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Isolation, Characterization, and cDNA Cloning of Chicken Turpentine-induced Protein, a New Member of the Scavenger Receptor Cysteine-rich (SRCR) Family of Proteins

2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês

10.1074/jbc.m011713200

1083-351X

Ken Iwasaki, Masami Morimatsu, Osamu Inanami, Eiji Uchida, Bunei Syuto, Mikinori Kuwabara, Masayoshi NIIYAMA,

Cell Adhesion Molecules Research

Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5−16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant activity and inhibits superoxide (O⨪2) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Spα, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species. Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5−16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant activity and inhibits superoxide (O⨪2) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Spα, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species. polyacrylamide disc gel electrophoresis bovine serum albumin concanavalin A wheat germ agglutinin ulex europeus agglutinin I soybean agglutinin dolichos biflorus agglutinin ricinus communis agglutinin I peanut agglutinin periodic acid-Schiff Hanks' balanced salt solution phorbol 12-myristate 13-acetate serum-opsonized zymosan superoxide copper- and zinc-containing superoxide dismutase electron spin resonance 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide protein kinase C 5,5-dimethyl-1-pyrroline N-oxide polymerase chain reaction rapid amplification of cDNA end base pair(s) scavenger receptor cysteine-rich Several acute-phase proteins in serum, such as C-reactive protein, hemopexin, fibrin, fibrinogen, transferrin, α1-acid glycoprotein, and α2-macroglobulin, have been reported in chicken (1Patterson L.T. Mora E.C. Tex. Rep. Biol. Med. 1965; 23: 600-606PubMed Google Scholar, 2Grieninger G. Liang T.J. Beuving G. Goldfarb V. Metcalfe S.A. Muller-Eberhard U. J. Biol. Chem. 1986; 261: 15719-15724Abstract Full Text PDF PubMed Google Scholar, 3Amrani D.L. Mauzy-Melitz D. Mosesson M.W. Biochem. J. 1986; 238: 365-371Crossref PubMed Scopus (48) Google Scholar, 4Grieninger G. Oddoux C. Diamond L. Weissbach L. Plant P.W. Ann. N. Y. Acad. Sci. 1989; 557: 257-270Crossref PubMed Scopus (10) Google Scholar, 5Hallquist N.A. Klasing K.C. Comp. Biochem. Physiol. Biochem. Mol. Biol. 1994; 108: 375-384Crossref PubMed Scopus (46) Google Scholar, 6Delers F. Domingo M. Engler R. Comp. Biochem. Physiol. 1983; 74: 619-622Crossref Scopus (6) Google Scholar, 7Klasing K.C. Poult. Sci. 1991; 70: 1176-1186Crossref PubMed Scopus (48) Google Scholar). These proteins are present in normal serum, but their levels increase in inflammatory diseases. We have been searching for new marker proteins to improve the diagnosis for the acute phase of chicken. Turpentine enhances the synthesis of acute-phase proteins and has been used as an inducer of acute inflammation (8Sinha B.K. Vegad J.L. Awadhiya R.P. Res. Vet. Sci. 1987; 42: 365-372Crossref PubMed Google Scholar, 9Jain N.K. Vegad J.L. Awadhiya R.P. Vet. Rec. 1982; 110: 421-422Crossref PubMed Scopus (10) Google Scholar).Tohjo et al. (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar) described two proteins, named 18-B and 18-C, that appeared in abundance after administration of turpentine. These proteins migrated in front of albumin in 4.5–16% polyacrylamide disc gel electrophoresis (PAGE).1 Since 18-B was not detected in the serum of healthy chickens, the protein was considered to be a new acute-phase protein. Tohjo et al. (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar) did not further characterize 18-B.On the other hand, Urban et al. (11Urban J. Chan D. Schreiber G. J. Biol. Chem. 1979; 254: 10565-10568Abstract Full Text PDF PubMed Google Scholar) reported that a new acute-phase α-1 protein in rat was induced by