 2 g/ml) in EMEM supplemented with 2% FCS for 48 hours at 37°C. Chemokines produced on a large scale were isolated from conditioned cell supernatants by a four-step purification procedure as previously described (Wuyts et al, 1997a).…”

Section: Production Of Chemokinesmentioning

confidence: 99%

“…Rabbit polyclonal anti-human ENA-78 (1:300) from PeproTech and goat polyclonal antihuman ENA-78 (1:300) from R&D Systems, respectively, were used as coating and capturing antibodies. Human IL-8 was measured with a classical sandwich ELISA using polyclonal goat anti-human IL-8 as coating antibody (1:800) and monoclonal mouse antihuman IL-8 (1:10 4 ) (R&D Systems) as capturing antibody, as described previously (Wuyts et al, 1999).…”

Section: Measurement Of Human Il-8 Ena-78 and Gcp-2 By Elisamentioning

confidence: 99%

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The CXC Chemokine GCP-2/CXCL6 Is Predominantly Induced in Mesenchymal Cells by Interleukin-1β and Is Down-Regulated by Interferon-γ: Comparison with Interleukin-8/CXCL8

Wuyts

Struyf

Gijsbers

et al. 2003

Laboratory Investigation

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SUMMARY:Human granulocyte chemotactic protein-2 (GCP-2)/CXCL6 is a CXC chemokine that functionally uses both of the IL-8/CXCL8 receptors to chemoattract neutrophils but that is structurally most related to epithelial cell-derived neutrophil attractant-78 (ENA-78)/CXCL5. This study provides the first evidence that GCP-2 protein is, compared with IL-8, weakly produced by some sarcoma, but less by carcinoma cells, and is tightly regulated in normal mesenchymal cells. IL-1␤ was the predominant GCP-2 inducer in fibroblasts, chondrocytes, and endothelial cells, whereas IL-8 was equally well up-regulated in these cells by TNF-␣, measles virus, or double-stranded RNA (dsRNA). In contrast, lipopolysaccharide (LPS) was a relatively better stimulus for GCP-2 versus IL-8 in fibroblasts. IFN-␥ down-regulated the GCP-2 production in fibroblasts induced by IL-1␤, TNF-␣, LPS, or dsRNA. The kinetics of GCP-2 induction by IL-1␤, LPS, or dsRNA in fibroblasts differed from those of IL-8. Freshly isolated peripheral blood mononuclear leukocytes, which are a good source of IL-8 and ENA-78, failed to produce GCP-2. However, lung macrophages and blood monocyte-derived macrophages produced GCP-2 in response to LPS. Quantitatively, secretion of GCP-2 always remained inferior to that of IL-8, despite the fact that the ELISA recognized all posttranslationally modified GCP-2 isoforms. The expression of GCP-2 was confirmed in vivo by immunohistochemistry. The patterns of producer cell types, inducers and kinetics and the quantities of GCP-2 produced, suggest a unique role for GCP-2 in physiologic and pathologic processes. (Lab Invest 2003, 83:23-34).

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“…CXCL5 is expressed in both lung and pancreatic cancer and is up-regulated in endometriosis and ovarian carcinoma where it can attract neutrophils (Wislez et al 2004, Furuya et al 2007, Frick et al 2008. Once present, neutrophils secrete a broad spectrum of proteases including cathepsin G that can cleave CXCL5; N-terminal truncation of CXCL5 significantly enhances the chemotactic potency of CXCL5 both in vitro and in vivo (Wuyts et al 1999, Mortier et al 2010. Neutrophils isolated from the peripheral blood of ovarian cancer patients also produce increased reactive oxygen species and display increased adhesion to ovarian cancer cells through enhanced CD11b/ CD18 expression on their cell surfaces (Klink et al 2008).…”

Section: Angiogenic Chemokine Ligands Can Promote Tumour Developmentmentioning

confidence: 99%

The emerging role of CXC chemokines in epithelial ovarian cancer

et al. 2012

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In recent years, chemokines have generated intense investigations due to their involvement in both physiological and pathological processes of inflammation, particularly in ovarian biology. The physiological process of ovulation in the normal ovary involves various chemokines that mediate the healing of the ruptured endometrium. It is now being reported that many of these chemokines are also associated with the cancer of the ovary. Chronic inflammation underlies the progression of ovarian cancer; therefore, it raises the possibility that chemokines are involved in the inflammatory process and mediate immune responses that may favour or inhibit tumour progression. Ovarian cancer is a gynaecological cancer responsible for highest rate of mortality in women. Although there have been several investigations and advances in surgery and chemotherapy, the survival rate for this disease remains low. This is mainly because of a lack of specific symptoms and biomarkers for detection. In this review, we have discussed the emerging role of the CXC chemokines in epithelial ovarian cancer (EOC). The CXC group of chemokines is gaining importance in the field of ovarian cancer for being angiostatic and angiogenic in function. While there have been several studies on the angiogenesis function, emerging research shows that ELR K CXC chemokines, CXCL9 and CXCL10, are angiostatic. Importantly, the angiostatic chemokines can inhibit the progression of EOC. Given that there are currently no biomarkers or specific therapeutic targets for the disease, these chemokines are emerging as promising targets for therapy.

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Isolation of the CXC chemokines ENA‐78, GROα and GROγ from tumor cells and leukocytes reveals NH₂‐terminal heterogeneity

Cited by 80 publications

References 40 publications

CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine

CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine

The CXC Chemokine GCP-2/CXCL6 Is Predominantly Induced in Mesenchymal Cells by Interleukin-1β and Is Down-Regulated by Interferon-γ: Comparison with Interleukin-8/CXCL8

The emerging role of CXC chemokines in epithelial ovarian cancer

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Isolation of the CXC chemokines ENA‐78, GROα and GROγ from tumor cells and leukocytes reveals NH2‐terminal heterogeneity

Cited by 80 publications

References 40 publications

CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine

CXCL1/MGSA Is a Novel Glycosaminoglycan (GAG)-binding Chemokine

The CXC Chemokine GCP-2/CXCL6 Is Predominantly Induced in Mesenchymal Cells by Interleukin-1β and Is Down-Regulated by Interferon-γ: Comparison with Interleukin-8/CXCL8

The emerging role of CXC chemokines in epithelial ovarian cancer

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Isolation of the CXC chemokines ENA‐78, GROα and GROγ from tumor cells and leukocytes reveals NH₂‐terminal heterogeneity