The N-terminal A domain of Staphylococcus aureus fibronectin-binding protein A binds to tropoelastin

Staphylococcus aureus is an important human pathogen. Its virulence factors include a variety of MSCRAMMs (microbial surface component recognizing adhesive matrix molecules), each capable of binding specifically to the host extracellular matrix. The fibronectin-binding protein, FnBPA, has been shown previously to bind immobilized fibronectin, fibrinogen, and alpha-elastin peptides. Here we show that region A of FnBPA (rAFnBPA) binds to recombinant human tropoelastin. Binding occurs to three separate truncates of tropoelastin, encompassing domains 2-18, 17-27, and 27-36, signifying that the interaction occurs at multiple sites. The greatest affinity was for the N-terminal truncate. We observed a pH dependency for the rAFnBPA-tropoelastin interaction with strong, nonsaturable binding at low pH. The interaction ceased at higher pH. These data support a model of surface-surface interactions between the negative charges present on rAFnBPA and the positive lysines of tropoelastin. A protein lacking the negatively charged C-terminal fibronectin-binding motif of the A domain of FnBPA and another construct lacking subdomain N1 were both capable of binding immobilized tropoelastin with a lower affinity. The binding properties of five site-directed mutants of rAFnBPA were compared with wild-type rAFnBPA. There was no decreased affinity for immobilized tropoelastin, in contrast to the defective binding of these mutants to alpha-elastin and fibrinogen. The data indicate novel interactions between tropoelastin and FnBPA that include the use of surface charges. These results demonstrate that FnBPA is capable of directly binding tropoelastin prior to its incorporation into elastin.     

Recombinant 70-kDa protein from the amino-terminal region of rat fibronectin inhibits binding of fibronectin to cells and bacteria

Binding of fibronectin to substrate-attached cells and to Staphylococcus aureus is mediated by the amino-terminal 70-kDa portion of fibronectin. The 70-kDa amino-terminus is composed of nine type I and two type II internal homology units, each containing two intrachain disulfide bonds. The exact structural features of the 70-kDa amino-terminus that are necessary for binding to cells and bacteria are not known. We characterized a recombinant 70-kDa protein from the amino-terminus of rat fibronectin using a baculovirus expression system. Recombinant 70-kDa (r70kDa) protein was easily purified in high amounts from the conditioned medium by affinity chromatography on gelatin-agarose. Secretion was much less when N-linked glycosylation was blocked by tunicamycin. Like the native fragment, the r70kDa protein contains intrachain disulfide bonds. In addition, the r70kDa protein was indistinguishable from the nonrecombinant 70-kDa fragment in its ability to compete for binding sites on fibroblasts and S. aureus. Thus, the r70kDa protein retains the important functional characteristics of the native fragment. This expression system is well adapted to studying the structural features important for the interaction of 70-kDa protein with cells.     

The N-terminal A domain of fibronectin-binding proteins A and B promotes adhesion of Staphylococcus aureus to elastin

The ability of Staphylococcus aureus to adhere to components of the extracellular matrix is an important mechanism for colonization of host tissues during infection. We have previously shown that S. aureus binds elastin, a major component of the extracellular matrix. The integral membrane protein, elastin-binding protein (EbpS), binds soluble elastin peptides and tropoelastin via its surface-exposed N-terminal domain. In this study, we demonstrate that some strains of S. aureus adhere strongly to immobilized human elastin and that this interaction is independent of EbpS but instead is mediated by the fibronectin-binding proteins, FnBPA and FnBPB. Our results show that EbpS mutant cells adhere to elastin-coated plates, whereas the cells negative for FnBPA and FnBPB do not adhere to the plates. Furthermore, only wild-type cells from the exponential phase of growth adhered when FnBPs were expressed maximally. We show that adherence to elastin promoted by FnBPA was not affected by soluble fibronectin, suggesting that the elastin binding domain is distinct from the fibronectin binding regions. Recombinant FnBPA(37-544) (rFnBPA(37-544)) protein corresponding to the A region of FnBPA and anti-FnBPA(37-544) antibodies inhibited FnBPA-mediated bacterial adherence to immobilized elastin. Finally, recombinant A domain proteins, rFnBPA(37-544) and rFnBPB(37-540), bound immobilized elastin dose-dependently and saturably. This interaction was inhibited by soluble elastin peptides, suggesting a specific receptor-ligand interaction.     

