Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes.

Glycosaminoglycans (GAGs), highly sulfated polymers built of hexosamine-uronic acid disaccharide units, are major components of the extracellular matrix, mostly in the form of proteoglycans. They interact with a large array of proteins, in particular of the blood coagulation cascade. Degradation of GAGs in mammalian systems occurs by the action of GAG hydrolases. Bacteria express a large number of GAG-degrading lyases that break the hexosamine-uronic acid bond to create an unsaturated sugar ring. Flavobacterium heparinum produces at least five GAG lyases of different specificity. Chondroitin AC lyase (chondroitinase AC, 75 kDa) is highly active toward chondroitin 4-sulfate and chondroitin-6 sulfate. Its crystal structure has been determined to 1.9 A resolution. The enzyme is composed of two domains. The N-terminal domain of approximately 300 residues contains mostly alpha-helices which form a doubly-layered horseshoe (a subset of the (alpha/alpha)6 toroidal topology). The approximately 370 residues long C-terminal domain is made of beta-strands arranged in a four layered beta-sheet sandwich, with the first two sheets having nine strands each. This fold is novel and has no counterpart in full among known structures. The sequence of chondroitinase AC shows low level of homology to several hyaluronate lyases, which likely share its fold. The shape of the molecule, distribution of electrostatic potential, the pattern of conservation of the amino acids and the results of mutagenesis of hyaluronate lyases, indicate that the enzymatic activity resides primarily within the N-terminal domain. The most likely candidate for the catalytic base is His225. Other residues involved in catalysis and/or substrate binding are Arg288, Arg292, Lys298 and Lys299.

Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes.

Identification of the atrial natriuretic factor-R1C receptor subtype (B-clone) in cultured rat aortic smooth muscle cells.

The present report demonstrates the presence in cultured rat aortic smooth muscle cells of a natriuretic factor receptor subtype with a specificity typical of the ANF-R1C (B-clone) receptor subtype. To prove the existence of this receptor subtype in this cell line we show that pCNP-(82-103) is the most potent activator of the intrinsic guanylate cyclase activity, and that [125I]pCNP-(82-103) binds to a specific receptor subtype which is insensitive to the ANF-R2 specific ligand, C-ANF. The investigation of its binding characteristics show the rank potency order of the natriuretic factors in competing for pCNP binding to be pCNP greater than pBNP greater than rANF. Furthermore it was possible to covalently photolabel this receptor subtype with underivatized]125I]pCNP and show that it is composed of a single subunit of 130 kDa with very high specificity for pCNP.

C-type natriuretic peptide in bovine chromaffin cells. The regulation of its biosynthesis and secretion.

We report here the regulation of the biosynthesis and the secretion of C-type natriuretic peptide (CNP) in cultured bovine chromaffin cells. The combined treatment with protein kinase A and -C activators induced a 6-fold increase of intracellular levels of CNP-(1-103). The biosynthesized CNP-(1-103) was co-released with its mature forms, typically CNP-(51-103), upon stimulation by nicotine or depolarizing agents. This confirms the neuropeptidic character of this third member of the natriuretic peptide family, which might act as a neuromodulator or neurotransmitter.

Highly sensitive and rapid radioimmunoassay for aldosterone in plasma and cell culture medium.

A radioimmunoassay for aldosterone was developed using a sensitive and specific antibody and 125Iodinated-aldosterone. This assay could be used for direct determination of aldosterone in cell culture medium or after extraction of aldosterone from plasma by solid phase procedure using C18 Sep-pak cartridges. The very low cross-reactivity of the antibody with cortisol and corticosterone (0.005% and 0.04% respectively) would allow the direct determination of aldosterone in cell culture medium without any prior extraction step. Since the incubation is performed at room temperature for 1 h and then, at 4 degrees C for 15 min, the results can be obtained in less than 3 h. The assay was linear from 0.5 fmol/tube to 1500 fmol/tube with an ED50 at 30 fmol/tube. The accuracy of the assay estimated using spiked plasma samples with a known amount of aldosterone give a coefficient of correlation of 0.97 (n = 10) between the aldosterone concentrations found and expected levels. The within-assay variability for plasma aldosterone varied from 4.7 to 11.1% and the between-assay variability ranged from 13.9 to 14.2%. The coefficient of correlation between plasma aldosterone measured by this new assay or by a current assay was 0.8 (n = 43). In summary, the combination of a shortened incubation time with a simple solid phase extraction for aldosterone in serum samples represents the major advantage of the present assay over the current methodology which usually requires a chromatographic separation of the mineralocorticoid prior to radioimmunoassay. Therefore this assay would be useful in experimental studies as well as in clinical laboratory.

