Alpha 2-macroglobulin bait region variants. A role for the bait region in tetramer formation

To test the hypothesis that a large portion of the bait region of human alpha 2-macroglobulin (alpha 2M) can be removed without adversely affecting the protein's structural and functional properties, we expressed two human alpha 2M variants with truncated bait regions and examined whether these variants folded normally and functioned as proteinase inhibitors. Each variant contains sites that are normal bait region cleavage sites in wild-type alpha 2M, including the primary trypsin cleavage site. The truncated bait regions are shorter by 23 and 27 residues, respectively, and lack the C-terminal portion as well as different parts of the N-terminal section of the bait region. We found that such bait region truncation permitted normal folding of the monomers as well as formation of the thiol ester and dimerization by disulfide cross-linking, although the resulting species bound 6-(p-toluidino)-2-naphthalenesulfonic acid in a manner more like thiol ester-cleaved alpha 2M than native alpha 2M. The variants' thiol esters reacted with nucleophiles at rates identical to wild-type alpha 2M. Surprisingly, however, the truncations prevented the noncovalent association of the covalent 360-kDa dimers that normally gives tetrameric alpha 2M, decoupled bait region cleavage from thiol ester activation, and resulted in the inability of the two variants to "trap" proteinase. This was despite apparent cleavage of the bait region by proteinase, albeit at very much reduced rates relative to wild-type tetrameric alpha 2M. The kinetics of thiol ester cleavage-dependent protein conformational changes also changed from sigmoidal to exponential. These findings indicate that residues in the bait region appear to be necessary for noncovalent association of 360-kDa disulfide-linked dimers to give tetrameric alpha 2M and suggest a role for the bait region in normal alpha 2M in coupling bait region cleavage to the sequence of conformational changes that result in thiol ester activation and ultimately proteinase trapping.     

Immunolocalization of transforming growth factor-beta 1 and transforming growth factor-beta 2 in the mouse ovary during gonadotrophin-induced follicular maturation

An immunohistochemical approach was utilized to evaluate the cellular distribution of transforming growth factor-beta 1 (TGF beta 1) and transforming growth factor beta 2 (TGF beta 2) at different stages of follicle development in the prepubertal mouse ovary under the following conditions: (i) after pregnant mare's serum gonadotrophin (PMSG) treatment; (ii) after PMSG and human chorionic gonadotrophin (HCG) treatment; (iii) after PMSG and HCG treatment plus mating. In the immature ovary, TGF beta 1 and TGF beta 2 immunoreactivities are localized in theca and granulosa cells and in oocytes. After PMSG treatment, TGF beta 1 and TGF beta 2 immunoreactivities are localized in granulosa cells; in addition, TGF beta 2 staining is noted in the matrix surrounding antral cells. Staining for both TGR beta 1 and TGF beta 2 drops in the theca but persists in the oocyte. PMSG plus HCG treatment results in a significant increase in TGF beta 1 and TGF beta 2 immunoreactivity in the theca and in the maintenance of TGF beta 1 staining in both basal granulosa cells and cumulus cells whereas TGF beta 2 immunoreactivity is essentially localized in the matrix surrounding cumulus cells. Staining for TGF beta 1 and TGF beta 2 persists in the oocyte. Following PMSG plus HCG treatment and mating, TGF beta 1 immunoreactivity is localized in the luteal cells of corpora lutea and TGF beta 2 shows a similar localization pattern. This study provides evidence that TGF beta 1 and TGF beta 2 peptides are expressed in specific cell types during induced follicular maturation in the mouse ovary.     

Sp1 is required for the early response of alpha2(I) collagen to transforming growth factor-beta1

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor beta1 (TGF-beta) stimulation of the human alpha2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-beta and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of alpha2(I) collagen mRNA accumulation by TGF-beta, whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-beta-treated cells. AP1 did bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, we conclude that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1, or the Sp1-related repressor, Sp3.     

