FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W1977781825> ?p ?o ?g. }

• W1977781825 endingPage "30634" @default.
• W1977781825 startingPage "30627" @default.
• W1977781825 abstract "C1q, the recognition subunit of the classical complement pathway, interacts with specific cell surface molecules via its collagen-like region (C1q-CLR). This binding of C1q to neutrophils triggers the generation of toxic oxygen species. To identify the site on C1q that interacts with the neutrophil C1q receptor, C1q was isolated, digested with pepsin to produce C1q-CLR, and further cleaved with either trypsin or endoproteinase Lys-C. The resulting fragments were separated by gel filtration chromatography and analyzed functionally (activation of the respiratory burst in neutrophils) and structurally. Cleavage of C1q-CLR with endoproteinase Lys-C did not alter its ability to trigger neutrophil superoxide production. However, when C1q-CLR was incubated with trypsin under conditions permitting optimal cleavage, the ability of C1q-CLR to stimulate superoxide production in neutrophils was completely abrogated. Fractionation of the digests obtained with the two enzymes and identification by amino acid sequencing permitted localization of the receptor interaction site to a specific region of the C1q-CLR. Circular dichroism analyses demonstrated that cleavage by trypsin does not denature the remaining uncleaved collagen-like structure, suggesting that after trypsin treatment, the loss of activity was not due to a loss of secondary structure of the molecule. However, irreversible heat denaturation of C1q-CLR also abrogated all activity. Thus, a specific conformation conferred by the collagen triple helix constitutes the functional receptor interaction site. These data should direct the design of future specific therapeutic reagents to selectively modulate this response. C1q, the recognition subunit of the classical complement pathway, interacts with specific cell surface molecules via its collagen-like region (C1q-CLR). This binding of C1q to neutrophils triggers the generation of toxic oxygen species. To identify the site on C1q that interacts with the neutrophil C1q receptor, C1q was isolated, digested with pepsin to produce C1q-CLR, and further cleaved with either trypsin or endoproteinase Lys-C. The resulting fragments were separated by gel filtration chromatography and analyzed functionally (activation of the respiratory burst in neutrophils) and structurally. Cleavage of C1q-CLR with endoproteinase Lys-C did not alter its ability to trigger neutrophil superoxide production. However, when C1q-CLR was incubated with trypsin under conditions permitting optimal cleavage, the ability of C1q-CLR to stimulate superoxide production in neutrophils was completely abrogated. Fractionation of the digests obtained with the two enzymes and identification by amino acid sequencing permitted localization of the receptor interaction site to a specific region of the C1q-CLR. Circular dichroism analyses demonstrated that cleavage by trypsin does not denature the remaining uncleaved collagen-like structure, suggesting that after trypsin treatment, the loss of activity was not due to a loss of secondary structure of the molecule. However, irreversible heat denaturation of C1q-CLR also abrogated all activity. Thus, a specific conformation conferred by the collagen triple helix constitutes the functional receptor interaction site. These data should direct the design of future specific therapeutic reagents to selectively modulate this response. C1q, the recognition subunit of the classical complement pathway, is a 460,000-Da serum protein that has an unusual macromolecular structure and contributes to a variety of functions in the response of the host to infection or injury. As part of the C1 complex, C1q binds to immune complexes or antibody-independent C1 activators resulting in the initiation of the classical complement cascade(1.Cooper N.R. Adv. Immunol. 1985; 37: 151-216Crossref PubMed Scopus (390) Google Scholar). However, upon dissociation from the C1r2C1s2 tetramer(2.Ziccardi R.J. Cooper N.R. J. Immunol. 1979; 123: 788-792PubMed Google Scholar), C1q can interact with cell surface molecules via its collagen-like domain, inducing cell-specific responses. One example of such a C1q receptor-mediated response is the enhancement of phagocytic capacity (3.Bobak D.A. Frank M.M. Tenner A.J. Eur. J. Immunol. 