Identification of a Region in the Vitamin D-binding Protein that Mediates Its C5a Chemotactic Cofactor Function (2004)

The vitamin D-binding protein (DBP), also known as group-specific component or Gc-globulin, is a multifunctional plasma protein that can significantly enhance the leukocyte chemotactic activity to C5a and C5a des-Arg. DBP is a member of the albumin gene family and has a triple domain modular structure with extensive disulfide bonding that is characteristic of this protein family. The goal of this study was to identify a region in DBP that mediates the chemotactic cofactor function for C5a. Full-length and truncated versions of DBP (Gc-2 allele) were expressed in Escherichia coli using a glutathione S-transferase fusion protein expression system. The structure of the expressed proteins was confirmed by SDS-PAGE and immunoblotting, whereas protein function was verified by quantitating the binding of [3H]vitamin D. Dibutyryl cAMP-differentiated HL-60 cells were utilized to test purified natural DBP and recombinant expressed DBP (reDBP) for their ability to enhance chemotaxis and intracellular Ca2+ flux to C5a. Natural and full-length reDBP (458 amino acid residues) as well as truncated reDBPs that contained the N-terminal domain I (domains I and II, residues 1–378; domain I, residues 1–191) significantly enhanced both cell movement and intracellular Ca2+ concentrations in response to C5a. Progressive truncation of DBP domain I localized the chemotactic enhancing region between residues 126–175. Overlapping peptides corresponding to this region were synthesized, and results indicate that a 20-amino-acid sequence (residues 130–149, 5′-EAFRKDPKEYANQFMWEYST-3′) in domain I of DBP is essential for its C5a chemotactic cofactor function. The vitamin D-binding protein (DBP), also known as group-specific component or Gc-globulin, is a multifunctional plasma protein that can significantly enhance the leukocyte chemotactic activity to C5a and C5a des-Arg. DBP is a member of the albumin gene family and has a triple domain modular structure with extensive disulfide bonding that is characteristic of this protein family. The goal of this study was to identify a region in DBP that mediates the chemotactic cofactor function for C5a. Full-length and truncated versions of DBP (Gc-2 allele) were expressed in Escherichia coli using a glutathione S-transferase fusion protein expression system. The structure of the expressed proteins was confirmed by SDS-PAGE and immunoblotting, whereas protein function was verified by quantitating the binding of [3H]vitamin D. Dibutyryl cAMP-differentiated HL-60 cells were utilized to test purified natural DBP and recombinant expressed DBP (reDBP) for their ability to enhance chemotaxis and intracellular Ca2+ flux to C5a. Natural and full-length reDBP (458 amino acid residues) as well as truncated reDBPs that contained the N-terminal domain I (domains I and II, residues 1–378; domain I, residues 1–191) significantly enhanced both cell movement and intracellular Ca2+ concentrations in response to C5a. Progressive truncation of DBP domain I localized the chemotactic enhancing region between residues 126–175. Overlapping peptides corresponding to this region were synthesized, and results indicate that a 20-amino-acid sequence (residues 130–149, 5′-EAFRKDPKEYANQFMWEYST-3′) in domain I of DBP is essential for its C5a chemotactic cofactor function. Vitamin D-binding protein (DBP) 1The abbreviations used are: DBP, vitamin D-binding protein; reDBP, recombinant expressed DBP; GST, glutathione S-transferase; HBSS, Hank's buffered saline solution; BSA, bovine serum albumin. is a multifunctional and highly polymorphic plasma protein synthesized primarily in the liver (1White P. Cooke N. Trends Endocrinol. Metab. 2000; 11: 320-327Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 2Gomme P.T. Bertolini J. Trends Biotechnol. 2004; 22: 340-345Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). DBP is expressed as a single polypeptide chain with a molecular mass of ∼56 kDa and circulates in plasma at 6–7 μm (1White P. Cooke N. Trends Endocrinol. Metab. 