turpentine administration and that its molecular mass based on SDS-PAGE was 68 kDa. 18-B in chicken is similar in electrophoretic characteristics and molecular size to Urban's protein in rat, but it is not clear whether both these proteins are identical or not.In this study, we attempted to identify a new acute-phase protein in chicken serum to establish a new marker for acute-phase inflammation. Thus, we purified a turpentine-induced protein from chicken serum and compared its molecular properties with those of the rat protein. In addition, we report the physiological function, cDNA cloning, and characteristics of 18-B.DISCUSSIONIn this study, 18-B was purified by salting-out and DEAE chromatography, finally yielding 0.4 mg of 18-B from 1 ml of the chicken acute-phase serum. This protein corresponds to 18-B reported by Tohjo et al. (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar) and was first isolated in the present study. This protein is a single peptide with a molecular mass of 54 kDa.Several chicken serum proteins, such as C-reactive protein, hemopexin, fibrin, fibrinogen, transferrin, α1-acid glycoprotein, and α2-macroglobulin, are shown to increase with increasing degree of inflammation (1−7). In addition, haptoglobin (9Jain N.K. Vegad J.L. Awadhiya R.P. Vet. Rec. 1982; 110: 421-422Crossref PubMed Scopus (10) Google Scholar), transferrin (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar), and α1-acid glycoprotein (6Delers F. Domingo M. Engler R. Comp. Biochem. Physiol. 1983; 74: 619-622Crossref Scopus (6) Google Scholar) are shown to be elevated in chicken by administration of turpentine. However, none of these proteins had all the molecular properties of 18-B (molecular size, N-terminal sequence, carbohydrate composition, and antigenicity).Urban et al. (11Urban J. Chan D. Schreiber G. J. Biol. Chem. 1979; 254: 10565-10568Abstract Full Text PDF PubMed Google Scholar) reported a novel turpentine-induced glycoprotein with a single peptide chain in rat. This protein has a molecular mass of 56 kDa and a pI value of 4.7. These properties are similar to those of 18-B, but the N-terminal amino acid sequences of these two proteins are not homologous. These results suggest that 18-B is a new turpentine-induced protein of chicken.To further understand the function of 18-B, the complete amino acid sequence was determined by cDNA cloning. The deduced amino acid sequence of 18-B has four repeated sequences. A search of all sequences in the GenBankTM data base was carried out to identify similar sequences, which resulted in homology with members of the SRCR family of proteins, such as hensin, macrophage scavenger receptor, WC1, CRP-ductin, ebnerin, CD163, Spα, CD5, and CD6. There are two types of SRCR family: SRCR group A domains contain six cysteine residues and are encoded by two exons, which include macrophage scavenger receptor and related proteins (26Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (836) Google Scholar), etc. SRCR group B domains contain eight cysteine residues and are encoded by a single exon, which include leukocyte antigens CD5, CD6, CD163 (M130), WC1, and Spα, etc. (27Jones N.H. Clabby M.L. Dialynas D.P. Huang H.J. Herzenberg L.A. Strominger J.L. Nature. 1986; 323: 346-349Crossref PubMed Scopus (185) Google Scholar, 28Huang H.J. Jones N.H. Strominger J.L. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 204-208Crossref PubMed Scopus (140) Google Scholar, 29Law S.K. Micklem K.J. Shaw J.M. Zhang X.P. Dong Y. Willis A.C. Mason D.Y. Eur. J. Immunol. 1993; 23: 2320-2325Crossref PubMed Scopus (211) Google Scholar, 30Wijngaard P.L. Metzelaar M.J. MacHugh N.D. Morrison W.I. Clevers H.C. J. Immunol. 