The cutaneous microfibrillar apparatus contains latent transforming growth factor-beta binding protein-1 (LTBP-1) and is a repository for latent TGF-beta1

A new 3D HCCH-COSY-TOCSY experiment is presented for the assignment of RNA sugar and protein side chains. The experiment, which combines COSY and TOCSY units, is more powerful than the sum of individual HCCH-COSY and HCCH-TOCSY pulse sequences. The experiment was applied to a 13C, 15N-labeled 26 mer RNA complexed with the antibiotic tobramycin, and a 12 kDa 13C, 15N-labeled FKBP12 protein sample. The power of HCCH-COSY-TOCSY is demonstrated through complete spin system assignments of sugars in the 26 mer RNA sample, which could not be assigned using a combination of HCCH-COSY, HCCH-TOCSY and 13C-edited NOESY experiments.     

Binding of the Epstein-Barr virus to human platelets causes the release of transforming growth factor-beta

Human platelets bear on their surface complement receptor type II (CR2), which is also the receptor for the EBV. Although the cross-linking of these receptors causes activation and aggregation of platelets, no immunologic consequence of the potential binding of EBV to these receptors on human platelets has ever been described. We report here that binding of EBV to human platelets causes the release of TGF-beta from the latter. Both infectious and UV-inactivated noninfectious viral particles can mediate this release. Anti-CR2 mAb OKB7, which blocks the binding of EBV to CR2, also blocks the EBV-mediated release of TGF-beta. Furthermore, platelets recovered from the initial incubation no longer release TGF-beta upon subsequent incubation with EBV. Since TGF-beta is a potent immunosuppressive agent, its release from platelets upon binding of EBV may play a role in the pathogenesis of EBV-associated diseases.     

Homodimers of the 60 kDa phosphatidylinositol-anchored transforming growth factor-beta 2 binding proteins in FBHEC and MG-63 cells

The recent finding that the clinical nitrovasodilator, glyceryl trinitrate (GTN), is mutagenic in Salmonella typhimurium strain TA1535 has been examined in closer detail, with emphasis on its mechanism of action. GTN increased the number of His+ revertants to a maximum of 4 times over background at a GTN dose of 5 mumol/plate. Hamster liver S9 depressed the toxicity of high GTN doses and increased the maximum number of revertants to 5 times over background at 10 mumol/plate. GTN did not cause significant reversion in any of the six other S. typhimurium strains tested (TA1975, TA102, TA1538, TA100, TA100NR, YG1026), although signs of toxicity were observed. Therefore, the mutagenicity of GTN was manifest only in the repair-deficient (uvrB and lacking in pKM101) strain which is responsive to single base changes. Oligonucleotide probe hybridization of TA1535 revertants showed that virtually all of the GTN-induced mutants contained C-->T transitions in either the first or second base of the hisG46 (CCC) target codon, with a preference for the latter. A similar mutational spectrum was seen previously with a complex of spermine and nitric oxide (NO) which releases nitric oxide. This suggests that NO, which can be derived from GTN via metabolic reduction, may be responsible for GTN's mutagenic action. The known NO scavenger oxymyoglobin did not substantially alter the dose response of GTN, indicating that extracellular NO was not mediating reversion. The data are consistent with the hypothesis that intracellular nitric oxide is responsible for the observed mutations.     

Expression of transforming growth factor-beta 1 messenger ribonucleic acid and the modulation of deoxyribonucleic acid synthesis by transforming growth factor-beta 1 in human endometrial cells