2000; 11: 320-327Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 2Gomme P.T. Bertolini J. Trends Biotechnol. 2004; 22: 340-345Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Due to its extensive polymorphisms, DBP initially was named the group-specific component of serum, later shortened to Gc-globulin (3Hirschfeld J. Acta Pathol. Microbiol. Scand. 1959; 47: 160Crossref PubMed Scopus (173) Google Scholar). DBP is a member of the albumin (ALB), α-fetoprotein (AFP), and α-albumin/afamin (AFM) gene family and thus has the characteristic multiple disulfide-bonded, triple domain modular structure (1White P. Cooke N. Trends Endocrinol. Metab. 2000; 11: 320-327Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Besides functioning as a circulating vitamin D transport protein, it has been demonstrated that plasma DBP effectively scavenges G-actin released at sites of necrotic cell death and prevents polymerization of actin in the circulation (1White P. Cooke N. Trends Endocrinol. Metab. 2000; 11: 320-327Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 2Gomme P.T. Bertolini J. Trends Biotechnol. 2004; 22: 340-345Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Distinct binding regions within the 458-amino-acid sequence of DBP have been identified: a vitamin D sterol binding segment in the N-terminal domain (amino acids 35–49) and a G-actin binding region in the C-terminal domain (amino acids 373–403) (4Haddad J.G. Hu Y.Z. Kowalski M.A. Laramore C. Ray K. Robzyk P. Cooke N.E. Biochemistry. 1992; 31: 7174-7181Crossref PubMed Scopus (78) Google Scholar, 5Swamy N. Dutta A. Ray R. Biochemistry. 1997; 36: 7432-7436Crossref PubMed Scopus (28) Google Scholar). More recent work on the crystal structure of DBP (bound to either vitamin D3 or actin) has confirmed the vitamin D sterol binding site but has demonstrated that actin interacts with distinct amino acid sequences in all three DBP domains (6Verboven C. Rabijns A. De Maeyer M. Van Baelen H. Bouillon R. De Ranter C. Nat. Struct. Biol. 2002; 9: 131-136Crossref PubMed Scopus (141) Google Scholar, 7Swamy N. Head J.F. Weitz D. Ray R. Arch. Biochem. Biophys. 2002; 402: 14-23Crossref PubMed Scopus (36) Google Scholar, 8Head J.F. Swamy N. Ray R. Biochemistry. 2002; 41: 9015-9020Crossref PubMed Scopus (54) Google Scholar, 9Otterbein L.R. Cosio C. Graceffa P. Dominguez R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8003-8008Crossref PubMed Scopus (131) Google Scholar). Complement C5a is a 74-amino-acid peptide generated by limited proteolytic cleavage of C5 during complement activation (10Kohl J. Mol. Immunol. 2001; 38: 175-187Crossref PubMed Scopus (157) Google Scholar). C5a is a very potent chemotactic factor for all leukocytes as well as several other cell types, and the peptide has several other proinflammatory functions as well (10Kohl J. Mol. Immunol. 2001; 38: 175-187Crossref PubMed Scopus (157) Google Scholar). C5a exerts these activities by binding to its high affinity receptor (C5aR or CD88) on the plasma membrane of target cells (11Gerard N.P. Gerard C. Nature. 1991; 349: 614-617Crossref PubMed Scopus (569) Google Scholar). Several groups have demonstrated that purified DBP can significantly enhance the neutrophil chemotactic activity (i.e. co-chemotatic activity) of C5a and its stable breakdown product C5a des-Arg (12Kew R.R. Webster R.O. J. Clin. Investig. 1988; 82: 364-369Crossref PubMed Scopus (127) Google Scholar, 13Perez H.D. Kelly E. Chenoweth D. Elfman F. J. Clin. Investig. 1988; 82: 360-363Crossref PubMed Scopus (59) Google Scholar, 14Petrini M. Azzara A. Carulli G. Ambrogi F. Galbraith R.M. J. Endocrinol. Investig. 1991; 14: 405-408Crossref PubMed Scopus (12) Google Scholar, 15Metcalf J.P. Thompson A.B. Gossman G.L. Nelson K.J. Koyama S. Rennard S.I. Robbins R.A. Am. Rev. Respir. Dis. 1991; 143: 844-849Crossref PubMed Google Scholar, 16Binder R. Kress A. Kan G. Herrmann K. Kirschfink M. Mol. Immunol. 1999; 36: 885-892Crossref PubMed Scopus (79) Google Scholar, 17Zwahlen R.D. Roth D.R. Inflammation. 