1992; 149: 3273-3277PubMed Google Scholar, 31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Each of the cysteine-rich domains of 18-B shares high degrees of sequence homology with that of SRCR group B family, in particular conserved sequence elements, including eight cysteine residues. These indicate that 18-B belongs to the SRCR group B family. The domain organization of 18-B is similar to, but different from, those of the previously reported proteins. Because 18-B is composed exclusively of SRCR domains, it can be distinguished from multidomain proteins that have both CUB (C1r/C1s Uegf Bmp 1) and ZP (zona pellucida) domains besides SRCR domain, such as hensin, CRP-ductin, and ebnerin (32Takito J. Yan L. Ma J. Hikita C. Vijayakumar S. Warburton D. Al-Awqati Q. Am. J. Physiol. 1999; 277: F277-F289PubMed Google Scholar, 33Cheng H. Bjerknes M. Chen H. Anat. Rec. 1996; 244: 327-343Crossref PubMed Scopus (93) Google Scholar, 34Li X.J. Snyder S.H. J. Biol. Chem. 1995; 270: 17674-17679Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, 18-B has neither transmembrane nor cytoplasmic domain, which indicates that 18-B is different from membrane proteins, such as CD5, CD6, and CD163 (27Jones N.H. Clabby M.L. Dialynas D.P. Huang H.J. Herzenberg L.A. Strominger J.L. Nature. 1986; 323: 346-349Crossref PubMed Scopus (185) Google Scholar, 35Aruffo A. Melnick M.B. Linsley P.S. Seed B. J. Exp. Med. 1991; 174: 949-952Crossref PubMed Scopus (127) Google Scholar, 29Law S.K. Micklem K.J. Shaw J.M. Zhang X.P. Dong Y. Willis A.C. Mason D.Y. Eur. J. Immunol. 1993; 23: 2320-2325Crossref PubMed Scopus (211) Google Scholar). Secreted protein, Spα, is the only protein that has such a simple domain organization, which is composed of SRCR domain only (31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, comparison of 18-B with Spα revealed two major differences: the primary structure of 18-B is longer by 123 residues than that of Spα, and 18-B has four domains, whereas Spα has three SRCR domains. On the basis of these findings, we propose that 18-B is a new member of the SRCR group B family of proteins.Leukocyte function is regulated by a discrete number of cell surface and secreted antigens that govern leukocyte activation, proliferation, survival, cell adhesion and migration, and effector function. Among the proteins that have been shown to regulate leukocyte function are members of the SRCR family (36Krieger M. Curr. Opin. Lipidol. 1997; 8: 275-280Crossref PubMed Scopus (252) Google Scholar, 37Aruffo A. Bowen M.A. Patel D.D. Haynes B.F. Starling G.C. Gebe J.A. Bajorath J. Immunol. Today. 1997; 18: 498-504Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 38Vijayakumar S. Takito J. Hikita C. Al-Awqati Q. J. Cell Biol. 1999; 144: 1057-1067Crossref PubMed Scopus (81) Google Scholar, 39Whitney G.S. Starling G.C. Bowen M.A. Modrell B. Siadak A.W. Aruffo A. J. Biol. Chem. 1995; 270: 18187-18190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The results of the chemiluminescence measurement and ESR spectroscopy proved 18-B to be an antioxidant. The suppressive effect of 18-B on the O⨪2production of heterophils is stronger in receptor-mediated response than of PKC-mediated response. This result was consistent in both chemiluminescence and ESR analysis, which indicate that 18-B inhibits O⨪2 mostly by activation of receptor and not by direct activation of PKC.The function of members of the SRCR group B family is not defined conclusively and fully understood yet. WC1 is involved in γδT cell regulation (30Wijngaard P.L. Metzelaar M.J. MacHugh N.D. Morrison W.I. Clevers H.C. J. Immunol. 1992; 149: 3273-3277PubMed Google Scholar). CD5 and CD6 modulate T cell activation. Spα is thought to regulate monocyte function (31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These proteins obviously exert their functions after binding to a specific ligand: CD5 binds to CD72 (40van de Velde H. von Hoegen I. Luo W. Parnes J.R. Thielemans K. Nature. 