Pharmacological evidence for the heterogeneity of atrial natriuretic factor-R1 receptor subtype.

The atrial natriuretic factor-R1 (ANF-R1) receptor is known to mediate the biological effects of diverse natriuretic/diuretic/vasorelaxant peptides. In order to investigate the differential selectivity of this class of receptor, we have compared its pharmacological profile for various natriuretic peptides in rat, bovine, and human kidney. In contrast to bovine and rat, human kidney glomeruli do not express significant amounts of ANF-R2 receptor. In addition, the binding of 125I-labeled rat ANF-(99-126) to the ANF-R1 receptor in human and bovine kidney glomeruli is not blocked by rat ANF-(103-123) (pK less than 6), whereas in rat kidney glomeruli this peptide displays high affinity for the ANF-R1 receptor (pK = 8.5). This observation reveals a species heterogeneity between the rat and the human and bovine kidney receptors. In addition, we have observed striking differences in the pharmacological profiles of rat papillary, bovine papillary, and human kidney glomerular receptors, which contain only the 130-kDa ANF-R1 monomer. The bovine and human profiles were similar but diverged from that of the rat. In addition to the species heterogeneity of the ANF-R1 class, we could detect a significant intraspecies heterogeneity. Two distinct profiles could be disclosed, one having high affinity for both ANF and brain natriuretic peptide (BNP) and being identified in all three species studied and the other displaying lower affinity for BNP and being found in rat and human kidneys. We also demonstrate that rat and bovine papillary ANF-R1 receptors are coupled to guanylate cyclase and that ANF and BNP could activate the enzyme with potency similar to their potency in competing for 125I-labeled rat ANF-(99-126) binding. The results presented demonstrate that the ANF-R1 receptor class can be subclassified, based on distinct pharmacological profiles, and indicate a wide diversity within the ANF-R1 receptor family.

Crystallization of an AMPA receptor binding domain without agonist: importance of carbohydrate content and flash-cooling conditions.

An AMPA-specific ionotropic glutamate receptor binding domain was overexpressed using the baculovirus system and purified by immunoaffinity and metal-affinity chromatography. Purified protein was enzymatically deglycosylated. Both glycosylated and deglycosylated proteins crystallized under the same conditions and in the same space group (P2). In both cases, it was observed that the use of MPD as a cryoprotectant induced a significant reduction in the unit-cell volume compared with glycerol or sucrose. For crystals of deglycosylated protein, cryoprotection with MPD also yielded a dramatic improvement in resolution.

Development of natriuretic peptide analogs selective for the atrial natriuretic factor-R1A receptor subtype.

A pharmacological characterization of subtypes of the atrial natriuretic factor (ANF) receptor ANF-R1, found in bovine adrenal cortex and rat papillary membrane preparations, has been carried out using various chimeric analogs based on rat ANF(99-126) [rANF(99-126)] and porcine brain natriuretic peptide 32 (pBNP32). Receptor binding and cGMP production assays in bovine adrenal cortex indicate that replacement of the amino-terminal segment of pBNP32 with that of rANF(99-126) enhances the affinity of the peptide for the ANF-R1A receptor subtype and its stimulation of associated guanylate cyclase activity. In rat kidney papillae, the substitution of amino- and/or carboxyl-terminal portions of pBNP32 with those of rANF(99-126) also results in a large increase in the affinity and agonistic potency for the ANF-R1A subtype but in only modest changes in those for the ANF-R1B receptor subtype. Interestingly, in this preparation the chimeric analogs could discriminate by their differential affinities and cGMP production potencies between the two receptor subtypes. In particular, pBNP1, obtained by combining the ring structure of pBNP32 with the amino- and carboxyl-terminal portions of rANF(99-126), is the most selective analog. pBNP1 displays higher affinity and agonistic potency for ANF-R1A receptor than for ANF-R1B receptor, with selectivity ratios between these two subtypes of 632- and 504-fold, respectively. Moreover, an excellent correlation is observed between the affinity of the peptides for the ANF-R1A receptor and their stimulation of particulate guanylate cyclase activity in bovine adrenal cortex (r = 0.99, p < 0.01) and rat papillary (r = 0.97, p < 0.01) membrane preparations. In addition, all the chimeric analogs in this study show affinities similar to those of rANF(99-126) and pBNP32 for the ANF-R2 receptor in NIH-3T3 membrane preparations. Importantly, the chimeric analogs pBNP1 and pBNP3, which contain the core of pBNP32 and the amino-terminal segment of rANF(99-126), display higher affinities for the ANF-R1A receptor type than for the ANF-R2 receptor type. These results indicate that the analogs combining the ring structure of pBNP32 with the amino- and/or carboxyl-terminal segments of rANF(99-126) are more selective for the ANF-R1A receptor subtype than are the natural peptides rANF(99-126) and pBNP32.