1990; 14: 109-123Crossref PubMed Scopus (34) Google Scholar). In addition to neutrophils, DBP can also augment the C5a chemotactic activity for monocytes and fibroblasts (18Piquette C.A. Robinson-Hill R. Webster R.O. J. Leukocyte Biol. 1994; 55: 349-354Crossref PubMed Scopus (69) Google Scholar, 19Senior R.M. Griffin G.L. Perez H.D. Webster R.O. J. Immunol. 1988; 141: 3570-3574PubMed Google Scholar). The chemotactic enhancing properties of DBP appear to be restricted to C5a/C5a des-Arg since this protein cannot enhance the chemotactic activity of formylated peptides, IL-8, leukotriene B4, or platelet-activating factor (12Kew R.R. Webster R.O. J. Clin. Investig. 1988; 82: 364-369Crossref PubMed Scopus (127) Google Scholar, 13Perez H.D. Kelly E. Chenoweth D. Elfman F. J. Clin. Investig. 1988; 82: 360-363Crossref PubMed Scopus (59) Google Scholar, 16Binder R. Kress A. Kan G. Herrmann K. Kirschfink M. Mol. Immunol. 1999; 36: 885-892Crossref PubMed Scopus (79) Google Scholar, 18Piquette C.A. Robinson-Hill R. Webster R.O. J. Leukocyte Biol. 1994; 55: 349-354Crossref PubMed Scopus (69) Google Scholar). The mechanisms by which DBP acts as a chemotactic cofactor for C5a are not known. The goal of this study was to determine whether a cochemotactic region could be located within DBP. The results demonstrate for the first time that DBP enhances both C5a-mediated chemotaxis and Ca2+ influx in differentiated HL-60 cells. In addition, a 20-amino-acid sequence within the N-terminal domain I of DBP (residues 130–149) was found to possess the C5a chemotactic cofactor activity. Reagents—Recombinant C5a was purchased from Sigma. Purified human DBP was obtained from Biodesign International (Kennebunkport, ME). Full-length human DBP cDNA (Gc-2 allele), clone number CS0DM004YF02, was purchased from Invitrogen. DNA restriction and modification enzymes were purchased from New England Biolabs (Beverley, MA). Oligonucleotides were synthesized by Invitrogen. Anti-human DBP was purchased from DiaSorin, (Stillwater, MN). pGEX-4T-2 expression vector was purchased from Amersham Biosciences. Dibutyryl-cAMP (Bt2cAMP), Me2SO, and protease inhibitor mixture were obtained from Sigma. Complement-activated serum using yeast cell walls (zymosan A) was prepared as described previously (20Trujillo G. Kew R.R. J. Immunol. 2004; 173: 4130-4136Crossref PubMed Scopus (23) Google Scholar). DBP-derived peptides, peptide 1 (residues 130–152, EAFRKDPKEYANQFMWEYSTNYG) and peptide 2 (residues 150–172, NYGQAPLSLLVSYTKSYLSMVGS), were synthesized and purified (>95% purity) by the American Peptide Company, Inc. (Sunnyvale, CA). Cells and Cell Culture—Human promyelocytic cell line HL-60 was obtained from ATCC (Rockville, MD) and cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum. Differentiation of HL-60 cells was initiated with 250 μm Bt2cAMP for 48 h. Construction of Full-length and Truncated Recombinant Expressed DBP (reDBP)—To construct GST fusion proteins, DNA sequences corresponding to the indicated regions of full-length human DBP or DBP domains were amplified by PCR, designed to generate products with 5′ BamHI and the 3′ XhoI restriction site. The Escherichia coli-expressed proteins were designated as follows: full-length reDBP (residues 1–458), DBP domains I and II (residues 1–378), DBP domains II and III (residues 192–458), DBP domain I (residues 1–191), DBP domain II (residues 192–378), DBP domain III (residues 379–458) were cloned into the corresponding site using pGEX-4T-2 expression plasmid as GST fusion partners in E. coli (sequences of all primers available from the authors upon request). E. coli strain XL-1 Blue (Stratagene, CA) served as host for DNA manipulation, and the E. coli strain BL-21 served the host for protein expression. Truncated versions of domain I (residues 1–175, 1–150, 1–125, and 1–112) also were expressed in E. coli as described above. Each construct was confirmed by DNA sequencing. Expression and Purification of Full-length and Truncated reDBP— DBP was expressed in E. coli according to the protocol described by Swamy et al. (21Swamy N. Ghosh S. Ray R. Protein Expression Purif. 