Renin increases mesangial cell transforming growth factor-beta1 and matrix proteins through receptor-mediated, angiotensin II-independent mechanisms.

Recent evidence suggesting a strong interplay between components of the renin-angiotensin system and key mediators of fibrosis led us to hypothesize that renin, independent of its enzymatic action to enhance angiotensin (Ang) II synthesis, directly increases production of the fibrogenic cytokine transforming growth factor (TGF)-beta. Human or rat mesangial cells (MCs) were treated with human recombinant renin (HrRenin) or rat recombinant renin (RrRenin) and the effects on TGF-beta1, plasminogen activator inhibitor-type 1 (PAI-1), fibronectin (FN) and collagen 1 mRNA and protein were investigated. Blockade of the rat MC renin receptor was achieved using siRNA. HrRenin or RrRenin, at doses shown to be physiologically relevant, induced marked dose- and time-dependent increases in TGF-beta1. These effects were not altered by adding an inhibitor of renin's enzymatic action (RO 42-5892), the Ang II receptor antagonist losartan or the Ang-converting enzyme inhibitor enalapril. RrRenin also induced PAI-1, FN and collagen 1 mRNA and PAI-1 and FN protein in a dose-dependent manner. Neutralizing antibodies to TGF-beta partially blocked these effects. Supernatant and cell lysate Ang I and Ang II levels were extremely low. MC angiotensinogen mRNA was undetectable both with and without added renin. Targeting of the rat renin receptor mRNA with siRNA blocked induction of TGF-beta1. We conclude that renin upregulates MC TGF-beta1 through a receptor-mediated mechanism, independent of Ang II generation or action. Renin-induced increases in TGF-beta1 in turn stimulate increases in PAI-1, FN and collagen I. Thus, renin may contribute to renal fibrotic disease, particularly when therapeutic Ang II blockade elevates plasma renin.

Crystal structure of Lyme disease antigen outer surface protein C from Borrelia burgdorferi.

The outer surface protein C (OspC) is one of the major host-induced antigens of Borrelia burgdorferi, the causative agent of Lyme disease. We have solved the crystal structure of recombinant OspC to a resolution of 2.5 A. OspC, a largely alpha-helical protein, is a dimer with a characteristic central four-helical bundle formed by association of the two longest helices from each subunit. OspC is very different from OspA and similar to the extracellular domain of the bacterial aspartate receptor and the variant surface glycoprotein from Trypanosoma brucei. Most of the surface-exposed residues of OspC are highly variable among different OspC isolates. The membrane proximal halves of the two long alpha-helices are the only conserved regions that are solvent accessible. As vaccination with recombinant OspC has been shown to elicit a protective immune response in mice, these regions are candidates for peptide-based vaccines.

Multiple binding sites in collagen type I for the integrins alpha1beta1 and alpha2beta1.

Integrins alpha(1)beta(1) and alpha(2)beta(1) are two major collagen receptors on the surface of eukaryotic cells. Binding to collagen is primarily due to an A-domain near the N terminus of the alpha chains. Previously, we reported that recombinant A-domain of alpha(1)beta(1) (alpha(1)A) had at least two affinity classes of binding sites in type I collagen (Rich, R. L., et al. (1999) J. Biol. Chem. 274, 24906-24913). Here, we compared the binding of the recombinant A-domain of alpha(2)beta(1) (alpha(2)A) to type I collagen with that of alpha(1)A using surface plasmon resonance and showed that alpha(2)A exhibited only one detectable class of binding sites in type I collagen, with a K(D) of approximately 10 microm at approximately 3 binding sites per collagen molecule. We further demonstrated that alpha(1)A and alpha(2)A competed with each other for binding to type I collagen in enzyme-linked immunosorbent assay (ELISA), suggesting that the binding sites in collagen for the two A-domains overlap or are adjacent to each other. By using rotary shadowing, the complexes of alpha(1)A- and alpha(2)A-procollagen were visualized. Morphometric analyses indicated three major binding regions (near the N terminus, in the central part, and near the C terminus) along the type I procollagen molecule for both A-domains. The positions of the respective binding regions for alpha(1)A and alpha(2)A were overlapping with or adjacent to each other, consistent with the ELISA results. Analysis of the sequences of type I collagen revealed that GER or GER-like motifs are present at each of the binding regions, and notably, the central region contains the GFOGER sequence, which was previously identified as a high affinity site for both alpha(1)A and alpha(2)A (Knight, C. G., et al. (2000) J. Biol. Chem. 275, 35-40). Peptides containing GLOGERGRO (peptide I, near the N terminus), GFOGERGVQ (peptide II, central), and GASGERGPO (peptide III, near the C terminus) were synthesized. Peptides I and II effectively inhibited the binding of alpha(1)A and alpha(2)A to type I collagen, while peptide III did so moderately. The N-terminal site in type I collagen has the sequence GLOGER in all three chains. Thus, it seems that peptide I represents a newly discovered native high affinity site for alpha(1)A and alpha(2)A.

