Glycoprotein Ib cross-linking/ligation on echicetin-coated surfaces or echicetin-IgMkappa in stirred suspension activates platelets by cytoskeleton modulated calcium release

Cross-linking platelet GPIb with the snake C-type lectin echicetin provides a specific technique for activation via this receptor. This allows GPIb-dependent mechanisms to be studied without the necessity for shear stress-induced binding of von Willebrand factor or primary alpha(IIb)beta(3) involvement. We already showed that platelets are activated, including tyrosine phosphorylation, by echicetin-IgMkappa-induced GPIb cross-linking. We now investigate the mechanism further and demonstrate that platelets, without modulator reagents, spread directly on an echicetin-coated surface, by a GPIb-specific mechanism, causing exocytosis of alpha-granule markers (P-selectin) and activation of alpha(IIb)beta(3). This spreading requires actin polymerization and release of internal calcium stores but is not dependent on external calcium nor on src family tyrosine kinases. Cross-linking of GPIb complex molecules on platelets, either in suspension or via specific surface attachment, is sufficient to induce platelet activation.

Structural model of the CopA copper ATPase of Enterococcus hirae based on chemical cross-linking

The CopA copper ATPase of Enterococcus hirae belongs to the family of heavy metal pumping CPx-type ATPases and shares 43% sequence similarity with the human Menkes and Wilson copper ATPases. Due to a lack of suitable protein crystals, only partial three-dimensional structures have so far been obtained for this family of ion pumps. We present a structural model of CopA derived by combining topological information obtained by intramolecular cross-linking with molecular modeling. Purified CopA was cross-linked with different bivalent reagents, followed by tryptic digestion and identification of cross-linked peptides by mass spectrometry. The structural proximity of tryptic fragments provided information about the structural arrangement of the hydrophilic protein domains, which was integrated into a three-dimensional model of CopA. Comparative modeling of CopA was guided by the sequence similarity to the calcium ATPase of the sarcoplasmic reticulum, Serca1, for which detailed structures are available. In addition, known partial structures of CPx-ATPase homologous to CopA were used as modeling templates. A docking approach was used to predict the orientation of the heavy metal binding domain of CopA relative to the core structure, which was verified by distance constraints derived from cross-links. The overall structural model of CopA resembles the Serca1 structure, but reveals distinctive features of CPx-type ATPases. A prominent feature is the positioning of the heavy metal binding domain. It features an orientation of the Cu binding ligands which is appropriate for the interaction with Cu-loaded metallochaperones in solution. Moreover, a novel model of the architecture of the intramembranous Cu binding sites could be derived.

Protein cross-linking mediated by tissue transglutaminase correlates with the maturation of extracellular matrices during lung development

At birth, the mammalian lung is still immature. The alveoli are not yet formed and the interairspace walls contain two capillary layers which are separated by an interstitial core. After alveolarization (first 2 postnatal weeks in rats) the alveolar septa mature: their capillary layers merge, the amount of connective tissue decreases, and the mature lung parenchyma is formed (second and third week). During the first 3 wk of life the role of tissue transglutaminase (tTG) was studied in rat lung by immunostaining of cryostat and paraffin sections, by Northern and Western blotting, and by a quantitative determination of gamma-glutamyl-epsilon-lysine. While enzyme activity and intracellular tTG were already present before term, the enzyme product (gamma-glutamyl-epsilon-lysine-crosslink) and extracellular tTG appeared between postnatal days 10 and 19 in the lung parenchyma. In large blood vessels and large airways, which mature earlier than the parenchyma, both the enzyme product and extracellular tTG had already appeared at the end of the first postnatal week. We conclude that tTG is expressed and externalized into the extracellular matrix of lung shortly before maturation of an organ area. Because tTG covalently and irreversibly crosslinks extracellular matrix proteins, we hypothesize that it may prevent or delay further remodeling of basement membranes and may stabilize other extracellular components, such as microfibrils.

Corneal cross-linking in keratoconus using the standard and rapid treatment protocol: differences in demarcation line and 12-month outcomes.