1997; 10: 115-122Crossref PubMed Scopus (8) Google Scholar). BL21 cells carrying pGEX-4T-2 plasmids expressed fusion protein GST linked to the DBP constructs. E. coli were grown at room temperature (22–24 °C) in Luria broth (LB) containing 100 μg/ml ampicillin and 50 μg/ml chloramphenicol until absorbance at 600 nm (A600) was 0.4–0.6. The expression of fusion proteins was induced by the addition of 0.5–1.0 mm isopropyl-1-thio-β-d-galactopyranoside for 4–8 h. Cells were collected by centrifugation at 5000 × g. The E. coli pellets (from 1 liter) were resuspended in 40 ml of Tris-buffered saline (50 mm Tris-HCl, pH mm containing and 2 ml of a protease inhibitor The cells were by and was by centrifugation at × for The cell containing the expressed fusion protein was with 1 ml of a of in Tris-buffered several with Tris-buffered the was with mm glutathione in 100 mm pH protein was with and to and GST was from reDBP using a affinity of the reDBPs were confirmed by N-terminal sequencing. Vitamin D3 binding of natural DBP full-length and truncated reDBP with were according to a with N. Dutta A. Ray R. Biochemistry. 1997; 36: 7432-7436Crossref PubMed Scopus (28) Google Scholar). In a containing natural DBP, reDBP in with of (50 mm Tris-HCl, pH mm mm were at for by with 100 of and centrifugation at 5000 × at from the were with the mixture and for Each was in were using the as described previously Biochem. J. 1990; PubMed Scopus Google Scholar). HL-60 cells × were resuspended in HBSS, containing 2 μm and at for 40 Cells the were used as a to the cells were and at × in containing of the cell was with 50 nm DBP for at room and the other served as the of cell or was to a and with concentrations of purified C5a at room temperature (22–24 was using a at for was by cells with μm concentrations were using the nm for according to the was as described previously R.R. Webster R.O. J. Leukocyte Biol. PubMed Scopus Google Scholar). In of C5a in the chemotaxis with BSA, mm was in in the of a The were with a and 50 of the cell × was into the The chemotactic was for 1 at in a with the was and on a Cell movement was as the in that the of the cells into the S. J. J. PubMed Scopus Google Scholar). and of several were for using of by multiple the CA). DBP and in HL-60 work in has demonstrated that with DBP movement to concentrations of the cochemotactic (12Kew R.R. Webster R.O. J. Clin. Investig. 1988; 82: 364-369Crossref PubMed Scopus (127) Google Scholar, R.R. Webster R.O. J. Leukocyte Biol. PubMed Scopus Google Scholar, R.R. Webster R.O. J. Immunol. Google Scholar). are differentiated cells and cannot be cell for is the promyelocytic cell line HL-60 that can be induced to into cells using as or Differentiation of HL-60 cells for 48 using 250 μm Bt2cAMP induced expression of the C5a receptor and both chemotaxis to C5a and significantly enhanced movement to C5a in the of purified natural DBP results are with using and indicate that differentiated HL-60 cells as a cell to the cochemotactic function of DBP. is a and well in response to C5a binding to its that cells with DBP for a significantly enhanced intracellular influx in response to C5a. In cells that DBP and C5a not influx not that DBP to to the cell for at enhanced chemotaxis is R.R. Webster R.O. J. Immunol. Google Scholar). the results in 1 that DBP can enhance chemotaxis and in differentiated HL-60 cells and indicate that this cell line as a cell to the cochemotactic function of DBP. of E. coli-expressed DBP by SDS-PAGE and available DBP was expressed in E. coli BL-21 in the plasmid vector pGEX-4T-2 to isopropyl-1-thio-β-d-galactopyranoside the fusion protein was as by SDS-PAGE SDS-PAGE of the protein corresponding to the molecular mass of full-length DBP to The at and kDa to the molecular mass of GST with truncated of DBP. have a of a a product of GST or the product of the protein cleavage of the purified fusion protein in of GST from DBP. The SDS-PAGE and cleavage and of GST a single protein of kDa for full-length DBP, 40 kDa for domains I and II, kDa for domains II and and kDa for domain I or domain II of of reDBP to D3 was to determine whether the expressed proteins could a binding of reDBP with a of and of demonstrate that all reDBP could the in a that the vitamin D sterol binding site of the reDBPs is to the natural DBP. In addition, full-length G-actin in a as by not The ability of reDBPs to