1988; 18: 2001-2007Crossref PubMed Scopus (49) Google Scholar, 4.Bobak D.A. Gaither T.A. Frank M.M. Tenner A.J. J. Immunol. 1987; 138: 1150-1156PubMed Google Scholar, 5.Sorvillo J.M. Gigli I. Pearlstein E. J. Immunol. 1986; 136: 1023-1026PubMed Google Scholar) that occurs when monocytes and culture-derived macrophages interact with C1q. Furthermore, interaction of C1q with neutrophils (6.Tenner A.J. Cooper N.R. J. Immunol. 1982; 128: 2547-2552PubMed Google Scholar), eosinophils(7.Hamada A. Greene B.M. J. Immunol. 1987; 138: 1240-1245PubMed Google Scholar), and vascular smooth muscle cells (8.Shingu M. Yoshioka K. Nobunaga M. Motomatu T. Inflammation. 1989; 13: 561-569Crossref PubMed Scopus (17) Google Scholar) triggers the generation of bactericidal oxygen species. C1q shares an unusual macromolecular structure with certain other molecules also known to enhance uptake of specific pathogenic material (9.Tenner A.J. Robinson S.L. Borchelt J. Wright J.R. J. Biol. Chem. 1989; 264: 13923-13928Abstract Full Text PDF PubMed Google Scholar, 10.Drickamer K. Dordal M.S. Reynolds L. J. Biol. Chem. 1986; 261: 6878-6887Abstract Full Text PDF PubMed Google Scholar, 11.Ezekowitz R.A.B. Day L.E. Herman G.A. J. Exp. Med. 1988; 167: 1034-1046Crossref PubMed Scopus (242) Google Scholar, 12.Voss T. Eistetter H. Schafer K.P. J. Mol. Biol. 1988; 201: 219-227Crossref PubMed Scopus (204) Google Scholar, 13.White R.T. Dammn D. Miller J. Spratt K. Schilling J. Hawgood S. Benson B. Cordell B. Nature. 1985; 317: 361-363Crossref PubMed Scopus (296) Google Scholar). Like C1q, both pulmonary surfactant protein A (SP-A) 1The abbreviations used are: SP-Apulmonary surfactant protein AC1q-CLRpepsin-resistant, collagen-like region of C1q; C1qRO and mannose-binding protein (MBP) have collagen-like sequences contiguous with noncollagen-like sequences, short NH2-terminal domains containing interchain disulfide bonds, and a single disruption in the Gly-X-Y repeat pattern within the collagen-like sequence (13.White R.T. Dammn D. Miller J. Spratt K. Schilling J. Hawgood S. Benson B. Cordell B. Nature. 1985; 317: 361-363Crossref PubMed Scopus (296) Google Scholar, 14.Bhattacharya S.N. Lynn W.S. Biochim. Biophys. Acta. 1980; 625: 343-355Crossref PubMed Scopus (14) Google Scholar, 15.Reid K.B.M. Biochem. Soc. Trans. 1983; 11: 1-12Crossref PubMed Scopus (127) Google Scholar) causing a characteristic “kink” or bend in the tertiary structure. Like C1q, SP-A (9.Tenner A.J. Robinson S.L. Borchelt J. Wright J.R. J. Biol. Chem. 1989; 264: 13923-13928Abstract Full Text PDF PubMed Google Scholar) and MBP (16.Tenner A.J. Robinson S.L. Ezekowitz R.A.B. Immunity. 1995; 3: 485-494Abstract Full Text PDF PubMed Scopus (151) Google Scholar) can enhance Fc receptor- and complement receptor-mediated phagocytosis. However, neither SP-A nor MBP by themselves stimulate superoxide (O) production by polymorphonuclear leukocytes (17.Goodman E.B. Tenner A.J. J. Immunol. 1992; 148: 3920-3928PubMed Google Scholar) in contrast to the readily detectable stimulation by C1q(6.Tenner A.J. Cooper N.R. J. Immunol. 1982; 128: 2547-2552PubMed Google Scholar, 17.Goodman E.B. Tenner A.J. J. Immunol. 1992; 148: 3920-3928PubMed Google Scholar). This suggests that the ligand interaction sites of the C1q receptor that triggers superoxide production (C1qRO) may differ from those sites of the receptor that enhance phagocytic capacity. Also consistent with the hypothesis that these receptors differ in some way is the observation that two monoclonal antibodies have been shown to inhibit the enhancement of phagocytosis mediated by C1q but do not block the production of superoxide by neutrophils (18.Guan E. Burgess W.H. Robinson S.L. Goodman E.B. McTigue K.J. Tenner A.J. J. Biol. Chem. 1991; 266: 20345-20355Abstract Full Text PDF PubMed Google Scholar,19.Guan E. Robinson S.L. Goodman E.B. Tenner A.J. J. Immunol. 1994; 152: 4005-4016PubMed Google Scholar). pulmonary surfactant protein A pepsin-resistant, collagen-like region of C1q; C1qRO Very little is currently known about the ligand requirements for functional interaction with the C1q receptor on any cell types. It is known that the 176,000-Da collagen-like fragment of Clq mediates the functional interaction with most cells(4.Bobak D.A. Gaither T.A. Frank M.M. Tenner A.J. J. Immunol. 