OBJECTIVE: The purpose of this study was (1) to evaluate the potential sites of transforming growth factor-beta 1 synthesis in human endometrium by analyzing separated endometrial glands and stromal cells for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis of total ribonucleic acid and (2) to investigate the effects of transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelial and stromal cells in culture. STUDY DESIGN: Endometrial glands and stroma from proliferative and secretory endometrium were isolated after collagenase treatment of endometrial tissue minces and were analyzed for transforming growth factor-beta 1 messenger ribonucleic acid by Northern analysis. We studied the effects of estradiol-17 beta and transforming growth factor-beta 1 on deoxyribonucleic acid synthesis in endometrial epithelium and transforming growth factor-beta 1 on stromal cells in culture by evaluating tritiated thymidine incorporation into trichloroacetic acid-precipitable material. RESULTS: Transforming growth factor-beta 1 messenger ribonucleic acid was detected for Northern analysis in separated endometrial stromal cells in levels that were greatest during the secretory phase and in greater levels than in epithelial cells from that same tissue. Transforming growth factor-beta 1 messenger ribonucleic acid in glandular epithelium in culture was not increased to detectable levels by treatment with transforming growth factor-beta 1. Deoxyribonucleic acid synthesis in endometrial glandular epithelium was inhibited by transforming growth factor-beta 1, but transforming growth factor-beta 1 stimulated deoxyribonucleic acid synthesis in endometrial stromal cells in culture. After treatment for 5 days with estradiol-17 beta (10(-8) mol/L), deoxyribonucleic acid synthesis in endometrial glands in culture was decreased by 40%. Transforming growth factor-beta 1 (1 ng/ml) did not alter this effect of estradiol-17 beta on deoxyribonucleic acid synthesis. CONCLUSIONS: Transforming growth factor-beta 1 acts to decrease deoxyribonucleic acid synthesis in epithelial cells and to increase it in stromal cells isolated from human endometrium and maintained in monolayer culture. Transforming growth factor-beta 1, potentially of stromal cell origin, could participate in the regulation of endometrial cell proliferation and differentiation in vivo.     

The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain

The motor protein kinesin is a heterotetramer composed of two heavy chains of approximately 120 kDa and two light chains of approximately 65 kDa protein. Kinesin motor activity is dependent on the presence of ATP and microtubules. The kinesin light chain-binding site in human kinesin heavy chain was determined by reconstituting in vitro a complex of recombinant heavy and light chains. The proteins expressed in bacteria included oligohistidine-tagged fragments of human ubiquitous kinesin heavy chain, spanning most of the stalk and all of the tail domain (amino acids 555-963); and untagged, essentially full-length human kinesin light chain (4-569) along with N-terminal (4-363) and C-terminal (364-569) light chain fragments. Heavy chain fragments were attached to Ni2+-charged beads and incubated with untagged light chain fragments. Analysis of eluted complexes by SDS-PAGE and immunoblotting mapped the light chain-binding site in heavy chain to amino acids 771-813, a region close to the C-terminal end of the heavy chain stalk domain. In addition, only the full-length and N-terminal kinesin light chain fragments bound to this heavy chain region. Within this heavy chain region are four highly conserved contiguous heptad repeats (775-802) which are predicted to form a tight alpha-helical coiled-coil interaction with the heptad repeat-containing N-terminus of the light chain, in particular region 106-152 of human light chain. This predicted hydrophobic, alpha-helical coiled-coil interaction is supported by both circular dichroism spectroscopy of the recombinant kinesin heavy chain fragment 771-963, which displays an alpha-helical content of 70%, and the resistance of the heavy/light chain interaction to high salt (0.5 M).     

Murine alpha-macroglobulins demonstrate divergent activities as neutralizers of transforming growth factor-beta and as inducers of nitric oxide synthesis. A possible mechanism for the endotoxin insensitivity of the alpha2-macroglobulin gene knock-out mouse

alpha2-Macroglobulin null mice demonstrate increased resistance to endotoxin challenge (Umans, L., Serneels, L., Overbergh, L., Van Leuven, F., and Van den Berghe, H. (1995) J. Biol. Chem. 270, 19778-19785). We hypothesized that this phenotype might reflect the function of murine alpha2M (malpha2M) as a neutralizer of transforming growth factor-beta (TGF-beta) and inducer of nitric oxide synthesis in vivo. When incubated with wild-type mouse plasma, TGF-beta1 and TGF-beta2 bound only to malpha2M. Alternative TGF-beta-binding proteins were not detected in plasma from alpha2M(-/-) mice. Wild-type mouse plasma, but not plasma from alpha2M(-/-) mice, inhibited TGF-beta1 binding to TGF-beta receptors on fibroblasts. Purified malpha2M bound TGF-beta1 and TGF-beta2 with similar affinity; the KD values were 28 +/- 4 and 33 +/- 4 nM, respectively. Murinoglobulin, the second murine alpha-macroglobulin, bound both TGF-beta isoforms with 30-fold lower affinity. Malpha2M counteracted the activities of TGF-beta1 and TGF-beta2 in an endothelial cell growth assay. Malpha2M also induced NO synthesis when incubated with RAW 264.7 cells, an activity which probably results from the neutralization of autocrine TGF-beta activity. Human alpha2M induced NO synthesis comparably to malpha2M; however, MUG had no effect. These studies demonstrate that the ability to neutralize TGF-beta is a property of malpha2M, which is not redundant in the murine alpha-macroglobulin family or in murine plasma. Malpha2M is the only murine alpha-macroglobulin that promotes NO synthesis. The absence of malpha2M, in alpha2M(-/-) mice, may allow TGF-beta to more efficiently suppress excessive iNOS expression following endotoxin challenge.     

