Binding of cystatin C to C4: the importance of sense-antisense peptides in their interaction.

Abstract

Hydropathic anticomplementarity of amino acids indicates that peptides derived from complementary DNA strands may form amphiphilic structures and bind one another. By using this concept, we have found that the antisense peptide Ser-Tyr-Asp-Leu complementary to the segment GlnIle-Val-Ala-Gly (residues 55-59) in cystatin C (an inhibitor of cysteine proteases) is located at positions 611-614 of the 18 chain of human C4, the fourth component of complement. Here we describe and characterize the specific interaction between cystatin C and C4 by ligand affinity chromatography and ELISA. Interaction between the two native proteins was mimicked on replacement of one of them with the corresponding sense-antisense peptide coupled to a carrier protein, and the binding was inhibited by these synthetic peptides in solution. Through the interaction with C4, cystatin C may play a regulatory role in complement activation that might be of particular importance at tissue sites where both proteins are produced by macrophages.Human cystatin C (formerly y trace) (1) is a basic protein of known primary structure (2) fully distributed in body fluids in a wide concentration range. It has been localized immunocytochemically in some cortical neurons, LH cells of the adenohypophysis, pancreatic A and thyroid C cells, and adrenal medulla (3). It is secreted into tissue culture medium by monocytes and other cells, and the down-regulation of its secretion may play a role in inflammation (4).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 proteins of "100 residues with no disulfide bonds. Type II cystatins (family that includes cystatin C) are molecules of 115-120 amino acids with two disulfide loops near the C terminus. Type III cystatins (kininogens) are high molecular weight proteins (Mr 68,000-110,000) composed of three cystatin type II-like domains (about 360 residues), the bradykinin moiety (9 amino acids), and a C-terminal polypeptide of variable length.Cystatins possess inhibitory activity against a broad spectrum of cysteine proteinases of plant origin (papain, chymopapain, ficin, and actinidin) as well as mammalian lysosomal proteases such as cathepsins B, H, and L (for review, see ref.7). The active site of inhibitory activity remains unknown, although some evidence suggests involvement of glycine at position 11 (8) and the segment 55-59 (cystatin C numbering) (6, 9, 10), both highly conserved in all known cystatins (7). Moreover, peptide Leu-Val-Gly (positions 9-11) inhibits growth of many bacteria, especially all group A streptococci, apparently due to inhibition of a cysteine protease produced by the bacteria (11).In addition to the inhibitory activity, the above-mentioned residues (almost identical in all members of the cystatin superfamily) may also be involved in protein-protein interactions. It is known that there is a tendency in the genetic code for codons of hydrophilic amino acids ...

Removal and Rebound Kinetics of Cystatin C in High-Flux Hemodialysis and Hemodiafiltration

“…There are no data on the relative pool size of dimerized cystatin C. It is likely that the assay that we used measured predominantly monomeric cystatin C, although there are no published data. Cystatin C may also be bound to the C4 component of complement (46) and other proteins, which may contribute to the reduction in apparent pool size. In our model, we iterated the relative sizes of the apparent intracellular and extracellular spaces, which will have taken into account the effect of any additional pools.…”

De Novo Design of Peptides Targeted to the EF Hands of Calmodulin

“…For Fig. 10, the percent inhibition was calculated using the formula: 100 N-labeled CaM was purified from cells grown in M9 medium using 15 NH 4 Cl as the sole nitrogen source. CaM (labeled and unlabeled) was purified by repeated chromatography on phenyl-Sepharose CL4B followed by ion exchange chromatography on DEAE-cellulose using a linear gradient (0 -0.5 M) of sodium chloride in 0.05 M sodium phosphate, pH 5.7.…”

“…1 and 12) have shown that in almost 40 different systems complementary peptides bind one another with specificity and moderate affinity. Additional evidence of complementary structure includes the following: the ability to locate the interactive sites of ligands and receptors by identification of complementary sequences (13)(14)(15)(16)(17); to generate interacting pairs of monoclonal idiotypic and anti-idiotypic antibodies with complementary combining sites by immunization with pairs of complementary peptides (18 -27); to produce antibodies to receptor-binding sites by immunization with complementary peptides for the receptor's ligand (11,18,(27)(28)(29)(30)(31)(32)(33)(34). Most recently, a novel hormone receptor was cloned, and its binding site was localized using this principle (17).…”

Affinity capture of [Arg8]vasopressin-receptor complex using immobilized antisense peptide.

“…The experimental work that has followed for the past 5 years has shown that AS polypeptide recognition is surprisingly selective and observable in many peptides (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Nevertheless, skepticism has persisted about the mechanistic and biological relevance of these interactions.…”

“…1 and the AS peptide recognition hypothesis upon which it is based. Previous studies with AS peptides (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(20)(21)(22)(23) have led to the hypothesis that AS peptide recognizes sense peptide by hydropathic pattern recognition, through multipoint contacts along interacting peptide chains that are conformationally extended rather than folded into compact three-dimensional structures. The pattern recognitiondegenerate conformation model for sense-AS peptide recognition led to the prediction given in Fig.…”