GCP-2. Alternative names. Discovery. Structure. Anja Wuyts*, Paul Proost and Jo Van Damme SUMMARY BACKGROUND

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1 Anja Wuyts*, Paul Proost and Jo Van Damme Laboratory of Molecular Immunology, Rega Institute ± University of Leuven, Minderbroedersstraat 10, Leuven, 3000, Belgium * corresponding author tel: , fax: , anja.wuyts@rega.kuleuven.ac.be DOI: /rwcy SUMMARY The primary structures of human, bovine, and murine granulocyte chemotactic protein 2 () (61±67% homology) were disclosed by amino acid sequence analysis of purified natural protein. chemoattracts neutrophilic granulocytes and induces enzyme release and an increase in [Ca 2+ ] i in these cells in vitro and has proinflammatory properties in vivo. Human, bovine, and murine occur as different N- terminally truncated forms. In contrast to human, for murine C-terminally extended forms (e.g. LIX) have also been isolated. For human and bovine, no difference in potency is observed between these isoforms, whereas for murine, N-terminal as well as C-terminal truncation result in increased specific activity. Similar to IL-8, but in contrast to the other ELR + CXC chemokines, human can efficiently activate cells by binding to CXCR1 and CXCR2. For human and murine, the cdna has been cloned. BACKGROUND Discovery Human granulocyte chemotactic protein 2 () was originally isolated as a granulocyte chemotactic factor from conditioned medium of human MG-63 osteosarcoma cells stimulated with a cytokine mixture (Proost et al., 1993a). It was separated from the simultaneously produced CXC chemokines IL-8, GRO, GRO, and IP-10 by four purification steps (adsorption to controlled pore glass beads, heparin- Sepharose affinity chromatography, cation-exchange fast protein liquid chromatography and reversedphase HPLC) (Proost et al., 1993a). Using the same purification procedure as for human, the bovine equivalent of was purified from phorbol 12-myristate 13-acetate (PMA)-stimulated bovine kidney (MDBK) cells (Proost et al., 1993b). Murine was identified as a neutrophil chemotactic factor produced by PMA-stimulated thymic epithelial (MTEC1) cells as well as by MO fibroblasts stimulated with a combination of LPS and poly(riboinosinic acid) poly(ribocytidylic acid) (poly(ri:rc)) Alternative names Bovine ENA-78, isolated from LPS-stimulated monocytes and alveolar macrophages, is identical to bovine (Allman-Iselin et al., 1994). The amino acid sequence of natural murine corresponds to the cdna-derived sequence of murine LIX (LPSinduced CXC chemokine), which was cloned from LPS-stimulated fibroblasts (Smith and Herschman, 1995). Structure Only the primary structure of has been determined. The proteins contain the four characteristic cysteines of chemokines and show the ELR- and CXC-motif in the N-terminal region (Proost et al., 1993b; Wuyts et al., 1996). As shown for other chemokines, disulfide bridges will be formed between the first and third and between the second and fourth cysteine residue.