Novel fold and assembly of the repetitive B region of the Staphylococcus aureus collagen-binding surface protein.

BACKGROUND: [corrected] The Staphylococcus aureus collagen-binding protein Cna mediates bacterial adherence to collagen. The primary sequence of Cna has a non-repetitive collagen-binding A region, followed by the repetitive B region. The B region has one to four 23 kDa repeat units (B(1)-B(4)), depending on the strain of origin. The affinity of the A region for collagen is independent of the B region. However, the B repeat units have been suggested to serve as a 'stalk' that projects the A region from the bacterial surface and thus facilitate bacterial adherence to collagen. To understand the biological role of these B-region repeats we determined their three-dimensional structure. RESULTS: B(1) has two domains (D(1) and D(2)) placed side-by-side. D(1) and D(2) have similar secondary structure and exhibit a unique fold that resembles but is the inverse of the immunoglobulin-like (IgG-like) domains. Comparison with similar immunoglobulin superfamily (IgSF) structures shows novel packing arrangements between the D(1) and D(2) domains. In the B(1)B(2) crystal structure, an omission of a single glycine residue in the D(2)-D(3) linker loop, compared to the D(1)-D(2) and D(3)-D(4) linker loops, resulted in projection of the D(3) and D(4) in a spatially new orientation. We also present a model for B(1)B(2)B(3)B(4). CONCLUSIONS: The B region of the Cna collagen adhesin has a novel fold that is reminiscent of but is inverse in nature to the IgG fold. This B region assembly could effectively provide the needed flexibility and stability for presenting the ligand binding A region away from the bacterial cell surface.

Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi.

VlsE is a 35-kDa surface-exposed lipoprotein of Borrelia burgdorferi that was shown previously to undergo antigenic variation through segmental recombination of silent vls cassettes with vlsE during experimental mouse infections. Previous data had indicated that sera from North American Lyme disease patients and experimentally infected animals contained antibodies reactive with VlsE. In this study, sera from patients with Lyme disease, syphilis, and autoimmune conditions as well as from healthy controls were examined for reactivity with VlsE by Western blotting and enzyme-linked immunosorbent assay (ELISA). Strong Western blot reactivity to a recombinant VlsE cassette region protein was obtained consistently with Lyme disease sera. Although sera from Lyme disease patients also reacted with a band corresponding to VlsE in B. burgdorferi B31-5A3, interpretation was complicated by low levels of VlsE expression in in vitro-cultured B. burgdorferi and by the presence of comigrating bands. An ELISA using recombinant VlsE was compared with an ELISA using sonically disrupted B. burgdorferi as the antigen. For a total of 93 Lyme disease patient sera examined, the VlsE ELISA yielded sensitivities of 63% for culture-confirmed erythema migrans cases and 92% for later stages, as compared to 61 and 98%, respectively, for the "whole-cell" ELISA. The specificities of the two assays with healthy blood donor sera were comparable, but the VlsE ELISA was 90% specific with sera from syphilis patients, compared to 20% specificity for the whole-cell ELISA with this group. Neither assay showed reactivity with a panel of sera from 20 non-Lyme disease arthritis patients or 20 systemic lupus erythematosus patients. Our results indicate that VlsE may be useful in the immunodiagnosis of Lyme disease and may offer greater specificity than ELISAs using whole B. burgdorferi as the antigen.

Ace is a collagen-binding MSCRAMM from Enterococcus faecalis.