Recycling of the 67-kDa Elastin Binding Protein in Arterial Myocytes Is Imperative for Secretion of Tropoelastin

We have shown previously that the 67-kDa elastin binding protein (EBP) colocalizes intracellularly and extracellularly with tropoelastin in fetal sheep aorta, suggesting that these two proteins associate along the secretory pathway. Moreover, we have established that association with EBP protects tropoelastin from serine proteinases and from intracellular coacervation, and is necessary for its proper extracellular assembly. Since the production of tropoelastin by aortic smooth muscle cells (Ao SMC) exceeds production of the EBP, we speculated that this binding protein might recycle back into the cell, associating again with newly synthesized tropoelastin. In this report we labeled cultured Ao SMC externally with the F(ab′)₂ fragments of immunoglobulin which recognizes sheep EBP and followed trafficking of EBP by immunofluorescence and electron microscopy. Our results indicate that the majority of the EBP residing on the cell surface can be internalized to endocytic compartments (but not to lysosomes) and recycled back to the plasma membrane within 45-60 min. We have also determined that reagents disturbing pH of distinct endocytic compartments (chloroquine and bafilomycin A₁, but not ammonium chloride) arrest recycling of the EBP and, at the same time, strongly inhibit deposition of insoluble elastin in cultures of sheep Ao SMC and in organ cultures of chicken aorta. In contrast, neither chloroquine nor bafilomycin A₁ inhibit total protein synthesis or synthesis of tropoelastin. Our results suggest that the EBP serves as a reusable shuttle protein for tropoelastin and that its recycling is essential for effective deposition of insoluble elastin.     

Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.     

Identification of enolase as a laminin-binding protein on the surface of Staphylococcus aureus

We have previously demonstrated that Staphylococcus aureus, a highly invasive bacteria, presents a 52-kDa surface protein that mediates its binding to laminin. In order to better characterize this receptor, we excised this putative laminin receptor from two-dimensional (2-D) PAGE and used it as antigen for raising a mouse hyperimmune serum which was for screening an S. aureus expression library. A single clone of 0.3 kb was obtained, and its sequence revealed 100% homology with S. aureus alpha-enolase. Moreover, amino acid sequencing of the 52-kDa protein eluted from the 2-D gel indicated its molecular homology with alpha-enolase, an enzyme that presents a high evolutionary conservation among species. In parallel, monoclonal antibodies raised against the S. aureus 52-kDa band also recognized yeast alpha-enolase in western blot analysis. These monoclonal antibodies were also able to promote capture of iodine-labeled bacteria when adsorbed to a solid phase, and this capture was inhibited by the addition of excess rabbit muscle alpha-enolase. Finally, the cell surface localization of S. aureus alpha-enolase was further confirmed by flow cytometry. Hence, alpha-enolase might play a critical role in the pathogenesis of S. aureus by allowing its adherence to laminin-containing extracellular matrix.     

Isolation of an amino-terminal fibronectin-binding protein on human U937 cells and rat peritoneal macrophages.

Several cell-mediated activities for the amino terminus of fibronectin have been documented. In the present study we describe a macrophage surface protein with binding activity directed to the amino terminus of the fibronectin molecule. The binding of a 29-kDa amino-terminal fibronectin fragment to macrophages reached steady state by 30 min and was half-maximal at approximately 2 x 10(-8) M. This binding was specifically inhibited by excess unlabeled 29-kDa fragment or intact fibronectin but not by a 180-kDa fibronectin fragment which lacks the amino terminus. Competitive binding studies of the 70-kDa amino-terminal fibronectin fragment to macrophages revealed a single binding site with KD = 7.14 x 10(-8) M and approximately 8 x 10(4) binding sites/cell. Radiolabeled surface proteins extracted from rat peritoneal macrophages and from the human U937 cell line were applied to an affinity column comprised of the 70-kDa amino-terminal fragment of fibronectin coupled to a solid support. A single trypsin-sensitive radiolabeled protein of 67 kDa, from either cell type, was eluted from this column with urea. This protein showed no immunologic identity with fibronectin, fibrin(ogen), or albumin. The 67-kDa protein exhibited identical apparent molecular weight under reducing and nonreducing conditions, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. We have localized the fibronectin binding activity of this protein to within the 29-kDa amino-terminal domain of fibronectin. The 67-kDa protein eluted from the 70-kDa column failed to bind to a column comprised of the 45-kDa gelatin-binding fragment of fibronectin. Additionally, the 67-kDa protein was specifically eluted from the 70-kDa column by the 29-kDa amino-terminal fragment but not by the 45-kDa gelatin-binding fragment. These data suggest that this 67-kDa protein is a macrophage cell surface binding protein for the amino terminus of fibronectin.     