enhance C5a-mediated chemotaxis and Ca2+ flux in differentiated HL-60 cells was that reDBPs containing the N-terminal domain I have the to enhance chemotaxis and intracellular Ca2+ flux in response to a C5a Cells with DBP response not In addition, HL-60 which very C5a not to C5a or C5a DBP not of reDBP on C5a-mediated in differentiated HL-60 cells. HL-60 cells were differentiated using 250 μm for 48 h. cells × were resuspended in chemotaxis and to to 100 C5a or C5a 50 nm indicated DBP for at cell movement in of cells with were resuspended in at × with or 50 nm indicated DBP and at room temperature for The in intracellular with 100 C5a was are expressed as the nm in the intracellular Ca2+ are the of that the is significantly results demonstrate that the C5a cochemotactic function of DBP in the N-terminal to identify the cochemotactic sequence within this a of truncated versions of domain I were containing either the N-terminal (amino acids 1–112) or C-terminal (amino acids of domain I were The recombinant protein the N-terminal of domain was expressed and purified but chemotactic enhancing activity for C5a and Several to the C-terminal of domain in E. not The of C-terminal of domain I was Full-length domain was truncated to residues since this protein chemotactic enhancing activity. the of domain I by SDS-PAGE and that domain I and enhancing whereas and possess the of activity as full-length natural DBP for both chemotaxis and Ca2+ flux of truncated reDBP of domain I on C5a-mediated in differentiated HL-60 cells. HL-60 cells were differentiated using 250 μm for 48 h. cells × were resuspended in chemotaxis to to 100 C5a or C5a 50 nm indicated DBP for at cell movement in of cells with were resuspended in at × with or 50 nm indicated DBP and at room temperature for The in intracellular with 100 C5a was are expressed as the nm in the intracellular Ca2+ are the of that the is significantly The results from indicate that a region within domain I of DBP, from amino acids to the C5a cochemotactic function. peptides within this region of DBP were synthesized to determine a cochemotactic Peptide 1 (residues 130–152, EAFRKDPKEYANQFMWEYSTNYG) and peptide 2 (residues 150–172, NYGQAPLSLLVSYTKSYLSMVGS), by or could not enhance C5a-mediated chemotaxis not of using these peptides to the cochemotactic function of full-length natural DBP by cells with peptide was The results from that peptide but not peptide could the cochemotactic function of purified full-length natural DBP In addition, peptide also could the HL-60 cell chemotactic response to serum, that this peptide can function to a potent chemotactic in a protein mixture study several is the first of differentiated HL-60 cells C5a-mediated chemotaxis to DBP, the first of a of Ca2+ influx in cell and the first to identify a sequence in DBP that mediates the C5a chemotactic cofactor function. HL-60 cells are the cell line to enhanced C5a chemotaxis to DBP. have demonstrated that cells with the C5a receptor can also be utilized as a cell to the cochemotactic function of DBP (20Trujillo G. Kew R.R. J. Immunol. 2004; 173: 4130-4136Crossref PubMed Scopus (23) Google Scholar). cell be to the mechanisms by which DBP chemotaxis to C5a. In addition, the of a in flux to C5a is it of intracellular by DBP to the cell is to that HL-60 cells to be with purified DBP for at to C5a addition to the of a DBP on the plasma membrane as previously (20Trujillo G. Kew R.R. J. Immunol. 2004; 173: 4130-4136Crossref PubMed Scopus (23) Google Scholar). the in this is the of a 20-amino-acid sequence in the N-terminal domain of DBP that C5a to full-length natural DBP. DBP disulfide a for the protein in a Swamy et al. (21Swamy N. Ghosh S. Ray R. Protein Expression Purif. 1997; 10: 115-122Crossref PubMed Scopus (8) Google previously expression of recombinant DBP in E. coli using GST as a fusion their generated a of truncated proteins that localized the C5a cochemotactic function to a region (residues in the C-terminal of domain peptides this region were synthesized and demonstrated that the cochemotactic function was to the 20-amino-acid sequence (residues sequence is the three of human DBP and with the that is in the C5a cochemotactic function between these DBP R. Kress A. Kan G. Herrmann K. Kirschfink M. Mol. Immunol. 