1987; 138: 1150-1156PubMed Google Scholar, 20.Tenner A.J. Cooper N.R. J. Immunol. 1980; 125: 1658-1664PubMed Google Scholar), that C1r2C1s2 blocks that interaction(6.Tenner A.J. Cooper N.R. J. Immunol. 1982; 128: 2547-2552PubMed Google Scholar), and that heat-aggregated C1q loses the ability to trigger B cell immunoglobulin secretion(21.Daha M.R. Klar N. Hoekzema R. van Es L.A. J. Immunol. 1990; 144: 1227-1232PubMed Google Scholar). In addition, it appears that a multivalent interaction is required for C1q to trigger a response upon binding to the cells(6.Tenner A.J. Cooper N.R. J. Immunol. 1982; 128: 2547-2552PubMed Google Scholar, 21.Daha M.R. Klar N. Hoekzema R. van Es L.A. J. Immunol. 1990; 144: 1227-1232PubMed Google Scholar). It is not known whether this is due to a requirement for receptor clustering or a surface- or aggregate-induced conformational alteration. This requirement is similar to that seen with CR1, CR2, FcγRIII, and many other receptors(22.Wener M.H. Uwatoko S. Mannik M. Arthritis Rheum. 1989; 32: 544-551Crossref PubMed Scopus (90) Google Scholar, 23.Brunswick M. June C.H. Finkelman F.D. Mond J.J. J. Immunol. 1989; 143: 1414-1421PubMed Google Scholar). If the receptor for C1q that triggers superoxide production differs from the one that enhances phagocytosis, it is probable that the interaction sites on the ligand, C1q, that mediate these responses are also distinct. Therefore, identification of the receptor interaction sites should permit the selective manipulation of the responses mediated by the different receptors; that is, to enhance phagocytosis without generating extracellular superoxide or to inhibit the production of superoxide without effecting monocyte phagocytic capacity. The present investigation characterizes a specific region on the collagen-like domain of C1q (C1q-CLR) that interacts with the neutrophil C1q receptor (C1qRO) to induce superoxide production. Enzymatic digestion, followed by functional analysis of the purified fragments and amino acid sequencing to unambiguously identify the cleavage sites generated, allowed the localization of a specific region of the C1q-CLR that is critical for the C1q-mediated neutrophil response. Furthermore, heat denaturation, as assessed by circular dichroism analyses, demonstrated that secondary structure of the collagen helix is required for the C1q-induced neutrophil superoxide response, indicating that a single linear amino acid sequence will not suffice for this interaction. Nevertheless, the data suggest that it may be possible to design synthetic antagonists to modulate this interaction and thus limit the production of toxic oxygen radicals. Pyrogen-free water (MilliQ-Plus) is used for all laboratory buffers and reagent preparation. TPCK-trypsin was purchased from Worthington (Freehold, NJ), and sequence grade endoproteinase Lys-C (endo Lys-C) was obtained from Boehringer Mannheim. Lymphocyte separation medium was purchased from Organon Teknika Corp. (Durham, N.C.). All other reagents used, except where noted otherwise, were obtained in the highest quality available from Sigma. C1q was isolated from plasma-derived human serum by the method of Tenner et al.(24.Tenner A.J. Lesavre P.H. Cooper N.R. J. Immunol. 1981; 127: 648-653PubMed Google Scholar) modified as described(25.Young K.R. Ambrus Jr., J.L. Malbran A. Fauci A.S. Tenner A.J. J. Immunol. 1991; 146: 3356-3364PubMed Google Scholar). The preparations used were fully active as determined by hemolytic titration and homogeneous as assessed by SDS-polyacrylamide gel electrophoresis (PAGE). C1q collagen-like fragments (C1q-CLR) were obtained by the digestion of C1q with pepsin using the procedure of Reid (26.Reid K.B.M. Biochem. J. 1976; 155: 5-17Crossref PubMed Scopus (120) Google Scholar) as modified by Siegel and Schumaker (27.Siegel R.C. Schumaker V.N. Mol. Immunol. 1983; 20: 53-66Crossref PubMed Scopus (65) Google Scholar). Protein concentration was determined using an extinction coefficient (E1%) at 280 nm of 6.8 for C1q (28.Reid K.B.M. Lowe D.M. Porter R.R. Biochem. J. 1972; 130: 749-763Crossref PubMed Scopus (182) Google Scholar) and 2.1 for C1q-CLR(27.Siegel R.C. Schumaker V.N. Mol. Immunol. 