Distribution and regulation of natriuretic factor-R1C receptor subtypes in mammalian cell lines.

The differential distribution of natriuretic peptide receptor subtypes and their distinct properties were assessed in mammalian cellular models which were screened for their ability to produce cGMP upon stimulation by different natriuretic peptides. The ANF-R1A receptor subtype was distinguished by its selective activation by atrial natriuretic factor (ANF) while the ANF-R1C was characterized by preferential stimulation by C-type natriuretic peptide (CNP). AT-620 pituitary cells, bovine adrenal chromaffin cells, and NIH-3T3 fibroblasts mainly express the ANF-R1C receptor subtype. Other cell lines such as PC12, RASM and GH3 express significant but varying amounts of both ANF-R1A and ANF-R1C subtypes. A10 and NIH cells which express high density of ANF-R2 receptor subtype, also demonstrate a higher sensitivity to CNP over ANF suggesting that they express significant amounts of ANF-R1C. Studies of the regulation by ATP of guanylyl cyclase activity indicate that both ANF-R1A and ANF-R1C subtypes are modulated in the same manner. In the presence of Mn2+, ATP inhibits the CNP-stimulated guanylyl cyclase activity while in the presence of Mg2+ adenine nucleotides potentiate the stimulation by CNP. In addition, we show that like the ANF-R1A, the ANF-R1C guanylyl cyclase activity can be regulated by phosphorylation since preincubation with TPA or FKL attenuates the subsequent stimulation by CNP in cultured cells. The results presented demonstrate that specific cell types express distinct natriuretic peptide receptor subtypes and also that the newly characterized ANF-R1C subtype is regulated by ATP and serine/threonine kinases in the same way as the ANF-R1A subtype.

Distinct properties of atrial natriuretic factor receptor subpopulations in epithelial and fibroblast cell lines.

We have characterized two atrial natriuretic factor (ANF) receptor subtypes, designated ANF-R1 and ANF-R2, in two established cell lines that express exclusively one receptor subtype. The ANF-R1 receptor is selectively expressed by the kidney epithelial cell line LLC-PK1. It is a 130-kDa protein that has a much higher affinity for the biologically active forms of ANF than for its metabolites. The binding of ANF to this subtype is potentiated by amiloride and by divalent cations. The activation of the ANF-R1 receptor leads to an accumulation of cyclic GMP that is only partially inhibited by methylene blue. The ANF-R2 receptor, which is expressed selectively by the fibroblast cell line NIH-3T3, is a 130-kDa protein composed of two disulfide-linked subunits of 64-kDa. Activation of this subtype by saturating concentrations of ANF does not appear to elicit cyclic GMP production. However, supraphysiological concentrations of ANF induce a nonsaturable accumulation of cyclic GMP with an apparent ED50 in the high micromolar range. In contrast to the ANF-R1 subtype, the stimulation of cyclic GMP production is completely abolished by methylene blue. This subtype recognizes the active forms of ANF as well as its metabolites, and the binding is insensitive to amiloride and is decreased by divalent cations. These two cell lines can serve as models for studying the differential regulatory properties of ANF-R1 and ANF-R2 subtypes. In addition, we have also characterized the two ANF receptor subtypes in rat kidney glomeruli, where they show the same structure and pharmacological characteristics as in the two model cell lines.