Localization of a single binding site for immunoglobulin light chains on human Tamm-Horsfall glycoprotein

Cast nephropathy is a severe complication of multiple myeloma. Binding of filtered monoclonal light chains (LC) with Tamm-Horsfall glycoprotein (THP) triggers heterotypic aggregation of these two proteins to form casts in the distal nephron of the kidney. To localize the LC binding site on THP, human THP was deglycosylated and underwent limited trypsin digestion in the presence or absence of a nephrotoxic LC known to bind THP. A 29.6-kD band was protected from trypsin digestion by the addition of LC. NH2-terminal amino acid sequence and amino acid analyses revealed this band was located between the 6th and 287th amino acid residues of THP. Six peptides located within this 29.6-kD fragment were synthesized and used as potential inhibitors of binding or aggregation of five different nephrotoxic LCs with THP. Peptide AHWSGHCCL (from amino acid 225 to 233) completely inhibited binding and aggregation of these proteins. Optimal inhibition required a cystine residue in this peptide. Truncation experiments demonstrated the entire sequence was necessary for ideal inhibition and the histidine residue explained the effects of pH on binding. These studies provided a basis for further study of LC-THP interaction and a potential approach toward the prevention of cast nephropathy.     

Bait region involvement in the dimer-dimer interface of human alpha 2-macroglobulin and in mediating gross conformational change. Evidence from cysteine variants that form interdimer disulfides

We have characterized four human alpha 2-macroglobulin (alpha 2M) bait region variants (G679C, M690C, V700C, and T705C) to test the hypothesis that the bait regions are involved in the interface between noncovalently associated dimers. All four variants folded correctly as judged by many normal properties. However, the presence of a cysteine resulted in disulfide formation between otherwise noncovalently associated dimers in all four variants. The extent of disulfide cross-linking varied with the location of the cysteine and gave a mixture of species that probably contained two, one, or zero interdimer disulfides in the tetramer. This was reflected in heterogeneity of conformational change upon thiol ester cleavage by methylamine, with the presence of crosslinks correlating with blockage of conformational change. The stoichiometry of trypsin inhibition was less in all cases than for wild-type alpha 2M. The M690C variant also showed evidence of some species with an intramolecular disulfide between bait regions of monomers within the same dimer. Taken together, the results are consistent with a location of the four bait regions in contact with, or in very close proximity to, one another. This suggests that they form all or part of the "cavity body" seen in the low resolution x-ray structure of transformed alpha 2M.     

Guanine nucleotide binding proteins mediate the chemotactic signal of transforming growth factor-beta 1 in rat IL-2 activated natural killer cells

Transforming growth factor (TGF)-beta 1 induced rat IL-2-activated natural killer (IANK) cell chemotaxis. Various doses of cholera toxin (CT) or pertussis toxin (PT) inhibited the activity of TGF-beta 1 suggesting a role for guanine nucleotide binding (G) proteins. ADP-ribosylation assay showed that rat IANK cell membranes possess a 39 kDa PT substrate and two, 41 and 42 kDa, CT substrates. ADP-ribosylation also showed that incubating IANK cell membranes with TGF-beta 1 in the presence of guanosine 5'-O-(3-thiotriphosphate) resulted in the disappearance of the PT substrate. Immunoblot analysis showed that rat IANK cell membranes possess one Gi (39 kDa), one G0 (39 kDa) and three Gs (40, 41, and 42 kDa) proteins. Pretreatment of IANK cell membranes with TGF-beta 1 in the presence of guanosine-5'-O-(3-thiotriphosphate) reduced the intensity of the 39 kDa G(0) and the 40 kDa Gs but not the 39 kDa Gi or the 41 kDa or 42 kDa Gs. Furthermore, TGF-beta 1 stimulated GTP binding and increased GTPase activity in IANK cell membranes. Both activities were inhibited by PT or CT. This inhibition was associated with the modification of G proteins by the toxins suggesting that bacterial toxin substrates are linked to TGF-beta 1 receptors. Our results suggest that G0 and Gs are involved in mediating the chemotactic signal of TGF-beta 1 in rat IANK cells.     