1983; 20: 53-66Crossref PubMed Scopus (65) Google Scholar). Alternatively, after enzymatic digestion, the protein concentration of the purified fragments was determined using the bicinchoninic acid protein assay (Pierce) with a C1q-CLR preparation of known concentration (determined using the above extinction coefficient) used as a standard. All proteins were stored at −70°C. Preliminary experiments demonstrated that optimal conditions for trypsin digestion of C1q-CLR were achieved when TPCK-trypsin was added to C1q-CLR (2.5-4.5 mg/ml in TBS, pH 7.5) to produce an enzyme:substrate ratio (E:S) of 1:10 (w:w) and incubated at 50°C for 30 min. Endo Lys-C was incubated with C1q-CLR at similar concentrations in TBS, pH 7.5 (E:S, 1:200, w:w) at 37°C for 2 h. Digestions were terminated by placing the samples on ice. Aliquots of the samples were then electrophoresed under nonreducing conditions using the Tricine SDS-PAGE system (10% acrylamide) described by Schägger and von Jagow(29.Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10460) Google Scholar). Fragments produced by enzymatic digestion were characterized and separated by gel filtration chromatography using a Superose 12 HR 10/30 (Pharmacia Biotech Inc.) column equilibrated in TBS, pH 7.4 (in the presence or the absence of 5 mM CaCl2). The void volume of the column was 8.1 ml, and the total column volume was 25 ml. Circular dichroism was recorded at ambient temperature using a Jasco J720 spectropolarimeter. Data were collected at 0.5-nm intervals, and 4 scans were averaged but not smoothed. A cell of 0.5-mm path length was used. Some circular dichroism spectra were obtained using a Jasco 500C circular dichrometer with a temperature-controlled cuvette. Protein concentrations used were 0.4-1 mg/ml. All data were converted to mean residue ellipticity. The Edman degradation method was carried out in a Hewlett-Packard Protein Sequencing System model G1005A with an on-line analyzer of the amino acid derivatives. The direct loading of samples, e.g. total digests, onto the sequencing cartridge without previous desalting ensured that all peptide components could be recovered and identified. 4-Hydroxyprolines but no hydroxylysines were identified during sequence analysis. Hydroxylysines predicted from the cDNA sequences (30.Sellar G.C. Blake D.J. Reid K.B.M. Biochem. J. 1991; 274: 481-490Crossref PubMed Scopus (174) Google Scholar) and previous amino acid sequencing (31.Reid K.B.M. Biochem. J. 1979; 179: 367-371Crossref PubMed Scopus (83) Google Scholar) were verified only as the absence of lysine. C1q-CLR was treated with pyroglutamate aminopeptidase (Boehringer Mannheim) to remove the modified glutamate from the NH2 terminus of the B chain (32.Lottspeich F. Henschen A. Hoppe-Seyler's Z. Physiol. Chem. 1978; 359: 1611-1616PubMed Google Scholar) to permit sequencing of that chain. Polymorphonuclear leukocytes are isolated from blood drawn from normal volunteers into EDTA-containing syringes or from buffy coats prepared from blood collected with CPDA1 as anticoagulant. Following centrifugation on lymphocyte separation media cushions and Dextran T500 (Pharmacia) sedimentation according to the method of Boyum(33.Boyum A. Scand. J. Immunol. 1976; 5 (5): 9-15Crossref PubMed Scopus (1330) Google Scholar), modified as described(4.Bobak D.A. Gaither T.A. Frank M.M. Tenner A.J. J. Immunol. 1987; 138: 1150-1156PubMed Google Scholar), the residual red blood cells were removed by hypotonic lysis. Cell preparations contain 93-97% neutrophils, 2-5% eosinophils, and 0-2% mononuclear cells. O was measured by the superoxide dismutase-inhibitable reduction of cytochrome c adapted to a microplate format(34.Mayo L.A. Curnutte J.T. Methods Enzymol. 1990; 186: 567-575Crossref PubMed Scopus (134) Google Scholar). 