Microfibril-associated glycoprotein-1 binding to tropoelastin: multiple binding sites and the role of divalent cations.

Microfibrils and elastin are major constituents of elastic fibers, the assembly of which is dictated by multimolecular interactions. Microfibril-associated glycoprotein-1 (MAGP-1) is a microfibrillar component that interacts with the soluble elastin precursor, tropoelastin. We describe here the adaptation of a solid-phase binding assay that defines the effect of divalent cations on the interactions between MAGP-1 and tropoelastin. Using this assay, a strong calcium-dependent interaction was demonstrated, with a dissociation constant of 2.8 +/- 0.3 nm, which fits a single-site binding model. Manganese and magnesium bestowed a weaker association, and copper did not facilitate the protein interactions. Three constructs spanning tropoelastin were used to quantify their relative contributions to calcium-dependent MAGP-1 binding. Binding to a construct spanning a region from the N-terminus to domain 18 followed a single-site binding model with a dissociation constant of 12.0 +/- 2.2 nm, which contrasted with the complex binding behavior observed for fragments spanning domains 17-27 and domain 27 to the C-terminus. To further elucidate binding sites around the kallikrein cleavage site of domains 25/26, MAGP-1 was presented with constructs containing C-terminal deletions within the region. Construct M1659, which spans a region from the N-terminus of tropoelastin to domain 26, inclusive, bound MAGP-1 with a dissociation constant of 9.7 +/- 2.0 nm, which decreased to 4.9 +/- 1.0 nm following the removal of domain 26 (M155n), thus displaying only half the total capacity to bind MAGP-1. These results demonstrate that MAGP-1 is capable of cumulative binding to distinct regions on tropoelastin, with different apparent dissociation constants and different amounts of bound protein.     

Elastin binds to a multifunctional 67-kilodalton peripheral membrane protein

Elastin binding proteins from plasma membranes of elastin-producing cells were isolated by affinity chromatography on immobilized elastin peptides. Three proteins of 67, 61, and 55 kDa were released from the elastin resin by guanidine/detergent, soluble elastin peptides, synthetic peptide VGVAPG, or galactoside sugars, but not by synthetic RGD-containing peptide or sugars not related to galactose. All three proteins incorporated radiolabel upon extracellular iodination and contained [3H]leucine following metabolic labeling, confirming that each is a synthetic product of the cell. The 67-kDa protein could be released from the cell surface with lactose-containing buffers, whereas solubilization of the 61- and 55-kDa components required the presence of detergent. Although all three proteins were retained on elastin affinity columns, the 61- and 55-kDa components were retained only in the presence of 67-kDa protein, suggesting that the 67-kDa protein binds elastin and the 61- and 55-kDa proteins bind to the 67-kDa protein. We propose that the 67-, 61-, and 55-kDa proteins constitute an elastin-receptor complex that forms a transmembrane link between the extracellular matrix and the intracellular compartment.     

Changes in elastin composition in aorta of spontaneously hypertensive rats (SHR)

Tropoelastin and elastin preparations obtained from aortae of spontaneously hypertensive rats (SHR) show an increased proportion of polar amino acids (aspartic acid, glutamic acid, arginine and tyrosine). The content of these amino acids is 1.43-3.04 times higher in SHR rats than in similar elastin or tropoelastin preparations obtained from normotensive animals. On the other hand elastin and tropoelastin preparations obtained from SHR rats show a lower frequency of the Val-Pro sequence; this was found to be 35.93 per 1000 amino acid residues in SHR rats as compared to 51.04 per 1000 amino acids in the preparations obtained from control animals. Since similar differences were found not only in elastin preparations but also in tropoelastin, contamination of these preparations with an acidic protein seems unlikely. In general the results obtained are similar to those seen in animals kept on a long term high fat diet. It appears feasible to suggest that these differences are caused by a changed proportion of two different elastin type.     