A putative collagen-binding MSCRAMM, Ace, of Enterococcus faecalis was identified by searching bacterial genome data bases for proteins containing domains homologous to the ligand-binding region of Cna, the collagen-binding MSCRAMM from Staphylococcus aureus. Ace was predicted to have a molecular mass of 71 kDa and contains features characteristic of cell surface proteins on Gram-positive bacteria, including a LPXTG motif for cross-linking to the cell wall. The N-terminal region of Ace contained a region (residues 174-319) in which 56% of the residues are identical or similar when compared with the minimal ligand-binding region of Cna (Cna 151-318); the remainder of the Ace A domain has 46% similarity with the corresponding region of the Cna A domain. Antibodies raised against recombinant Ace A domain were used to verify the cell surface expression of Ace on E. faecalis. These antibodies also effectively inhibited the adhesion of enterococcal cells to a collagen substrate, suggesting that Ace is a functional collagen-binding MSCRAMM. Structural modeling of the conserved region in Ace (residues 174-319) suggested a structure very similar to that reported for residues 151-318 of the Cna collagen-binding domain in which the ligand-binding site was identified as a trench transversing a beta-sheet face (Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 10, 833-838). Biochemical analyses of recombinant Ace and Cna A domains supported the modeling data in that the secondary structures were similar as determined by CD spectroscopy and both proteins bound at multiple sites in type I collagen with micromolar affinities, but with different apparent kinetics. We conclude that Ace is a collagen-binding MSCRAMM on enterococci and is structurally and functionally related to the staphylococcal Cna protein.

Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, alpha(1)beta(1) integrin and Staphylococcus aureus cna MSCRAMM.

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin alpha(2)beta(1) and a Staphylococcus aureus adhesin reveal a "trench" on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833-838). We report here the crystal structure of the alpha subunit I domain from the alpha(1)beta(1) integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human alpha(1) integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.

Crystallization and preliminary X-ray analysis of B-domain fragments of a Staphylococcus aureus collagen-binding protein.

Recombinant proteins of monomeric and dimeric B-domain repeats of a Staphylococcus aureus FDA 574 collagen-binding adhesin have been crystallized. The single repeat unit (B1) was crystallized in a body-centered orthorhombic lattice with a = 96.9, b = 101.3, c = 120. 8 A in either the I222 or I212121 space group. These crystals diffracted to 2.5 A resolution and the calculated Vm values of 3.2 and 2.2 A3 Da-1 suggest the possibility of a dimer or a trimer in the asymmetric unit. The two-repeat fragment (B1B2) crystallized in the orthorhombic space group P212121 with cell dimensions a = 42.4, b = 79.4, c = 130.4 A and diffracted to 2.3 A resolution. For this species, the calculated Vm value of 2.2 A3 Da-1 indicates the presence of a monomer in the asymmetric unit.

Crystallization of ClfA and ClfB fragments: the fibrinogen-binding surface proteins of Staphylococcus aureus.

Recombinant constructs encoding the fibrinogen-binding domains of ClfA and ClfB from Staphylococcus aureus have been crystallized. ClfA was crystallized in the orthorhombic space group P212121 with unit-cell parameters a = 39.58, b = 81.39 and c = 112.65 A. A complete data set was recorded to 2.1 A resolution and had a Vm of 2. 3 A3 Da-1 with 46.5% solvent, suggesting one molecule per asymmetric unit. Co-crystals of ClfA with the 17 amino-acid C-terminal peptide of fibrinogen gamma-chain diffracted to 2.1 A resolution and had unit-cell parameters a = 39.11, b = 81.39 and c = 109.51 A in the space group P212121. ClfB was crystallized in the tetragonal space group P41212 or P43212 with unit-cell parameters a = 96.31, b = 96. 31 and c = 84.13 A and diffracted to 2.45 A resolution. The estimated Vm of 2.6 A3 Da-1 with 53% solvent indicated one molecule in the asymmetric unit.

Is the sensitivity of cells for FGF-1 and FGF-2 regulated by cell surface heparan sulfate proteoglycans?

We have examined the importance of cell surface heparan sulfate proteoglycans (HSPG) in fibroblast growth factor (FGF) signaling. 3T3 cells grown under conventional conditions were much more sensitive to FGF-2 compared to FGF-1. However, cells were equally sensitive to FGF-1 and FGF-2 using conditions which reduced the effect of endogenous HSPG. Addition of heparin, or treatment with chlorate, an inhibitor of proteoglycan sulfation, resulted in enhanced or reduced growth factor response, respectively, and eliminated the differences between FGF-1 and FGF-2. HSPGs isolated from trypsin digests of 3T3 cells had a much higher affinity for FGF-2 compared to that for FGF-1 when analyzed by affinity chromatography. Glycosaminoglycan chains or core protein fragments derived from the HSPG failed to show the same high apparent affinity for FGF-2, suggesting that an intact proteoglycan structure was important for the high FGF-2 affinity. Addition of HSPG ectodomains, isolated from cultured 3T3 cells or produced as recombinant molecules, to chlorate-treated cultures of 3T3 cells inhibited the mitogenic activity of FGF-2 and eliminated the effect of heparin as a potentiator of either growth factor. These results support the idea that the cell-associated HSPG is an integral component of the FGF signaling system and in 3T3 cells contributes to the increased sensitivity of these cells to FGF-2 compared to FGF-1. Since isolated ectodomains of HSPG inhibited rather than stimulated the mitogenic response of the FGFs, the proper anchoring of the HSPG in the cell membrane appears to be important for a stimulatory effect.