1991; 351: 662-665Crossref PubMed Scopus (278) Google Scholar), CD6 to ALCAM (39Whitney G.S. Starling G.C. Bowen M.A. Modrell B. Siadak A.W. Aruffo A. J. Biol. Chem. 1995; 270: 18187-18190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). No ligand has been defined yet for 18-B and the related CD163, WC1, and Spα. However, our results showing that 18-B inhibits O⨪2 mostly by activation of receptor suggest that there may be a 18-B-binding protein on heterophils, and 18-B may regulate heterophil function in a manner of inhibition of the overproduction of the reactive oxygen species.In conclusion, our studies demonstrate that turpentine-induced 18-B is a new member of SRCR family of proteins, which may be implicated in regulation of cell function. Several acute-phase proteins in serum, such as C-reactive protein, hemopexin, fibrin, fibrinogen, transferrin, α1-acid glycoprotein, and α2-macroglobulin, have been reported in chicken (1Patterson L.T. Mora E.C. Tex. Rep. Biol. Med. 1965; 23: 600-606PubMed Google Scholar, 2Grieninger G. Liang T.J. Beuving G. Goldfarb V. Metcalfe S.A. Muller-Eberhard U. J. Biol. Chem. 1986; 261: 15719-15724Abstract Full Text PDF PubMed Google Scholar, 3Amrani D.L. Mauzy-Melitz D. Mosesson M.W. Biochem. J. 1986; 238: 365-371Crossref PubMed Scopus (48) Google Scholar, 4Grieninger G. Oddoux C. Diamond L. Weissbach L. Plant P.W. Ann. N. Y. Acad. Sci. 1989; 557: 257-270Crossref PubMed Scopus (10) Google Scholar, 5Hallquist N.A. Klasing K.C. Comp. Biochem. Physiol. Biochem. Mol. Biol. 1994; 108: 375-384Crossref PubMed Scopus (46) Google Scholar, 6Delers F. Domingo M. Engler R. Comp. Biochem. Physiol. 1983; 74: 619-622Crossref Scopus (6) Google Scholar, 7Klasing K.C. Poult. Sci. 1991; 70: 1176-1186Crossref PubMed Scopus (48) Google Scholar). These proteins are present in normal serum, but their levels increase in inflammatory diseases. We have been searching for new marker proteins to improve the diagnosis for the acute phase of chicken. Turpentine enhances the synthesis of acute-phase proteins and has been used as an inducer of acute inflammation (8Sinha B.K. Vegad J.L. Awadhiya R.P. Res. Vet. Sci. 1987; 42: 365-372Crossref PubMed Google Scholar, 9Jain N.K. Vegad J.L. Awadhiya R.P. Vet. Rec. 1982; 110: 421-422Crossref PubMed Scopus (10) Google Scholar). Tohjo et al. (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar) described two proteins, named 18-B and 18-C, that appeared in abundance after administration of turpentine. These proteins migrated in front of albumin in 4.5–16% polyacrylamide disc gel electrophoresis (PAGE).1 Since 18-B was not detected in the serum of healthy chickens, the protein was considered to be a new acute-phase protein. Tohjo et al. (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar) did not further characterize 18-B. On the other hand, Urban et al. (11Urban J. Chan D. Schreiber G. J. Biol. Chem. 1979; 254: 10565-10568Abstract Full Text PDF PubMed Google Scholar) reported that a new acute-phase α-1 protein in rat was induced by turpentine administration and that its molecular mass based on SDS-PAGE was 68 kDa. 18-B in chicken is similar in electrophoretic characteristics and molecular size to Urban's protein in rat, but it is not clear whether both these proteins are identical or not. In this study, we attempted to identify a new acute-phase protein in chicken serum to establish a new marker for acute-phase inflammation. Thus, we purified a turpentine-induced protein from chicken serum and compared its molecular properties with those of the rat protein. In addition, we report the physiological function, cDNA cloning, and characteristics of 18-B. DISCUSSIONIn this study, 18-B was purified by salting-out and DEAE chromatography, finally yielding 0.4 mg of 18-B from 1 ml of the chicken acute-phase serum. This protein corresponds to 18-B reported by Tohjo et al. (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar) and was first isolated in the present study. This protein is a single peptide with a molecular mass of 54 kDa.Several chicken serum proteins, such as C-reactive protein, hemopexin, fibrin, fibrinogen, transferrin, α1-acid glycoprotein, and α2-macroglobulin, are shown to increase with increasing degree of inflammation (1−7). In addition, haptoglobin (9Jain N.K. Vegad J.L. Awadhiya R.P. Vet. Rec. 1982; 110: 421-422Crossref PubMed Scopus (10) Google Scholar), transferrin (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar), and α1-acid glycoprotein (6Delers F. Domingo M. Engler R. Comp. Biochem. Physiol. 