1999; 36: 885-892Crossref PubMed Scopus (79) Google Scholar). of this sequence other DBP, in that this region is to DBP and that it not function by a sequence in a In addition, of this human peptide with the corresponding sequences in N.E. J. Biol. Full Text PDF PubMed Google F. 1990; PubMed Scopus (34) Google and DBP M. N. S. Biochem. Mol. Biol. 1994; Google the other sequences a very high of amino acid of the residues are and the are or it is to that this region in domain I of DBP functions as the cochemotactic sequence in all Several recent have described the crystal structure of DBP, either or to G-actin or vitamin D (6Verboven C. Rabijns A. De Maeyer M. Van Baelen H. Bouillon R. De Ranter C. Nat. Struct. Biol. 2002; 9: 131-136Crossref PubMed Scopus (141) Google Scholar, 7Swamy N. Head J.F. Weitz D. Ray R. Arch. Biochem. Biophys. 2002; 402: 14-23Crossref PubMed Scopus (36) Google Scholar, 8Head J.F. Swamy N. Ray R. Biochemistry. 2002; 41: 9015-9020Crossref PubMed Scopus (54) Google Scholar, 9Otterbein L.R. Cosio C. Graceffa P. Dominguez R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8003-8008Crossref PubMed Scopus (131) Google Scholar). of the structure has that the protein is of a of albumin Nature. 1992; PubMed Scopus Google but in to DBP into a structure that as the G-actin binding site J. PubMed Scopus (23) Google Scholar). The cochemotactic sequence in DBP (residues 130–149) is located in number (residues but in number (residues in domain I (6Verboven C. Rabijns A. De Maeyer M. Van Baelen H. Bouillon R. De Ranter C. Nat. Struct. Biol. 2002; 9: 131-136Crossref PubMed Scopus (141) Google Scholar, 7Swamy N. Head J.F. Weitz D. Ray R. Arch. Biochem. Biophys. 2002; 402: 14-23Crossref PubMed Scopus (36) Google Scholar, 8Head J.F. Swamy N. Ray R. Biochemistry. 2002; 41: 9015-9020Crossref PubMed Scopus (54) Google Scholar, 9Otterbein L.R. Cosio C. Graceffa P. Dominguez R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8003-8008Crossref PubMed Scopus (131) Google Scholar). of DBP crystal structure using the that this region is not DBP G-actin or vitamin D and is to with cells. well with recent that demonstrated that of DBP with vitamin or both not the C5a cochemotactic activity of DBP. S. J. G. and R. R. this cochemotactic peptide is a distinct sequence and the region are vitamin and the structure of to be in the DBP. The binding of DBP to cells is for the protein to its a chemotactic cofactor for a or of by the and of vitamin D and acids to cells (1White P. Cooke N. Trends Endocrinol. Metab. 2000; 11: 320-327Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 2Gomme P.T. Bertolini J. Trends Biotechnol. 2004; 22: 340-345Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). The cell DBP binding site is but this essential in DBP is DBP to with affinity to multiple cell Kew R.R. J. Immunol. 1999; Google receptor family and A. D. D. H. C. J. F. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. C. M. A. Ray R. J. Proc. Natl. Acad. Sci. U. S. A. 2001; PubMed Scopus Google and S. H. N. J. Natl. 2002; PubMed Scopus Google Scholar). have that the known for is for the C5a cochemotactic activity of DBP and be in the or of the DBP binding site (20Trujillo G. Kew R.R. J. Immunol. 2004; 173: 4130-4136Crossref PubMed Scopus (23) Google Scholar). The results in this not the of whether is a distinct cell binding and cochemotactic region in DBP or whether the sequence (residues 130–149) both DBP has been to to and this protein has several binding sequences in domains II and In sequence in domain III is a very region that be a binding site. the cell binding site on DBP to be The results also not the that are other sequences in DBP that be in its cochemotactic function. The cochemotactic sequence (residues 130–149) can effectively the of full-length natural DBP to enhance chemotaxis to but the sequence cannot augment that other regions of DBP be in this function. the results that amino acids essential in the C5a chemotactic cofactor function of DBP could that from this sequence have to C5a-mediated