2 1070 Anja Wuyts, Paul Proost and Jo Van Damme Main activities and pathophysiological roles chemoattracts and activates (intracellular calcium increase, enzyme release) neutrophilic granulocytes in vitro. The protein induces local neutrophil accumulation and plasma extravasation in vivo, indicating a role in inflammation (Proost et al., 1993a,b; Wuyts et al., 1996, 1997a, 2000). GENE AND GENE REGULATION Accession numbers Human : U83303, Y08770 Murine : U27267 Chromosome location The human gene is localized on the long arm of chromosome 4 (Modi and Chen, 1998). Regulatory sites and corresponding transcription factors The human gene consists of four exons and three introns. The region 3 0 of the -coding region contains three polyadenylation signals and multiple copies of the ATTTA motif, which are associated with rapid message degradation. The promoter has potential binding sites for AP-2, NF-IL6, and NFB transcription factors (Rovai et al., 1997). Cells and tissues that express the gene IL-1 and LPS are inducers of human mrna expression in diploid fibroblasts, MG-63 osteosarcoma cells, and monocytic THP-1 cells. In contrast, IFN has no effect or downregulates mrna in these cell types (Table 1) (Rovai et al., 1997; Froyen et al., 1997; Van Damme et al., 1997). Whereas PMA and dsrna stimulate expression in MG-63 cells, they downregulate mrna in THP-1 cells (Froyen et al., 1997; Van Damme et al., 1997). Dexamethasone attenuates the TNF-induced expression in MG-63 cells(rovai et al., 1997). Human mrna is upregulated in Chlamydia trachomatis-infected endometrial epithelial cells (Wyrick et al., 1999) and is constitutively expressed in heart, lung, liver, and Table 1 Regulation of human mrna expression in different cell types Cell type IL-1 IFN TNF LPS dsrna PMA Fibroblasts + 0 nd MG-63 cells THP-1 cells + nd + nd, not determined; +, increased expression; 0, no effect;, decreased expression. pancreas, but not or weakly in brain, kidney, and placenta tissue (Van Damme et al., 1997). Murine mrna is induced in fibroblasts by LPS and by TGF1, but it is not expressed in LPSstimulated macrophages. Dexamethasone attenuates the LPS-induced mrna expression in fibroblasts (Smith and Herschman, 1995). In mice, mrna can be detected in the lung, but not in other organs. However, after intravenous administration of LPS, mrna is expressed by a variety of tissues. LPS-induced expression is strongest in the heart, intermediate in lung, spleen, bowel, kidney, and skeletal muscle, and weakest in brain and liver. This pattern of expression is different from that of the other murine ELR + CXC chemokines KC and MIP-2. The induction of mrna in acute endotoxemia is delayedcomparedtothatofkcandmip-2,and mrnaremainselevatedforalongerperiodoftime.the difference in tissue distribution of expression and in kinetics of induction indicate that these three murine ELR + CXC chemokines are regulated differently (Rovai et al., 1998). The LPS-induced expression of murine mrna in lung, small bowel, heart, liver, and spleen is attenuated by endogenous glucocorticoids. However, in brain, the expression is increased by dexamethasone, indicating that might mediate a function in brain distinct from its proinflammatory role as a neutrophil chemoattractant. In contrast to, endotoxemia-induced lung expression of KC and MIP-2 is insensitive to glucocorticoids (Rovai et al., 1998). Murine mrna in the lung is also increased during staphylococcal enterotoxin B-induced acute lung inflammation (Neumann et al., 1998). PROTEIN Accession numbers Human : P80162 Bovine : P80221 Murine : P50228

3 1071 Sequence The primary structure of (75 residues) (Figure 1) was first determined by N-terminal and internal amino acid sequence analysis of purified natural protein (Proost et al., 1993b). The sequence was confirmed by cloning of the human cdna and gene, except for two additional amino acids at the C-terminus (Froyen et al., 1997; Rovai et al., 1997). The cdna encodes a 114 residue protein, including a 37 amino acid signal peptide (Rovai et al., 1997). contains four cysteine residues and shows the ELR and CXC motif. Bovine contains 75 amino acids and has 67% identical amino acids with human (Proost et al., 1993b). The murine cdna encodes a protein of 92 