1983; 74: 619-622Crossref Scopus (6) Google Scholar) are shown to be elevated in chicken by administration of turpentine. However, none of these proteins had all the molecular properties of 18-B (molecular size, N-terminal sequence, carbohydrate composition, and antigenicity).Urban et al. (11Urban J. Chan D. Schreiber G. J. Biol. Chem. 1979; 254: 10565-10568Abstract Full Text PDF PubMed Google Scholar) reported a novel turpentine-induced glycoprotein with a single peptide chain in rat. This protein has a molecular mass of 56 kDa and a pI value of 4.7. These properties are similar to those of 18-B, but the N-terminal amino acid sequences of these two proteins are not homologous. These results suggest that 18-B is a new turpentine-induced protein of chicken.To further understand the function of 18-B, the complete amino acid sequence was determined by cDNA cloning. The deduced amino acid sequence of 18-B has four repeated sequences. A search of all sequences in the GenBankTM data base was carried out to identify similar sequences, which resulted in homology with members of the SRCR family of proteins, such as hensin, macrophage scavenger receptor, WC1, CRP-ductin, ebnerin, CD163, Spα, CD5, and CD6. There are two types of SRCR family: SRCR group A domains contain six cysteine residues and are encoded by two exons, which include macrophage scavenger receptor and related proteins (26Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (836) Google Scholar), etc. SRCR group B domains contain eight cysteine residues and are encoded by a single exon, which include leukocyte antigens CD5, CD6, CD163 (M130), WC1, and Spα, etc. (27Jones N.H. Clabby M.L. Dialynas D.P. Huang H.J. Herzenberg L.A. Strominger J.L. Nature. 1986; 323: 346-349Crossref PubMed Scopus (185) Google Scholar, 28Huang H.J. Jones N.H. Strominger J.L. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 204-208Crossref PubMed Scopus (140) Google Scholar, 29Law S.K. Micklem K.J. Shaw J.M. Zhang X.P. Dong Y. Willis A.C. Mason D.Y. Eur. J. Immunol. 1993; 23: 2320-2325Crossref PubMed Scopus (211) Google Scholar, 30Wijngaard P.L. Metzelaar M.J. MacHugh N.D. Morrison W.I. Clevers H.C. J. Immunol. 1992; 149: 3273-3277PubMed Google Scholar, 31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Each of the cysteine-rich domains of 18-B shares high degrees of sequence homology with that of SRCR group B family, in particular conserved sequence elements, including eight cysteine residues. These indicate that 18-B belongs to the SRCR group B family. The domain organization of 18-B is similar to, but different from, those of the previously reported proteins. Because 18-B is composed exclusively of SRCR domains, it can be distinguished from multidomain proteins that have both CUB (C1r/C1s Uegf Bmp 1) and ZP (zona pellucida) domains besides SRCR domain, such as hensin, CRP-ductin, and ebnerin (32Takito J. Yan L. Ma J. Hikita C. Vijayakumar S. Warburton D. Al-Awqati Q. Am. J. Physiol. 1999; 277: F277-F289PubMed Google Scholar, 33Cheng H. Bjerknes M. Chen H. Anat. Rec. 1996; 244: 327-343Crossref PubMed Scopus (93) Google Scholar, 34Li X.J. Snyder S.H. J. Biol. Chem. 1995; 270: 17674-17679Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, 18-B has neither transmembrane nor cytoplasmic domain, which indicates that 18-B is different from membrane proteins, such as CD5, CD6, and CD163 (27Jones N.H. Clabby M.L. Dialynas D.P. Huang H.J. Herzenberg L.A. Strominger J.L. Nature. 