96-well Immulon 2 plates (Dynatech Laboratories, Chantilly, VA) were coated with 10-100 μg/ml C1q-CLR or test protein/fragments diluted in phosphate-buffered saline for 2-3 h at room temperature. After washing the plate with phosphate-buffered saline, the reaction was started by the addition of 100 μl of neutrophil suspension (3.5 × 106/ml) to the microtiter wells containing 100 μl of cytochrome c reaction mixture (200 μM cytochrome c in Hanks' balanced salt solution containing 1 mM Ca2+ and 1 mM Mg2+). A550 was recorded every 30 s at 37°C using a ThermoMax kinetic microplate reader (Molecular Devices, Inc., Menlo Park, CA) equipped with a 1.0-nm band pass filter. The initial absorbance value was subtracted from each subsequent reading, and this value converted to nmol of O using an extinction coefficient of 0.022 μM−1 cm−1. Phorbol dibutyrate (200 ng/ml) was used as a positive control stimulus. Control samples containing superoxide dismutase (final concentration, 40 μg/ml) were always run in parallel with each sample and showed no change in A550 under any condition tested (not shown). It has been previously well established that the collagen-like portion of the C1q molecule binds to neutrophils and triggers superoxide generation(6.Tenner A.J. Cooper N.R. J. Immunol. 1982; 128: 2547-2552PubMed Google Scholar, 17.Goodman E.B. Tenner A.J. J. Immunol. 1992; 148: 3920-3928PubMed Google Scholar). Therefore, in all the present studies, C1q was treated with pepsin, and the pepsin-resistant C1q-CLR isolated by gel filtration was used as the functional ligand. To investigate parameters that could determine structural requirements for the functional interaction of C1q with its receptor, conditions that would lead to the loss of secondary structure as assessed by CD were explored. C1q-CLR in either 10 mM acetic acid, TBS, or TBS with 5 mM Ca2+, pH 7.2, was heated to various temperatures, and the CD spectra were recorded. Fig. 1A shows that when heated in the acidic buffer used traditionally for collagen-like molecules, secondary structure, as assessed by the magnitude of the positive band at 224 nm characteristic of collagen triple helix, was rapidly lost in the 46-52°C range, similar to that previously reported by Brodsky-Doyle et al.(35.Brodsky-Doyle B. Leonard K.R. Reid K.B.M. Biochem. J. 1976; 159: 279-286Crossref PubMed Scopus (120) Google Scholar). In contrast, when C1q-CLR was heated at pH 7.2 (in either the presence or the absence of Ca2+), the loss of structure of the C1q-CLR was more gradual between 46 and 70°C. To determine to what extent, if any, this denaturation was reversible and if this denaturation altered the ability of C1q-CLR to trigger neutrophil superoxide production, C1q-CLR was incubated at pH 7.2 for 30 min at 56, 79, and 100°C, temperatures known to induce increasingly greater loss of specific secondary structure, and then cooled to ambient temperature. The CD of the cooled material was then compared with that of an unheated control sample to determine if renaturation had occurred. In contrast to that seen with the acidic conditions used by Brodsky-Doyle and colleagues (35.Brodsky-Doyle B. Leonard K.R. Reid K.B.M. Biochem. J. 1976; 159: 279-286Crossref PubMed Scopus (120) Google Scholar) (and repeated by us), the CD spectra of C1q-CLR heated to 56°C at pH 7.2 and subsequently recooled, was essentially identical to that of the untreated control, with the exception of some variability in the negative peak at 197 nm (Fig. 1B). This renaturation after heating was complete after an incubation of 45-60 min on ice. Heating the protein to 79°C, however, resulted in a greater and largely irreversible loss of C1q-CLR secondary structure. Samples heated at 79°C and above did not renature even after days at 4°C (data not shown). The effect of loss of secondary structure on the ability of C1q-CLR to trigger the generation of superoxide by neutrophils was then examined. Whereas irreversible loss of secondary structure by heating to 79 or 100°C resulted in complete loss of the ability of C1q-CLR to trigger neutrophil O production, C1q-CLR heated to 56°C, followed by the recovery of secondary structure upon cooling, retained total functional activity as compared with the untreated C1q-CLR in its capacity to trigger O (Fig. 2). In this experiment and all superoxide assays performed, neutrophils in uncoated control wells produced no superoxide, with a recording essentially identical to the 79°C/100°C samples (data not shown). To investigate which region of the C1q-CLR molecule was required for C1q receptor-mediated triggering of the superoxide response in neutrophils, C1q-CLR was subjected to enzymatic cleavage. The fact that the C1q-CLR molecule could be reversibly denatured by heating at 50°C was used to promote enzymatic cleavage by trypsin, which normally is not