amino acids after cleavage of a 40- residue signal peptide (Smith and Herschman, 1995). Description of protein Human is a 6 kda protein which occurs as four different N-terminally truncated forms (77, 75, 72, and 69 amino acids). These isoforms are separated by reversed-phase HPLC (Proost et al., 1993a,b). Similarly, in addition to intact bovine (5 kda, 75 amino acids), isoforms missing 6, 7, and 8 N- terminal amino acids have been purified from bovine kidney cells (Proost et al., 1993b). Bovine, isolated from LPS-stimulated monocytes and alveolar macrophages, is missing five amino acids at the N- terminus (Allman-Iselin et al., 1994). Murine, isolated from epithelial cells and fibroblasts, occurs as 28 different N- and/or C-terminally truncated isoforms, containing from 69 ((10±78)) up to 92 ((1±92)) amino acids. These isoforms correspond to protein bands of 6 to 9.5 kda on SDS- PAGE In contrast to human and bovine, the murine Figure 1 Amino acid sequence for human, bovine, and murine. Signal sequences are underlined. Human MSLPSSRAAR VPGPSGSLCA LLALLLLLTP PGPLASAGPV SAVLTELRCT CLRVTLRVNP KTIGKLQVFP AGPQCSKVEV VASLKNGKQV CLDPEAPFLK KVIQKILDSG NKKN Bovine GPVAAVVREL RCVCLTTTPG IHPKTVSDLQ VIAAGPQCSK VEVIATLKNG REVCLDPEAP LIKKIVQKIL DSGKN Murine MSLQLRSSAH IPSGSSSPFM RMAPLAFLLL FTLPQHLAEA APSSVIAATE LRCVCLTVTP KINPKLIANL EVIPAGPQCP TVEVIAKLKN QKEVCLDPEA PVIKKIIQKI LGSDKKKAKR NALAVERTAS VQ isoforms are not completely separated by reversedphase HPLC. This final purification step yields fractions containing mixtures of forms truncated at the N- and/or C-terminus. These naturally truncated forms shortened (S) at the N-terminus or C- terminus, compared to longer (L) forms, are designated (SS), (SL), (LS), and (LL), respectively Discussion of crystal structure The crystal structure of has not been determined, but is supposed to be similar to that of IL-8. Important homologies Human is highly homologous to human ENA-78 (77% identical amino acids), whereas it shows only low homology to IL-8 (30% identical amino acids) (Table 2). Bovine has 67% identical amino acids with human and 72% with human ENA-78 (Proost et al., 1993b). However, bovine shows a similar elution profile on cation-exchange chromatography and reversed-phase HPLC as human and is therefore considered to be the equivalent of human. Murine GCP- 2(1±78) has 64%, 61%, and 55% identical amino acids with bovine, human, and human ENA-78, respectively (Wuyts et al., 1996) (Table 2). Posttranslational modifications does not contain N-glycosylation sites. The calculated molecular mass for human and bovine (8312 and 7927 Da, respectively) is higher than their relative molecular mass (6 and 5 kda, respectively) deduced from SDS-PAGE (Proost et al., 1993b). Furthermore, synthetic nonglycosylated Table 2 Structural comparison (% identical residues) of the amino acid sequence of, IL-8, and ENA-78 Human Bovine Murine Human IL-8 Human 100 Bovine Murine Human IL Human ENA

4 1072 Anja Wuyts, Paul Proost and Jo Van Damme human and murine show identical biochemical (elution profiles during purification, relative molecular mass on SDS-PAGE) and biological properties to that of the natural protein, indicating that there is probably no glycosylation (Wuyts et al., 1997a, 2000). For human, bovine, and murine, different N-terminally processed forms have been identified (Proost et al., 1993a,b; Wuyts et al., 1996). In addition, several C-terminally extended forms of murine have been isolated from natural cellular sources CD26/dipeptidylpeptidase IV (DPP IV) has been shown to remove the dipeptide from human, yielding (3±77) (Proost et al., 1998). CELLULAR SOURCES AND TISSUE EXPRESSION Cellular sources that produce Human protein was isolated from cytokinestimulated MG-63 osteosarcoma cells (Proost et al., 1993a). The bovine equivalent is produced by kidney cells, monocytes, alveolar macrophages and endometrial epithelial cells (Proost et al., 1993b; Allman- Iselin et al., 1994; Staggs et al., 1998; Austin et al., 1999). Murine was purified from conditioned medium of stimulated thymic epithelial cells and fibroblasts Eliciting and inhibitory stimuli including exogenous and endogenous modulators production by osteosarcoma cells is induced with a mixture of