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Binding of Cystatin C to C4

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Binding of Cystatin C to C4 Proc. Natl. Acad. Sci. USA Vol. 87, pp. 1288-1291, February 1990 Biochemistry Binding of cystatin C to C4: The importance of sense-antisense peptides in their interaction (cysteine-proteinase inhibitor/complement/peptide-peptide interaction) JORGE GHISO*, ESTER SABALLt, JULIANA LEONI*, AGUEDA ROSTAGNO*, AND BLAS FRANGIONE* *Department of Pathology, New York University Medical Center, New York, NY 10016; and tCatedra de Inmunobiologia, Facultad de Cs. Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario, Argentina Communicated by Michael Heidelberger, December 6, 1989 ABSTRACT Hydropathic anticomplementarity of amino In addition to the inhibitory activity, the above-mentioned acids indicates that peptides derived from complementary DNA residues (almost identical in all members of the cystatin strands may form amphiphilic structures and bind one an- superfamily) may also be involved in protein-protein inter- other. By using this concept, we have found that the antisense actions. It is known that there is a tendency in the genetic peptide Ser-Tyr-Asp-Leu complementary to the segment Gln- code for codons of hydrophilic amino acids to be comple- Ile-Val-Ala-Gly (residues 55-59) in cystatin C (an inhibitor of mented by codons of hydrophobic amino acids, resulting in cysteine proteases) is located at positions 611-614 ofthe 18 chain peptide structures that might interact specifically through of human C4, the fourth component of complement. Here we amphiphilic conformations (12). It was shown that peptides describe and characterize the specific interaction between generated from the noncoding strand of DNA specifically cystatin C and C4 by ligand affinity chromatography and recognize segments coded by the complementary strand (13). ELISA. Interaction between the two native proteins was mim- This concept was successfully used in the demonstration of icked on replacement of one of them with the corresponding the interaction between different peptide pairs (14) as well as sense-antisense peptide coupled to a carrier protein, and the in the purification of several receptors, among them corti- binding was inhibited by these synthetic peptides in solution. cotropin (ACTH) (13), fibronectin (15), and angiotensin II Through the interaction with C4, cystatin C may play a (16). Practical application of this approach is restricted to regulatory role in complement activation that might be of systems in which DNA sequence information is available. We particular importance at tissue sites where both proteins are have taken advantage of our recent report on the nucleotide produced by macrophages. sequence of the gene coding for cystatin C (17); from the sequence coding for the segment Gln-Ile-Val-Ala-Gly (resi- dues 55-59), we have deduced the complementary DNA Human cystatin C (formerly y trace) (1) is a basic protein of strand and, hence, the amino acid sequence of the antisense known primary structure (2) fully distributed in body fluids in peptide. We now present evidence of the specificity and a wide concentration range. It has been localized immuno- saturability of the interaction between cystatin C and the cytochemically in some cortical neurons, LH cells of the complementary deduced peptide Ser-Tyr-Asp-Leu and dem- adenohypophysis, pancreatic A and thyroid C cells, and onstrate that a protein containing the antisense peptide adrenal medulla (3). It is secreted into tissue culture medium (human C4, the fourth component of complement) interacts by monocytes and other cells, and the down-regulation of its with cystatin C and that this binding is inhibited by the secretion may play a role in inflammation (4). antisense peptide. Structural and genetic studies indicate that cystatin C is part of a superfamily that comprises three families, types I, II, and III (5, 6). Type I cystatins (also called stefins) are MATERIALS AND METHODS proteins of "100 residues with no disulfide bonds. Type II Proteins, Synthetic Peptides, and Antibodies. Human cys- cystatins (family that includes cystatin C) are molecules of tatin C was isolated from urine as described (18). C4 was 115-120 amino acids with two disulfide loops near the C purified from human plasma according to Gresham et al. (19) terminus. Type III cystatins (kininogens) are high molecular with modifications: 20 ml of whole plasma containing 10 mM weight proteins (Mr 68,000-110,000) composed of three cys- EDTA, 10 mM benzamidine hydrochloride, 5% L-lysine (free tatin type II-like domains (about 360 residues), the bradykinin base), and 1 mM phenylmethylsulfonyl fluoride was precip- moiety (9 amino acids), and a C-terminal polypeptide of itated with PEG 6000 (final concentration, 5%) for 1 hr at 