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 ...

Bacterial adherence to extracellular matrix proteins (ECMp) plays important roles during host-pathogen interaction, however its genetic regulation remains poorly understood. yloA of the model bacterium Bacillus subtilis shows high homology to genes encoding fibronectin-binding proteins of Gram-positive pathogens. Here, we characterized the regulatory network of YloA-dependent adhesive properties of the probiotic B. subtilis natto (Bsn). YloA-proficient, but not YloA-deficient, Bsn specifically bound to ECMp in a concentration-dependent manner and were proficient in biofilm formation. yloA expression showed a continuous increase in activity during the growth phase and decreased during the stationary phase. The transcription factors AbrB and DegU downregulated yloA expression during the logarithmic and stationary growth phases respectively. Analysis of the yloA promoter region revealed the presence of AT-rich direct and inverted repeats previously reported to function as DegU-recognized binding sites. In spo0A cells, yloA expression was completely turned off because of upregulation of AbrB throughout growth. Accordingly, DNase I footprinting analysis confirmed that AbrB bound to the promoter region of yloA. Interestingly, Bsn bound fibronectin with higher affinity, lower Kd, than several bacterial pathogens and competitively excluded them from binding to immobilized-fibronectin, a finding that might be important for the anti-infective properties of B. subtilis and its relatives.

Many glomerular diseases are associated with changes in the expression and distribution in the components of extracellular matrix. A remarkable feature in acute renal failure induced by mercuric chloride in rats was large fibronectin (Fn) deposits in kidneys 1 h post-HgCl2 injection (5 mg/kg body wt., s.c.). Our study examined some mechanisms as potential explanation of the early Fn deposits in mercuric chloride induced acute renal failure. Total tissue mRNA of livers and kidneys of control and treated rats were used in Northern blot to determine whether accumulation of Fn in kidney is associated with increases in the expression of this protein in the kidney and/or in the liver. Analysis of Fn levels by Western blot were also performed. Northern blot did not show significant difference between control and treated rats, while the abundance of polymerized-Fn in kidney tissue was increased 1 h and 5 h post HgCl2 injection. HgCl2 influence on Fn folding was studied in vitro to detect possible conformational changes that could altered its normal pattern of matrix assembly and/or binding to different ligands. In this context HgCl2 binding to Fn was measured following native tryptophan fluorescence of Fn in the presence of HgCl2 (0.5-250 mM). Binding parameters for the HgCl2-Fn complex formation were Kd = (1.6 +/- 0.2) 10(-4) M; n = 1 +/- 0.3, indicating a low apparent affinity and one type binding site. Thermal denaturation of Fn showed, between 30-60 degrees C, a soft reversible conformational change, while between 75-80 degrees C a highly and irreversible transition is produced suggesting a modification of the tertiary structure. HgCl2 abolished this transition. The kinetic of thermal unfolding of Fn was also measured and the effects observed due to HgCl2 presence reinforced the previous data. Finally, the effect of HgCl2 on Fn binding to denatured collagen (gelatin) was also measured as an index of the effect of this cation on biological properties of Fn. Fn binds gelatin strongest in the presence of HgCl2. Our results suggest that higher Fn deposits in kidney-treated rats seems not to be associated to augmented mRNA-Fn neither in kidney nor in liver. On the other hand, increased levels of polymerized Fn abundance was observed in kidney tissue from mercury-treated rats. We also describe that HgCl2 promotes, in vitro, conformational changes on Fn structure, inducing its denaturation and increasing its binding to gelatin, all events that could be related to the Fn deposits in renal tissues of HgCl2 treated rats, and could be expected in other situations that promoted interstitial fibrosis, not associated to overexpression of matrix-proteins.