cytokines, derived from mitogenstimulated mononuclear cells (Proost et al., 1993a). However, the exact nature of protein induction has not yet been studied due to the lack of a specific ELISA. Bovine kidney cells and monocytes produce after stimulation with PMA and LPS, respectively (Proost et al., 1993b; Allman-Iselin et al., 1994). IFN, IFN, PMA, and pregnancy-specific protein B are inducers of bovine in endometrial epithelial cells (Staggs et al., 1998; Austin et al., 1999). Bovine has been demonstrated immunohistochemically in inflamed lung tissues in cases of bovine pneumonic pasteurellosis; it was detected in the alveolar epithelial cells, mesothelial cells, endothelial cells and leukocytes (Allman-Iselin et al., 1994). PMA and LPS plus dsrna induce the production of murine in epithelial cells and fibroblasts, respectively RECEPTOR UTILIZATION Human induces an increase in [Ca 2+ ] i in human neutrophils, which is completely prevented by pertussis toxin, whereas cholera toxin does not inhibit this increase. This was the first indication that acts on neutrophils through pertussis toxin-sensitive G protein-coupled receptors. The increase in [Ca 2+ ] i in response to is abolished or strongly reduced after stimulation of neutrophils with equimolar concentrations of or the other ELR + CXC chemokines IL-8, GRO, GRO, and ENA-78. Alternatively, desensitizes the calcium response of neutrophils induced by ENA-78, GRO, GRO, and IL-8, indicating that shares its receptor(s) and/or signal transduction pathways with the other ELR + CXC chemokines (Wuyts et al., 1997a). The precise receptor usage of has further been studied using CXCR1 and CXCR2 transfectants (Wuyts et al., 1997a, 1998). is equally potent and efficient at inducing a calcium rise in both CXCR1- and CXCR2-transfected cells, whereas the related ENA-78 is a better stimulus for CXCR2- than for CXCR1-transfected cells (Figure 2a,b) (Wuyts et al., 1998). In contrast, IL-8 is more potent to induce a calcium increase in CXCR1 than in CXCR2 transfectants. inhibits the calcium increase induced by IL-8 in both transfected cell types and vice versa, whereas ENA-78 can only inhibit the IL-8-induced response in CXCR2-transfected cells. This indicates that CXCR2 is shared by IL-8,, and ENA-78, whereas the CXCR1-mediated calcium mobilization is efficiently activated by IL-8 and, but not by ENA-78. In addition,, like IL-8, chemoattracts CXCR1- and CXCR2-transfected cells with a similar potency, whereas ENA-78 is more potent to attract CXCR2 transfectants (Figure 2c,d). Furthermore, can displace 125 I-labeled IL-8 from both CXCR1 and CXCR2, whereas ENA-78 can only displace IL-8 from CXCR2 (Figure 2e,f ). In conclusion, can activate cells through both CXCR1 and CXCR2, whereas ENA-78 is an efficient ligand for CXCR2 only (Wuyts et al., 1998). Comparison of the primary structures of and IL-8 revealed that both chemokines contain a basic amino acid (Arg20 and Lys15, respectively) at position 6 after the second cysteine, whereas no basic residue is present in the other ELR + CXC chemokines. Lys15 has previously been shown to be important for IL-8 binding to CXCR1 (SchraufstaÈ tter et al.,

5 1073 Figure 2 CXCR1 and CXCR2 receptor usage by human. The ability of human to induce an increase in [Ca 2+ ] i (a, b) and chemotaxis (c, d) of CXCR1- (a, c) and CXCR2- (b, d) transfected cells and to displace 125 I-labeled IL-8 from CXCR1 (e) and CXCR2 (f ) was compared with that of ENA-78 and IL- 8. Results show that in addition to IL-8, also can activate cells through both CXCR1 and CXCR2, whereas ENA-78 is a much better ligand for CXCR ). Substitution of Arg20 of by Gly (GCP- 2(R20G)) shows that this basic residue is indeed important for activation of cells through CXCR1. (R20G) and wild-type induce similar responses (calcium increase, migration) in CXCR2 transfectants, whereas CXCR1-expressing cells do not respond to the variant (Wolf et al., 1998). According to these findings, murine and MIP-2, both containing a basic residue at position 6 after the second cysteine residue (Lys21 and Arg17, respectively), induce a calcium rise in both CXCR1- and CXCR2-transfected cells. However, both proteins are more potent to signal through CXCR2 than through CXCR1 (Wuyts et al., 2000).