Heparin-dependent fibroblast growth factor activities: effects of defined heparin oligosaccharides.

Heparin and related molecules have been identified as important participants in fibroblast growth factor (FGF) signaling although the mechanisms of action remain unclear. We have used heparin oligosaccharides to examine steps in the signaling process which could be affected by the polysaccharide. Immobilized FGF-1 and FGF-2 bound all sizes of oligosaccharides tested, ranging from tetrasaccharide to decasaccharide, at physiological salt concentration. Each group of oligosaccharide was eluted from the FGF affinity columns in several peaks, and larger oligosaccharides showed higher apparent affinity for the immobilized growth factors compared to the shorter ones. Heparin hexasaccharides were the smallest fragments providing complete protection of FGF-1 and FGF-2 against trypsin digestion. Tetrasaccharides, however, were able to provide partial protection. The requirement of heparin for ligand-receptor interaction was evaluated in receptor binding assays using Sf9 insect cells engineered to overexpress different recombinant FGF receptor (FGFR) species including FGFR1 beta, FGFR1 alpha or FGFR4 at the cell surface. In these assays hexasaccharides were the smallest fragments capable of stimulating FGF-receptor interaction. Over the range of concentrations examined, neither hexasaccharides nor octasaccharides were able to stimulate receptor binding to the level attained by intact heparin. In fact, these oligosaccharides interfered with the ability of intact heparin in promoting FGF-receptor binding. The presence of both stimulatory and inhibitory activities in hexasaccharide and octasaccharide populations could be attributed to structural heterogeneity within the oligosaccharide preparations. However, similar observations were obtained with "highly-sulfated" structurally homogeneous preparations of hexasaccharide and octasaccharide, although these molecules generally had greater stimulatory and less inhibitory activity than their structurally heterogeneous counterparts. Hexasaccharides were found to be the smallest fragments able to potentiate the FGF-1-induced 3T3 cell proliferation while their effect on FGF-2 signaling was less clear. These observations suggest that heparin can modulate FGF-signaling at several stages with different end results.

Chondroitin sulfate proteoglycan and heparan sulfate proteoglycan production by cultured pigeon peritoneal macrophages.

Proteoglycan production was examined in cultures of thioglycollate-elicited peritoneal macrophages obtained from White Carneau and Show Racer pigeons. Following a 24-h incubation in the presence of [35S]sulfate and [3H]serine, total production and distribution of 35S-labeled proteoglycan into media (60-65%), pericellular (21-27%), and intracellular (13-14%) compartments was similar in White Carneau and Show Racer macrophage cultures. Media proteoglycans consisted of high-molecular-weight chondroitin sulfate proteoglycan, low-molecular-weight chondroitin sulfate proteoglycan, and heparan sulfate proteoglycan. High-molecular-weight chondroitin sulfate proteoglycan was predominantly 6-sulfated (80%) and contained a core protein larger than 200 kd, whereas low-molecular-weight chondroitin sulfate proteoglycan was 4-sulfated and contained a 28-kd core protein. Pericellular proteoglycan was similar in size to low-molecular-weight proteoglycan and consisted of a predominantly 6-sulfated (75%) chondroitin sulfate proteoglycan and heparan sulfate proteoglycan. Intracellular 35S-labeled chondroitin sulfate and heparan sulfate were smaller than media and pericellular proteoglycans, suggestive of intracellular degradative processing.

Proteoglycans produced by cholesterol-enriched macrophages bind plasma low density lipoprotein.