Processing of soluble elastin in cultured neonatal rat smooth muscle cells

The synthesis and extracellular deposition of elastin by cultured neonatal rat aorta smooth muscle cells has been followed. The addition of beta-aminopropionitrile to the culture medium promotes accumulation of soluble precursors of elastin. Under such conditions, a protein possessing characteristics of a soluble elastin precursor with an apparent molecular weight of 77,000 was detected and partially purified. Pulse-chase studies suggested that this 77-kDa protein undergoes an extracellular, enzymatically catalyzed process to a 71-kDa protein. This 71-kDa protein is strikingly similar to tropoelastins isolated from other tissue systems, in which no evidence for higher molecular weight soluble precursors is at present available. Data presented in this communication suggest that the 77-kDa protein, which we have designated protropoelastin, represents a precursor to the tropoelastin moiety produced in the neonatal rat smooth muscle cell culture.     

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.     

Purification of homogeneity and amino acid sequence analysis of a receptor protein for interleukin 1

The interleukin 1 (IL-1) receptor from mouse EL-4 thymoma cells was purified to homogeneity by a method which utilized ligand affinity chromatography and classical chromatographic techniques. After solubilization of the receptor from intact cells with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the IL-1 binding activity was purified greater than 23,000-fold. Analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ligand blot demonstrated that a single protein of molecular mass of approximately 80 kDa is the IL-1 binding polypeptide. The purified protein bound IL-1 with a dissociation constant of approximately 1.1 X 10(-10) M, which is indistinguishable from the affinity of the cell-bound receptor. The amino acid composition of this protein is strikingly similar to the composition deduced from the sequence of a cDNA coding for an IL-1 receptor from EL-4 cells. Protein sequence analysis of Staphylococcus aureus V-8 protease-derived peptides yields data consistent with the sequence proposed from cloned cDNA. These studies have demonstrated that the high affinity IL-1 receptor on EL-4 cells is the 80-kDa protein.     

Isolation and characterization of a 14-kDa albumin-binding fragment of streptococcal protein G

Protein G, a streptococcal cell wall protein, has separate binding sites for human albumin and IgG. Streptococci expressing protein G were treated with the bacteriolytic agent mutanolysin. Several IgG- and human serum albumin (HSA)-binding peptides were identified in the material thus solubilized and one of these, a 14-kDa peptide, was found to bind HSA but not IgG in Western blot experiments. This molecule was purified by affinity chromatography on Sepharose coupled with HSA followed by gel filtration on Sepharose 6B and a final affinity chromatography on IgG-Sepharose, by which low Mr W(15 to 20 kDa)IgG-binding peptides were removed. In different binding experiments the purified 14-kDa peptide bound exclusively HSA and the equilibrium constant between the peptide and HSA was determined to be 3.4 X 10(8) M-1. The relation between the 14-kDa molecule and protein G was studied by analyzing the N-terminal amino acid sequence of the peptide and comparing it with the previously determined protein G sequence. The 40 N-terminal amino acids were found to be identical with an amino acid sequence starting at position 62 in the protein G molecule. These and previous data enabled us to locate the albumin binding to the repetitively arranged domains in the N-terminal half of the protein G molecule.     

In vitro degradation of human tropoelastin by MMP-12 and the generation of matrikines from domain 24

Degradation of elastic fibers in tissues can result in the development of disorders that include aneurysms, atherosclerosis, and loss of skin elasticity. Tropoelastin is the precursor of the cross-linked elastin and its expression is triggered by elastin-degrading factors as a response to damage. Factors like UV radiation not only increase the expression of tropoelastin but also potent metalloelastases such as macrophage elastase (MMP-12). The development of elastin-degrading diseases, moreover, is a chronic process during which elastin and tropoelastin are repeatedly exposed to attacks by MMP-12. Hence, in this work we report the in vitro susceptibility of tropoelastin and the potential of MMP-12 to generate matrikines. This work provides evidence that tropoelastin is substantially and rapidly degraded by MMP-12 even at very dilute enzyme concentrations. MMP-12 cleaves at least 86 sites in tropoelastin. Analysis of the generated peptides revealed that some small peptides contained the motif GXXPG that may enable them to bind with the elastin binding protein (EBP). Furthermore, using synthesized peptides it was confirmed that several sites in the sequence encoded by exon 24 which contains repetitive units of biologically active VGVAPG domains are susceptible to attack by MMP-12, provided that the active subsites in MMP-12 (S₄ to S₄′) are occupied. Such cleavage events have lead to the generation of ligands that may bind to EBP.     