Design of potent bivalent thrombin inhibitors based on hirudin sequence: incorporation of nonsubstrate-type active site inhibitors.

Hirudin from medicinal leech is the most potent and specific thrombin inhibitor from medicinal leech with a K(i) value of 2.2 x 10(-14) M. It consists of an active site blocking moiety, hirudin1-48, a fibrinogen-recognition exo-site binding moiety, hirudin55-65, and a linker, hirudin49-54, connecting these inhibitor moieties. Synthetic inhibitors were designed based on the C-terminal portion of hirudin. The bulky active site blocking moiety, hirudin1-48, was replaced by small nonsubstrate-type active site inhibitors of thrombin, e.g., dansyl-Arg-(D-pipecolic acid). The linker moiety was replaced by omega-amino acids of (12-aminododecanoic acid)-(4-aminobutyric acid), and hirudin55-65 was used as a fibrinogen-recognition exo-site binding moiety in most of the inhibitors. The crystal structure of the inhibitor in complex with human alpha-thrombin showed that dansyl, Arg, and D-pipecolic acid of the active site blocking moiety occupy S3, S1, and S2 subsites of thrombin, respectively, and were therefore designated as P3, P1, and P2 residues. The use of dansyl-Arg-(D-pipecolic acid) improved the affinity (K(i)) of the inhibitor 10-100-fold (down to 1.70 x 10(-11) M) compared to that of the similar compounds having D-Phe-Pro-Arg as their substrate-type inhibitor moiety (K(i) = 10(-9)-10(-10) M). The linker connected to P2 residue eliminated the scissile peptide bond. The inhibitor was also stable against human plasma proteases. Further inhibitor design revealed that the toxic dansyl group could be replaced by 4-tert-butylbenzenesulfonyl group and 1- or 2-naphthalenesulfonyl group for in vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Nonpolar interactions of thrombin S' subsites with its bivalent inhibitor: methyl scan of the inhibitor linker.

We have designed bivalent thrombin inhibitors, consisting of a nonsubstrate type active site blocking segment, a hirudin-based fibrinogen recognition exosite blocking segment, and a linker connecting these segments. The inhibition provided by the bivalent inhibitors with various linker lengths revealed that a minimum of 15 atoms was required for simultaneous binding of the two blocking segments of the inhibitor to thrombin without significant distortion. The crystal structure of the inhibitors with a 16-atom linker showed some conformational flexibility in the linker portion which still lies deep in the groove joining the active site and the fibrinogen recognition exosite. Since the thrombin S' subsites are not well characterized, we designed a new strategy to search for possible nonpolar interactions between the linker and the thrombin S' subsites. This strategy, the "methyl scan", is based on the incorporation of a methyl side chain at each atom position of the linker by using sarcosine, D,L-alanine, D,L-3-aminoisobutyric acid, or N-methyl-beta-alanine. The methyl groups on the second and the eighth atom positions of the linker, which correspond to the side chains of the P1' and the P3' residues, respectively, improved the affinity of the inhibitors significantly. Further study of the stereospecificity showed that L-Ala at the P1' residue and D-Ala at the P3' residue preferably improved the affinity of the inhibitors 20- and 25-fold, respectively. Molecular modeling calculations using a methyl probe were also carried out to identify favorable nonpolar interacting sites on the thrombin surface. Two sites were identified in the vicinity of the P1' and the P3' residues, supporting the validity of the methyl scan method. Thus, this study has improved our understanding of the interactions taking place in this groove. In particular, we have been able to show that some specific structural features, such as hydrophobic complementarity between the linker and the thrombin S' subsites, could be exploited and make these inhibitors trivalent.

Crystallization and preliminary analysis of chondroitinase AC from Flavobacterium heparinum.

Chondroitinase AC (E.C. 4.2.2.5) overexpressed in its host, Flavobacterium heparinum, was crystallized by vapor diffusion using polyethylene glycol methyl ether as precipitant. It crystallizes in the space group P43212 or its enantiomorph with a = b = 87.1 and c = 193.1 A and one molecule in the asymmetric unit. Crystals diffract to a maximum of 2.5 A resolution on a rotating-anode source. Screening for heavy-atom derivatives identified a lead compound that binds to a single site on the protein. Further screening is in progress.