1986; 323: 346-349Crossref PubMed Scopus (185) Google Scholar, 35Aruffo A. Melnick M.B. Linsley P.S. Seed B. J. Exp. Med. 1991; 174: 949-952Crossref PubMed Scopus (127) Google Scholar, 29Law S.K. Micklem K.J. Shaw J.M. Zhang X.P. Dong Y. Willis A.C. Mason D.Y. Eur. J. Immunol. 1993; 23: 2320-2325Crossref PubMed Scopus (211) Google Scholar). Secreted protein, Spα, is the only protein that has such a simple domain organization, which is composed of SRCR domain only (31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, comparison of 18-B with Spα revealed two major differences: the primary structure of 18-B is longer by 123 residues than that of Spα, and 18-B has four domains, whereas Spα has three SRCR domains. On the basis of these findings, we propose that 18-B is a new member of the SRCR group B family of proteins.Leukocyte function is regulated by a discrete number of cell surface and secreted antigens that govern leukocyte activation, proliferation, survival, cell adhesion and migration, and effector function. Among the proteins that have been shown to regulate leukocyte function are members of the SRCR family (36Krieger M. Curr. Opin. Lipidol. 1997; 8: 275-280Crossref PubMed Scopus (252) Google Scholar, 37Aruffo A. Bowen M.A. Patel D.D. Haynes B.F. Starling G.C. Gebe J.A. Bajorath J. Immunol. Today. 1997; 18: 498-504Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 38Vijayakumar S. Takito J. Hikita C. Al-Awqati Q. J. Cell Biol. 1999; 144: 1057-1067Crossref PubMed Scopus (81) Google Scholar, 39Whitney G.S. Starling G.C. Bowen M.A. Modrell B. Siadak A.W. Aruffo A. J. Biol. Chem. 1995; 270: 18187-18190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The results of the chemiluminescence measurement and ESR spectroscopy proved 18-B to be an antioxidant. The suppressive effect of 18-B on the O⨪2production of heterophils is stronger in receptor-mediated response than of PKC-mediated response. This result was consistent in both chemiluminescence and ESR analysis, which indicate that 18-B inhibits O⨪2 mostly by activation of receptor and not by direct activation of PKC.The function of members of the SRCR group B family is not defined conclusively and fully understood yet. WC1 is involved in γδT cell regulation (30Wijngaard P.L. Metzelaar M.J. MacHugh N.D. Morrison W.I. Clevers H.C. J. Immunol. 1992; 149: 3273-3277PubMed Google Scholar). CD5 and CD6 modulate T cell activation. Spα is thought to regulate monocyte function (31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These proteins obviously exert their functions after binding to a specific ligand: CD5 binds to CD72 (40van de Velde H. von Hoegen I. Luo W. Parnes J.R. Thielemans K. Nature. 1991; 351: 662-665Crossref PubMed Scopus (278) Google Scholar), CD6 to ALCAM (39Whitney G.S. Starling G.C. Bowen M.A. Modrell B. Siadak A.W. Aruffo A. J. Biol. Chem. 1995; 270: 18187-18190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). No ligand has been defined yet for 18-B and the related CD163, WC1, and Spα. However, our results showing that 18-B inhibits O⨪2 mostly by activation of receptor suggest that there may be a 18-B-binding protein on heterophils, and 18-B may regulate heterophil function in a manner of inhibition of the overproduction of the reactive oxygen species.In conclusion, our studies demonstrate that turpentine-induced 18-B is a new member of SRCR family of proteins, which may be implicated in regulation of cell function. In this study, 18-B was purified by salting-out and DEAE chromatography, finally yielding 0.4 mg of 18-B from 1 ml of the chicken acute-phase serum. This protein corresponds to 18-B reported by Tohjo et al. (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar) and was first isolated in the present