Cloning of a cDNA encoding the human transforming growth factor-beta type II receptor: heterogeneity of the mRNA

A combined study of anterograde axonal degeneration and Golgi electron microscopic technique was designed to examine the distribution and density of axon terminals from the mediodorsal thalamic nucleus (MD) over layer III pyramidal cells in the prelimbic cortex of the rat. The reconstructive analysis of serial ultrathin sections of gold-toned apical and basal dendrites of layer III pyramidal cells showed that degenerating thalamocortical axon terminals from MD formed asymmetrical synaptic contacts predominantly with dendritic spines of the identified basal dendrites as well as apical dendrites. There was little difference in the numerical density of thalamocortical synapses from MD per unit length of both apical and basal dendrites.     

Oxidized alpha2-macroglobulin (alpha2M) differentially regulates receptor binding by cytokines/growth factors: implications for tissue injury and repair mechanisms in inflammation

Alpha2M binds specifically to TNF-alpha, IL-1beta, IL-2, IL-6, IL-8, basic fibroblast growth factor (bFGF), beta-nerve growth factor (beta-NGF), platelet-derived growth factor (PDGF), and TGF-beta. Since many of these cytokines are released along with neutrophil-derived oxidants during acute inflammation, we hypothesize that oxidation alters the ability of alpha2M to bind to these cytokines, resulting in differentially regulated cytokine functions. Using hypochlorite, a neutrophil-derived oxidant, we show that oxidized alpha2M exhibits increased binding to TNF-alpha, IL-2, and IL-6 and decreased binding to beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Hypochlorite oxidation of methylamine-treated alpha2M (alpha2M*), an analogue of the proteinase/alpha2M complex, also results in decreased binding to bFGF, beta-NGF, PDGF-BB, TGF-beta1, and TGF-beta2. Concomitantly, we observed decreased ability to inhibit TGF-beta binding and regulation of cells by oxidized alpha2M and alpha2M*. We then isolated alpha2M from human rheumatoid arthritis synovial fluid and showed that the protein is extensively oxidized and has significantly decreased ability to bind to TGF-beta compared with alpha2M derived from plasma and osteoarthritis synovial fluid. We, therefore, propose that oxidation serves as a switch mechanism that down-regulates the progression of acute inflammation by sequestering TNF-alpha, IL-2, and IL-6, while up-regulating the development of tissue repair processes by releasing bFGF, beta-NGF, PDGF, and TGF-beta from binding to alpha2M.     

Immunohistochemical localization of transforming growth factor-beta 1 beta 2 during folliculogenesis in the quail ovary

Immunohistochemical methods were used to show the presence and distribution of transforming growth factor-beta 1 and beta 2 during folliculogenesis in quail ovarian tissues. The results indicated that both transforming growth factor-beta subtypes are present. Immunolabelling for transforming growth factor-beta 1 demonstrated that prelampbrush oocytes are immunoreactive in the Balbiani complex, and developing and pre-ovulatory oocytes in the zona radiata. Immunolabelling was also associated with granulosa cells. The number of stained granulosa cells decreased during folliculogenesis. In the pre-ovulatory follicles, immunolabelling was found predominantly in the theca interna. Immunolabelling for transforming growth factor-beta 2 was associated with the zona radiata of developing and pre-ovulatory follicles, and with stromal interstitial cells. Moderate immunoreactivity was found in the Balbiani complex of prelampbrush oocytes. Weak immunolabelling was localized in the granulosa cells of prelampbrush follicles, and in a few cells of the theca interna of pre-ovulatory follicles. The immunolocalization of transforming growth factor-beta 1 and -beta 2 in the quail ovary supports their autocrine and/or paracrine role in avian ovarian processes.