an efficient protease for collagen-like structures. C1q-CLR was heated to 50°C, trypsin was added, and the incubation was continued at 50°C for 30 min. SDS-PAGE suggested that all chains of C1q-CLR were cleaved under these conditions (Fig. 3, lanes 3 and 7) compared with no enzyme controls (Fig. 3, lanes 2 and 6). To separate and characterize the resultant fragments, the digestion mixture was applied to a Superose 12 column, and the elution of the peak fractions was recorded (Fig. 4). When compared with the elution of undigested C1q-CLR sample (Fig. 4A, arrow; elution volume, 8.6 ml), the main fragment generated by trypsin cleavage was distinctly retained and thus significantly smaller in size (Fig. 4B, arrow; elution volume, 11.6 ml). This elution behavior is consistent with the evidence of cleavage seen by SDS-PAGE analysis but indicates that some of the multichain structure of C1q is maintained (albumin elutes at 12.6 ml).Fig. 4Gel filtration demonstrates that C1q-CLR is digested with trypsin. Typical Superose 12 column profile of C1q-CLR (1 mg) incubated at 50°C for 30 min without enzyme (A) or with trypsin (B) at E:S of 1:10. C is the profile of trypsin alone at the same concentration used in the digestion. The main fragment (designated by the open arrows) was used in the structural and functional assays. The areas labeled a, b, and c in B indicate the fractions pooled for amino acid sequencing and referred to in Table 1. (The second peak in A is a nonprotein contaminant variably detected in fast protein liquid chromatography profiles.) The flow rate of the column was 0.5 ml/min.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The major peak of both the undigested C1q-CLR and the trypsin-generated NH2-terminal fragment of C1q-CLR (noted with the arrows in Fig. 4) were then assayed for the ability to trigger polymorphonuclear leukocyte superoxide production. The fragment resulting from trypsin digestion had very little activity as compared with the untreated or mock digested control (Fig. 5). This loss of activity did not reflect an inability of the trypsin-generated fragment to bind to the microtiter well surface as nearly equal molar amounts of the trypsin-digested, and untreated C1q-CLR were detected both by protein assay and by a more sensitive enzyme-linked immunosorbent assay using an anti-C1q monoclonal antibody. The data were consistent with loss of a critical sequence(s) required for the interaction with C1qRO. Amino acid sequencing demonstrated that the earliest eluting peak from the trypsin-treated sample (Fig. 4B, pool a) contained the intact NH2 termini of the A and C chains. The lack of the B chain NH2 terminus is consistent with the previous identification of pyroglutamate at the NH2 terminus of this chain(36.Reid K.B.M. Thompson E.O.P. Biochem. J. 1978; 173: 863-868Crossref PubMed Scopus (19) Google Scholar). Treatment with pyroglutamate-aminopeptidase prior to NH2-terminal sequence analysis allowed the quantitative identification of the predicted B chain residues Leu-Ser-(Cys)-Thr and thus verified that the B chain NH2 terminus was present but blocked by this modified glutamine. Thus, the loss of activity was not due to digestion of the amino terminus of any of the three chains of the C1q molecule. Furthermore, no evidence of any cleavage products associated with the main (NH2-terminal) fragment after purification was detected, nor were trypsin autodigestion fragments contaminating this main protein peak. No cleavages by trypsin occurred in the first 31 amino acids of the A or C chain as sequencing through the first NH2-terminal 31 residues on the A and C chains demonstrated a quantitative yield of each amino acid following the arginine and lysine residues (Fig. 6). Amino acid sequencing of the other fractions separated by the Superose column containing peptides of smaller size (Fig. 4B, pools b and c) allowed identification of multiple trypsin cleavage sites in the C1q-CLR and provided evidence that the most NH2-terminal cleavage occurred on the A chain after Arg38, on the B chain after Lys61, and on the C chain after Arg41 (Table 1 and Fig. 6). When tested for the ability to trigger superoxide production using the standard assay conditions, none of the fractions containing the smaller peptide fragments demonstrated any