study. This protein is a single peptide with a molecular mass of 54 kDa. Several chicken serum proteins, such as C-reactive protein, hemopexin, fibrin, fibrinogen, transferrin, α1-acid glycoprotein, and α2-macroglobulin, are shown to increase with increasing degree of inflammation (1−7). In addition, haptoglobin (9Jain N.K. Vegad J.L. Awadhiya R.P. Vet. Rec. 1982; 110: 421-422Crossref PubMed Scopus (10) Google Scholar), transferrin (10Tohjo H. Miyoshi F. Uchida E. Niiyama M. Syuto B. Poult. Sci. 1995; 74: 648-655Crossref PubMed Scopus (33) Google Scholar), and α1-acid glycoprotein (6Delers F. Domingo M. Engler R. Comp. Biochem. Physiol. 1983; 74: 619-622Crossref Scopus (6) Google Scholar) are shown to be elevated in chicken by administration of turpentine. However, none of these proteins had all the molecular properties of 18-B (molecular size, N-terminal sequence, carbohydrate composition, and antigenicity). Urban et al. (11Urban J. Chan D. Schreiber G. J. Biol. Chem. 1979; 254: 10565-10568Abstract Full Text PDF PubMed Google Scholar) reported a novel turpentine-induced glycoprotein with a single peptide chain in rat. This protein has a molecular mass of 56 kDa and a pI value of 4.7. These properties are similar to those of 18-B, but the N-terminal amino acid sequences of these two proteins are not homologous. These results suggest that 18-B is a new turpentine-induced protein of chicken. To further understand the function of 18-B, the complete amino acid sequence was determined by cDNA cloning. The deduced amino acid sequence of 18-B has four repeated sequences. A search of all sequences in the GenBankTM data base was carried out to identify similar sequences, which resulted in homology with members of the SRCR family of proteins, such as hensin, macrophage scavenger receptor, WC1, CRP-ductin, ebnerin, CD163, Spα, CD5, and CD6. There are two types of SRCR family: SRCR group A domains contain six cysteine residues and are encoded by two exons, which include macrophage scavenger receptor and related proteins (26Kodama T. Freeman M. Rohrer L. Zabrecky J. Matsudaira P. Krieger M. Nature. 1990; 343: 531-535Crossref PubMed Scopus (836) Google Scholar), etc. SRCR group B domains contain eight cysteine residues and are encoded by a single exon, which include leukocyte antigens CD5, CD6, CD163 (M130), WC1, and Spα, etc. (27Jones N.H. Clabby M.L. Dialynas D.P. Huang H.J. Herzenberg L.A. Strominger J.L. Nature. 1986; 323: 346-349Crossref PubMed Scopus (185) Google Scholar, 28Huang H.J. Jones N.H. Strominger J.L. Herzenberg L.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 204-208Crossref PubMed Scopus (140) Google Scholar, 29Law S.K. Micklem K.J. Shaw J.M. Zhang X.P. Dong Y. Willis A.C. Mason D.Y. Eur. J. Immunol. 1993; 23: 2320-2325Crossref PubMed Scopus (211) Google Scholar, 30Wijngaard P.L. Metzelaar M.J. MacHugh N.D. Morrison W.I. Clevers H.C. J. Immunol. 1992; 149: 3273-3277PubMed Google Scholar, 31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Each of the cysteine-rich domains of 18-B shares high degrees of sequence homology with that of SRCR group B family, in particular conserved sequence elements, including eight cysteine residues. These indicate that 18-B belongs to the SRCR group B family. The domain organization of 18-B is similar to, but different from, those of the previously reported proteins. Because 18-B is composed exclusively of SRCR domains, it can be distinguished from multidomain proteins that have both CUB (C1r/C1s Uegf Bmp 1) and ZP (zona pellucida) domains besides SRCR domain, such as hensin, CRP-ductin, and ebnerin (32Takito J. Yan L. Ma J. Hikita C. Vijayakumar S. Warburton D. Al-Awqati Q. Am. J. Physiol. 