The elastin receptor shows structural and functional similarities to the 67-kDa tumor cell laminin receptor

Laminin- and elastin-binding proteins were isolated by ligand affinity chromatography from plasma membranes of fetal bovine auricular chondroblasts and human A2058 melanoma cells. From both cell types, a 67-kDa protein was identified which bound to either elastin or laminin affinity resins. Structural and functional similarities between the elastin and laminin-binding proteins were suggested by 1) cross-reactivity between antibodies directed against the two proteins; 2) elution of the laminin receptor from laminin columns with soluble elastin peptides; and 3) modulation of substrate binding by galactoside sugars. In addition, extraction properties indicate that both receptors are peripheral membrane proteins whose association with the cell surface is mediated by their lectin properties. Mapping of the binding site on laminin suggests that the 67-kDa chondroblast receptor interacts with a hydrophobic elastin-like sequence in domain V of the B1 chain, and chemotaxis studies indicate that cell migration to elastin peptides and laminin involves the same receptor.     

Cloning and functional characterization of an NAD(+)-dependent DNA ligase from Staphylococcus aureus

A Staphylococcus aureus mutant conditionally defective in DNA ligase was identified by isolation of complementing plasmid clones that encode the S. aureus ligA gene. Orthologues of the putative S. aureus NAD(+)-dependent DNA ligase could be identified in the genomes of Bacillus stearothermophilus and other gram-positive bacteria and confirmed the presence of four conserved amino acid motifs, including motif I, KXDG with lysine 112, which is believed to be the proposed site of adenylation. DNA sequence comparison of the ligA genes from wild type and temperature-sensitive S. aureus strain NT64 identified a single base alteration that is predicted to result in the amino acid substitution E46G. The S. aureus ligA gene was cloned and overexpressed in Escherichia coli, and the enzyme was purified to near homogeneity. NAD(+)-dependent DNA ligase activity was demonstrated with the purified enzyme by measuring ligation of (32)P-labeled 30-mer and 29-mer oligonucleotides annealed to a complementary strand of DNA. Limited proteolysis of purified S. aureus DNA ligase by thermolysin produced products with apparent molecular masses of 40, 22, and 21 kDa. The fragments were purified and characterized by N-terminal sequencing and mass analysis. The N-terminal fragment (40 kDa) was found to be fully adenylated. A fragment from residues 1 to 315 was expressed as a His-tagged fusion in E. coli and purified for functional analysis. Following deadenylation with nicotinamide mononucleotide, the purified fragment could self-adenylate but lacked detectable DNA binding activity. The 21- and 22-kDa C-terminal fragments, which lacked the last 76 amino acids of the DNA ligase, had no adenylation activity or DNA binding activity. The intact 30-kDa C terminus of the S. aureus LigA protein expressed in E. coli did demonstrate DNA binding activity. These observations suggest that, as in the case with the NAD(+)-dependent DNA ligase from B. stearothermophilus, two independent functional domains exist in S. aureus DNA ligase, consisting of separate adenylation and DNA binding activities. They also demonstrate a role for the extreme C terminus of the ligase in DNA binding. As there is much evidence to suggest that DNA ligase is essential for bacterial survival, its discovery in the important human pathogen S. aureus indicates its potential as a broad-spectrum antibacterial target for the identification of novel antibiotics.     

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.     

Steroid binding activity is retained in a 16-kDa fragment of the steroid binding domain of rat glucocorticoid receptors

The steroid binding domain of the rat glucocorticoid receptor is considered as extending from amino acids 550 to 795. However, such a synthetic protein (i.e. amino acids 547-795; Mr approximately 31,000) has been reported to show very little affinity for the potent synthetic glucocorticoid dexamethasone. We now disclose that digestion of steroid-free rat glucocorticoid receptors with low concentrations of trypsin yields a single species, of Mr = 16,000, that is specifically labeled by dexamethasone 21-mesylate. This 16-kDa fragment retains high affinity binding for [3H]dexamethasone that is only approximately 23-fold lower than that seen with the intact 98-kDa receptor. Analysis of the protease digestion patterns obtained both with trypsin and with lysylendopeptidase C allowed us to deduce the proteolytic cleavage maps of the receptor with these enzymes. From these protease maps, the sequence of the 16-kDa fragment was identified as being threonine 537 to arginine 673. These results show that glucocorticoid receptor fragments smaller than 34 kDa do bind steroids and that the amino acids Thr537-Arg673 constitute a core sequence for ligand binding within the larger steroid binding domain. The much slower kinetics in generating the 16-kDa fragment from affinity-labeled receptors suggests that steroid binding causes a conformation change in the receptor near the cleavage sites.