superoxide generating activity (data not shown).Tabled 1 Open table in a new tab Digestion of C1q-CLR with lower concentrations of trypsin (E:S, 1:100) resulted in a less degraded fragment as demonstrated by both Superose chromatography and SDS-PAGE (data not shown) and variable but significant activity in the neutrophil superoxide assay. Comparison of the sequence data of the fragments generated in each case revealed an apparent inverse relationship between the amount of superoxide generating activity and the percent of C1q-CLR that was cleaved at the most internal arginine residues (Arg38 in the A chain and Arg41 in the C chain). Therefore, digestion with endo Lys-C, which, like trypsin, cleaves at lysine residues but, unlike trypsin, does not cleave at arginine, was assessed to determine if a population of homogeneously cleaved molecules could be generated and subsequently assessed for the ability to trigger superoxide products. Analysis by SDS-PAGE demonstrated that specific peptide bonds of the C1q-CLR molecules were quantitatively and homogeneously cleaved by endo Lys-C at an E:S of 1:200 (Fig. 7). No additional cleavage occurred when higher amounts (E:S, 1:20) of endo Lys-C were added (data not shown) or when C1q-CLR was preheated to 50°C prior to digestion (Fig. 7, lane 4). The major proteolytic components were then separated and purified by Superose size exclusion chromatography. NH2-terminal sequencing of the first eluting peak (Fig. 8B, pool a, corresponding to Fig. 7, lane 1) demonstrated that as in the trypsin digest, this peak contained an unaltered NH2 terminus with no cleavage fragments remaining associated with the major C1q-CLR fragment generated. Sequencing of the later eluting peak areas (Fig. 8B, pools b and c) as well as the total digest identified all of the endo Lys-C fragments generated (Table 1 and Fig. 6). The most NH2-terminal cleavage sites that identify the COOH terminus of the major fragment generated by endo Lys-C and purified by gel chromatography were at Lys59 of the A chain, Lys61 of the B chain, and Lys58 of the C chain. The NH2-terminal C1q-CLR fragment generated by endo Lys-C retained complete activity in the neutrophil superoxide assay (Fig. 9), in contrast to the observed loss of activity of the fragment generated by trypsin digestion (Fig. 5).Fig. 8Gel filtration of C1q-CLR after endo Lys-C digestion. C1q-CLR incubated at 37°C for 2 h without enzyme (A) or with endo Lys-C at an E:S of 1:200 (B) was applied to Superose 12 column and eluted as described. The main fragment (designated by the arrows), eluting at 10.7 ml, was used in all structural and functional assays. The areas labeled a, b, and c in B indicate the fractions pooled for amino acid sequencing and referred to in Table 1. Flow rate of the column was 0.5 ml/min.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 9Endo Lys-C digestion has no inhibitory effect on the stimulation of O generation by C1q-CLR. Superoxide production by neutrophils was assayed by measuring reduction of cytochrome c. The fragment obtained from C1q-CLR digested with endo Lys-C (E:S, 1:200) (dotted line) retains full activity relative to the untreated C1q-CLR (solid line) or mock digested C1q-CLR (dashed and dotted line). Protein concentration used to coat the wells was 30 μg/ml.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The loss of ability of C1q-CLR to trigger neutrophil superoxide production after trypsin treatment could be related to the loss of specific amino acid residues (positions A 38-59 and/or C 41-58). Al" @default.
• W1977781825 created "2016-06-24" @default.
• W1977781825 creator A5034468688 @default.
• W1977781825 creator A5036034012 @default.
• W1977781825 creator A5090374075 @default.
• W1977781825 date "1995-12-01" @default.
• W1977781825 modified "2023-10-10" @default.
• W1977781825 title "Localization of the Site on the Complement Component C1q Required for the Stimulation of Neutrophil Superoxide Production" @default.
• W1977781825 cites W127377800 @default.
• W1977781825 cites W1500664838 @default.
• W1977781825 cites W1517991643 @default.
• W1977781825 cites W1523168730 @default.
• W1977781825 cites W1526755525 @default.
• W1977781825 cites W1551693981 @default.
• W1977781825 cites W1552769647 @default.
• W1977781825 cites W1552947467 @default.