Proteoglycans (PG) produced by [35S]sulfate and [3H]serine labeled cultures of cholesterol-enriched macrophages obtained from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons were characterized and assessed for their capacity to bind low density lipoprotein (LDL). The majority of 35S-labeled PG was released into the culture media in both WC and SR macrophage cultures and consisted of large and small size PG as determined by Sepharose CL-4B chromatography. Large PG were identified as chondroitin sulfate-PG comprised of 4-sulfated disaccharides whereas small PG consisted of primarily 4-sulfated chondroitin sulfate-PG and lesser amounts of heparin sulfate-PG. Experiments demonstrated that 32-34% of 35S-labeled large PG and 86-93% of small PG bound to LDL-substituted Sepharose. Interactions between PG and LDL-substituted Sepharose were inhibited in the presence of heparin or soluble LDL. Glycosaminoglycans derived from macrophage PG had a decreased binding affinity demonstrating the importance of an intact PG. The results suggest that macrophage PG may facilitate trapping of LDL in the intimal intima and promote foam cell formation through a mechanism involving the uptake of PG-LDL complexes.

Metabolism and turnover of cell surface-associated heparan sulfate proteoglycan and chondroitin sulfate proteoglycan in normal and cholesterol-enriched macrophages.

Analysis of sulfur-35-labeled proteoglycans indicated that cholesterol-enriched pigeon peritoneal macrophages synthesized 42% more 35S-labeled proteoglycan when compared with control macrophages during a 24-hour incubation. Proteoglycan turnover was subsequently studied in radiolabeled macrophage cultures after a 1-, 3-, 6-, 12-, or 24-hour chase with fresh media. During the chase, intracellular proteoglycan disappeared rapidly, whereas there was a small accumulation of 35S-labeled proteoglycan in the media that plateaued at about 6 hours and remained relatively constant thereafter. Pericellular heparan sulfate proteoglycan and chondroitin sulfate proteoglycan disappeared throughout the chase and did not appear to accumulate in the media or in the intracellular compartment. The rapid disappearance of intracellular proteoglycans along with the relative lack in metabolism of media proteoglycans indicated that the majority of pericellular proteoglycans were metabolized via an intracellular degradative pathway. Kinetic analysis of pericellular proteoglycans revealed the presence of a single pool of heparan sulfate proteoglycan (half-life [t1/2] = 6.9 hours) and a single pool of chondroitin sulfate proteoglycan (t1/2 = 11.5 hours) in control macrophage cultures. Cholesterol-enriched macrophage cultures also contained a single pool of pericellular heparan sulfate proteoglycan (t1/2 = 7.3 hours) but contained two pools of chondroitin sulfate proteoglycan (t1/2 = 0.8 hour and 25.9 hours).

Macrophage secretory products selectively stimulate dermatan sulfate proteoglycan production in cultured arterial smooth muscle cells.

Arterial dermatan sulfate proteoglycan has been shown to increase with atherosclerosis progression, but factors responsible for this increase are unknown. To test the hypothesis that smooth muscle cell proteoglycan synthesis may be modified by macrophage products, pigeon arterial smooth muscle cells were exposed to the media of either cholesteryl ester-loaded pigeon peritoneal macrophages or a macrophage cell line P388D1. Proteoglycans radiolabeled with [35S]sulfate and [3H]serine were isolated from culture media and smooth muscle cells and purified following precipitation with 1-hexadecylpyridinium chloride and chromatography. Increasing concentrations of macrophage-conditioned media were associated with a dose-response increase in [35S]sulfate incorporation into secreted proteoglycans, but there was no change in cell-associated proteoglycans. Incorporation of [3H]serine into total proteoglycan core proteins was not significantly different (5.2 X 10(5) dpm and 5.5 X 10(5) disintegrations per minute (dpm) in control and conditioned media-treated cultures, respectively), but selective effects were observed on individual proteoglycan types. Twofold increases in dermatan sulfate proteoglycan and limited degradation of chondroitin sulfate proteoglycan were apparent based on core proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Immunoinhibition studies indicated that interleukin-1 was involved in the modulation of proteoglycan synthesis by macrophage-conditioned media. These data provide support for the role of macrophages in alteration of the matrix proteoglycans synthesized by smooth muscle cells and provide a mechanism to account for the reported increased dermatan sulfate/chondroitin sulfate ratios in the developing atherosclerotic lesion.

A simplified method for monitoring and calibrating refrigerator alarm systems.

A method to modify existing temperature alarm systems on blood bank refrigerators is described. This provides an accurate and convenient method for monitoring the temperature alarm system outside of the refrigerator.