1999; 277: F277-F289PubMed Google Scholar, 33Cheng H. Bjerknes M. Chen H. Anat. Rec. 1996; 244: 327-343Crossref PubMed Scopus (93) Google Scholar, 34Li X.J. Snyder S.H. J. Biol. Chem. 1995; 270: 17674-17679Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). Furthermore, 18-B has neither transmembrane nor cytoplasmic domain, which indicates that 18-B is different from membrane proteins, such as CD5, CD6, and CD163 (27Jones N.H. Clabby M.L. Dialynas D.P. Huang H.J. Herzenberg L.A. Strominger J.L. Nature. 1986; 323: 346-349Crossref PubMed Scopus (185) Google Scholar, 35Aruffo A. Melnick M.B. Linsley P.S. Seed B. J. Exp. Med. 1991; 174: 949-952Crossref PubMed Scopus (127) Google Scholar, 29Law S.K. Micklem K.J. Shaw J.M. Zhang X.P. Dong Y. Willis A.C. Mason D.Y. Eur. J. Immunol. 1993; 23: 2320-2325Crossref PubMed Scopus (211) Google Scholar). Secreted protein, Spα, is the only protein that has such a simple domain organization, which is composed of SRCR domain only (31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). However, comparison of 18-B with Spα revealed two major differences: the primary structure of 18-B is longer by 123 residues than that of Spα, and 18-B has four domains, whereas Spα has three SRCR domains. On the basis of these findings, we propose that 18-B is a new member of the SRCR group B family of proteins. Leukocyte function is regulated by a discrete number of cell surface and secreted antigens that govern leukocyte activation, proliferation, survival, cell adhesion and migration, and effector function. Among the proteins that have been shown to regulate leukocyte function are members of the SRCR family (36Krieger M. Curr. Opin. Lipidol. 1997; 8: 275-280Crossref PubMed Scopus (252) Google Scholar, 37Aruffo A. Bowen M.A. Patel D.D. Haynes B.F. Starling G.C. Gebe J.A. Bajorath J. Immunol. Today. 1997; 18: 498-504Abstract Full Text PDF PubMed Scopus (129) Google Scholar, 38Vijayakumar S. Takito J. Hikita C. Al-Awqati Q. J. Cell Biol. 1999; 144: 1057-1067Crossref PubMed Scopus (81) Google Scholar, 39Whitney G.S. Starling G.C. Bowen M.A. Modrell B. Siadak A.W. Aruffo A. J. Biol. Chem. 1995; 270: 18187-18190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). The results of the chemiluminescence measurement and ESR spectroscopy proved 18-B to be an antioxidant. The suppressive effect of 18-B on the O⨪2production of heterophils is stronger in receptor-mediated response than of PKC-mediated response. This result was consistent in both chemiluminescence and ESR analysis, which indicate that 18-B inhibits O⨪2 mostly by activation of receptor and not by direct activation of PKC. The function of members of the SRCR group B family is not defined conclusively and fully understood yet. WC1 is involved in γδT cell regulation (30Wijngaard P.L. Metzelaar M.J. MacHugh N.D. Morrison W.I. Clevers H.C. J. Immunol. 1992; 149: 3273-3277PubMed Google Scholar). CD5 and CD6 modulate T cell activation. Spα is thought to regulate monocyte function (31Gebe J.A. Kiener P.A. Ring H.Z. Li X. Francke U. Aruffo A. J. Biol. Chem. 1997; 272: 6151-6158Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). These proteins obviously exert their functions after binding to a specific ligand: CD5 binds to CD72 (40van de Velde H. von Hoegen I. Luo W. Parnes J.R. Thielemans K. Nature. 1991; 351: 662-665Crossref PubMed Scopus (278) Google Scholar), CD6 to ALCAM (39Whitney G.S. Starling G.C. Bowen M.A. Modrell B. Siadak A.W. Aruffo A. J. Biol. Chem. 1995; 270: 18187-18190Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). No ligand has been defined yet for 18-B and the related CD163, WC1, and Spα. However, our results showing that 18-B inhibits O⨪2 mostly by activation of receptor suggest that there may be a 18-B-binding protein on heterophils, and 18-B may regulate heterophil function in a manner of inhibition of the overproduction of the reactive oxygen species. In conclusion, our studies demonstrate that turpentine-induced 18-B is a new member of SRCR family of proteins, which may be implicated in regulation of cell function.