• W1977781825 cites W1560807874 @default.
• W1977781825 cites W1561665048 @default.
• W1977781825 cites W1567944784 @default.
• W1977781825 cites W1568481877 @default.
• W1977781825 cites W1569111738 @default.
• W1977781825 cites W1573118011 @default.
• W1977781825 cites W1586297781 @default.
• W1977781825 cites W1591127773 @default.
• W1977781825 cites W1597180465 @default.
• W1977781825 cites W1600557486 @default.
• W1977781825 cites W1618869301 @default.
• W1977781825 cites W1619222389 @default.
• W1977781825 cites W1820570900 @default.
• W1977781825 cites W183757317 @default.
• W1977781825 cites W1868525735 @default.
• W1977781825 cites W1912866866 @default.
• W1977781825 cites W1963984598 @default.
• W1977781825 cites W1967130686 @default.
• W1977781825 cites W1975085160 @default.
• W1977781825 cites W1979221090 @default.
• W1977781825 cites W1987050309 @default.
• W1977781825 cites W1988352491 @default.
• W1977781825 cites W2010554932 @default.
• W1977781825 cites W2012679621 @default.
• W1977781825 cites W2030356483 @default.
• W1977781825 cites W2033450022 @default.
• W1977781825 cites W2035681019 @default.
• W1977781825 cites W2036735626 @default.
• W1977781825 cites W2047774312 @default.
• W1977781825 cites W2054292263 @default.
• W1977781825 cites W2076130162 @default.
• W1977781825 cites W2076841009 @default.
• W1977781825 cites W2093178095 @default.
• W1977781825 cites W2095553999 @default.
• W1977781825 cites W2121783932 @default.
• W1977781825 cites W2155453220 @default.
• W1977781825 cites W2158620829 @default.
• W1977781825 cites W2169806852 @default.
• W1977781825 cites W2273521055 @default.
• W1977781825 cites W2343236643 @default.
• W1977781825 cites W2401675758 @default.
• W1977781825 cites W2470975657 @default.
• W1977781825 cites W936023199 @default.
• W1977781825 doi "https://doi.org/10.1074/jbc.270.51.30627" @default.
• W1977781825 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/8530499" @default.
• W1977781825 hasPublicationYear "1995" @default.
• W1977781825 type Work @default.
• W1977781825 sameAs 1977781825 @default.
• W1977781825 citedByCount "30" @default.
• W1977781825 countsByYear W19777818252013 @default.
• W1977781825 crossrefType "journal-article" @default.
• W1977781825 hasAuthorship W1977781825A5034468688 @default.
• W1977781825 hasAuthorship W1977781825A5036034012 @default.
• W1977781825 hasAuthorship W1977781825A5090374075 @default.
• W1977781825 hasBestOaLocation W19777818251 @default.
• W1977781825 hasConcept C104317684 @default.
• W1977781825 hasConcept C112313634 @default.
• W1977781825 hasConcept C121332964 @default.
• W1977781825 hasConcept C127716648 @default.
• W1977781825 hasConcept C139719470 @default.
• W1977781825 hasConcept C162324750 @default.
• W1977781825 hasConcept C168167062 @default.
• W1977781825 hasConcept C169760540 @default.
• W1977781825 hasConcept C181199279 @default.
• W1977781825 hasConcept C185592680 @default.
• W1977781825 hasConcept C188082640 @default.
• W1977781825 hasConcept C203014093 @default.
• W1977781825 hasConcept C24998067 @default.
• W1977781825 hasConcept C2778348673 @default.
• W1977781825 hasConcept C2780795997 @default.
• W1977781825 hasConcept C55493867 @default.
• W1977781825 hasConcept C86803240 @default.
• W1977781825 hasConcept C95444343 @default.
• W1977781825 hasConcept C97355855 @default.
• W1977781825 hasConceptScore W1977781825C104317684 @default.
• W1977781825 hasConceptScore W1977781825C112313634 @default.
• W1977781825 hasConceptScore W1977781825C121332964 @default.
• W1977781825 hasConceptScore W1977781825C127716648 @default.
• W1977781825 hasConceptScore W1977781825C139719470 @default.
• W1977781825 hasConceptScore W1977781825C162324750 @default.
• W1977781825 hasConceptScore W1977781825C168167062 @default.
• W1977781825 hasConceptScore W1977781825C169760540 @default.

• next