4°C. variable length. After centrifugation at 40,000 x g for 30 min, the pellet was Cystatins possess inhibitory activity against a broad spec- discarded and the supernatant was made 21% in PEG 6000 to trum of cysteine proteinases of plant origin (papain, chymo- precipitate C3 and C4. The pellet was redisolved in 3 mM papain, ficin, and actinidin) as well as mammalian lysosomal phosphate buffer (pH 7.3) containing 6.5 mM EDTA, 6.5 mM proteases such as cathepsins B, H, and L (for review, see ref. benzamidine hydrochloride, 33 mM E-amino caproic acid, 7). The active site of inhibitory activity remains unknown, and 61.5 mM NaCl and applied to a 60-ml DEAE-Sephacel although some evidence suggests involvement of glycine at (Pharmacia) column equilibrated with the same buffer. After position 11 (8) and the segment 55-59 (cystatin C numbering) washing off the unbound material, the column was sequen- (6, 9, 10), both highly conserved in all known cystatins (7). tially eluted with 200 ml of a linear NaCI gradient (100 ml of Moreover, peptide Leu-Val-Gly (positions 9-11) inhibits 3 mM phosphate/65 mM NaCI, pH 7.3, and 100 ml of 3 mM growth of many bacteria, especially all group A streptococci, phosphate/133 mM NaCI, pH 7.3), washed with 100 ml of 3 apparently due to inhibition of a cysteine protease produced mM phosphate/133 mM NaCl, pH 7.3, and eluted again with by the bacteria (11). 200 ml of a linear NaCl gradient (100 ml of 3 mM phosphate/ 133 mM NaCl, pH 7.3, and 100 ml of 3 mM phosphate/300 mM NaCl, pH 7.3). As a final purification step, C4 was The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: BSA, bovine serum albumin. 1288 Downloaded by guest on September 24, 2021 Biochemistry: Ghiso et al. Proc. Natl. Acad. Sci. USA 87 (1990) 1289 precipitated with PEG 6000 (final concentration, 16%), redi- 0.1% BSA/0.05% Tween 20, pH 7.5) were allowed to react solved in phosphate-buffered saline [(PBS) 20 mM phos- for 1 hr at room temperature. Bound ligand was detected with phate/150 mM NaCl, pH 7.0], and stored at -70TC. Identi- the suitable antibody followed by alkaline phosphatase- fication of C4 in the column fractions was achieved by conjugated goat F(ab')2 anti-rabbit IgG. Between all steps in immunodiffusion (20) and purity was assessed by SDS/ the ELISA, wells were washed three times with Tris/ PAGE (21). BSA/Tween. The reaction was developed for 30 min with Synthetic peptides Gln-Ile-Val-Ala-Gly, Ser-Tyr-Asp-Leu, p-nitrophenyl phosphate (1 mg/ml) in diethanolamine buffer Gin-Ile-Val-Ala-Gly-Cys, Ser-Tyr-Asp-Leu-Cys, Thr-Tyr- (Bio-Rad), stopped with 0.4 M NaOH, and quantified in a Lys-Phe-Phe-Glu-Gln-Met-Gln-Asn-Cys, Asn-Trp-Cys- Microplate reader, model MR600 (Dynatech), at 410 nm. Lys-Arg-Gly-Arg-Lys-Gln, and Asp-Glu-Leu-Leu-Gln-Lys- Inhibition Assays. Synthetic peptides Ser-Tyr-Asp-Leu and Glu-Gln-Asn-Tyr-Ser-Asp were synthesized in the Center for Gln-Ile-Val-Ala-Gly as well as unrelated peptides Thr- the Analysis and Synthesis of Macromolecules (State Uni- Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln-Asn-Cys, Asn-Trp- versity of New York, Stony Brook) by solid-phase tech- Cys-Lys-Arg-Gly-Arg-Lys-Gln, and Asp-Glu-Leu-Leu- niques (22) and further purified by high-performance liquid Gln-Lys-Glu-Gln-Asn-Tyr-Ser-Asp were screened for inhi- chromatography. Their purity was ascertained by amino acid bition of binding by C4 and cystatin C. Different amounts of analysis using a Waters Pico Tag system and by Edman the synthetic peptides (0.1-10 ,ug) dissolved in 50/ul of50 mM degradation analysis on a 477A Applied Biosystems se- Tris (pH 7.4) were added to 50 ng of either cystatin C or C4 quencer. in 50 ,ul of the same buffer and incubated 1 hr at room Peptides Gln-Ile-Val-Ala-Gly-Cys, Ser-Tyr-Asp-Leu-Cys, temperature. Aliquots of each mixture were transferred into and Thr-Tyr-Lys-Phe-Phe-Glu-Gln-Met-Gln-Asn-Cys were Ser-Tyr-Asp-Leu-BSA-, C4-, cystatin C-, Gln-Ile-Val-Ala- coupled to bovine serum albumin (BSA) by way of their Gly-BSA-, unrelated peptide-BSA-, or BSA-coated wells and C-terminal cysteine, by use of the heterobifunctional reagent the bound cystatin C or C4 was measured as above (see figure m-maleimido-benzoyl-N-hydroxysuccinimide ester (23). legends for details). Anti-human cystatin C was produced in rabbits (24); rabbit anti-human C4 was purchased from Dako (Santa Barbara, CA); alkaline phosphatase-labeled goat F(ab')2 anti-rabbit RESULTS AND DISCUSSION IgG was acquired from Tago. The nucleotide and amino acid sequences of the segment Affinity Chromatography Assays. Cystatin C and BSA were 55-59 in cystatin C (the most conserved in all cystatins), the coupled to CNBr-activated Sepharose 4B (Pharmacia) at 5 nucleic acid sequence ofcomplementary DNA strand, and the mg/ml of beads according to manufacturer's instructions.
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