PURPOSE To compare the occurrence rate and depth of the demarcation line and topographical outcome after corneal cross-linking (CXL) for keratoconus using two different treatment protocols. METHODS A retrospective analysis of 131 eyes with progressive keratoconus treated with CXL using riboflavin and UV-A was performed. Eyes were treated either with the standard Dresden protocol (30 minutes irradiation, 3 mW/cm(2), UV-XTM 1000) or a rapid protocol (10 minutes irradiation, 9 mW/cm(2), UV-XTM 2000). The presence and depth of the corneal demarcation line was assessed with an anterior segment optical coherence tomography device 1 month after CXL by a masked observer. Corneal topography and tomography was performed at baseline and at 12-month follow-up with Pentacam and the TMS (Topographic Modeling System) device. RESULTS In the standard protocol group, 76.5% (62/81) of treated corneas revealed a demarcation line 1 month after CXL, whereas such a demarcation line was observed in only 22% (11/50) of eyes treated with the rapid protocol (P < 0.0001). The demarcation line was significantly more superficial in the rapid protocol group (P = 0.004). Corneal topography values between baseline and 12 months after CXL showed a mean change of -0.76 diopters (D) in Kmax (SD ± 2.7) in the standard protocol group versus a mean change of +0.72 D in Kmax (SD ± 1.5) in the rapid protocol (P = 0.007). CONCLUSIONS The rapid CXL protocol negatively influences the occurrence and depth of the demarcation line 1 month after CXL. Our results show a negative effect on the topographical outcome 1 year after CXL.

Impact of corneal cross-linking on topical drug penetration in humans.

PURPOSE To analyse the influence of corneal cross-linking (CXL) with ultraviolet-A (UV-A) and riboflavin on drug permeability in human subjects. METHODS Keratoconus patients (n = 23; mean age 26.9 ± 5.8 years) undergoing a standard CXL procedure with UV-A (5.4 J/cm(2) , 30 min) and riboflavin in one eye were included in the study. The pupillary diameter, measured before and every 3 min for 30 min after the topical application of one drop of 2% pilocarpine, was used as an indirect measure of the corneal permeability. The pupillary diameter was measured with an infrared pupillometer device before (baseline) and 4 months after CXL. RESULTS Prior to pilocarpine application, no significant difference in the pupillary diameter was detected before CXL and 4 months later. The mean decrease in the pupillary diameter after the application of pilocarpine was similar at baseline and the 4-month follow-up visit: mean decreases of 3.9 and 3.7 mm were observed 30 min after pilocarpine application, respectively (p > 0.05). CONCLUSIONS No significant influence of CXL on the corneal penetration of topically applied pilocarpine was observed in this clinical study.

Corneal Cross-Linking in a 4-Year-Old Child With Keratoconus and Down Syndrome

PURPOSE To describe the clinical outcome of corneal cross-linking (CXL) in a young child with keratoconus. METHODS This is a case report of a young girl with keratoconus with ophthalmologic findings and 3-year follow-up. Follow-up visits included visual acuity measurement, retinoscopy, corneal tomography, and topography. RESULTS A girl with Down syndrome was diagnosed with bilateral keratoconus and relative amblyopia at the age of 4 years. The best-corrected near visual acuity was 20/100 binocularly. Corneal tomography showed the following parameters: OD K(max) 47.2 diopters (D), thinnest location 442 μm; OS K(max) 49.6 D, thinnest location 432 μm. Three months later, the keratoconus in the left eye progressed (K(max) 50.2 D, thinnest location 424 μm), and CXL was performed. One year later, CXL was necessary also in the right eye because of progression. The girl was most recently reexamined at the age of 7 years. The corrected near visual acuity was 20/80 in both eyes. The corneal curvature slightly flattened, and the corneal thickness stabilized (OD K(max) 46.8 D, thinnest location 389 μm; OS K(max) 49.4 D, thinnest location 360 μm). CONCLUSIONS Onset of keratoconus can occur in early childhood, especially in patients with Down syndrome. In this case, CXL was performed at 4 and 5 years of age without complications and stopped further keratoconus progression.

Interacting helical faces of subunits a and c in the F1Fo ATP synthase of Escherichia coli defined by disulfide cross-linking

Subunits a and c of Fo are thought to cooperatively catalyze proton translocation during ATP synthesis by the Escherichia coli F1Fo ATP synthase. Optimizing mutations in subunit a at residues A217, I221, and L224 improves the partial function of the cA24D/cD61G double mutant and, on this basis, these three residues were proposed to lie on one face of a transmembrane helix of subunit a, which then interacted with the transmembrane helix of subunit c anchoring the essential aspartyl group. To test this model, in the present work Cys residues were introduced into the second transmembrane helix of subunit c and the predicted fourth transmembrane helix of subunit a. After treating the membrane vesicles of these mutants with Cu(1,10-phenanthroline)2SO4 at 0°, 10°, or 20°C, strong a–c dimer formation was observed at all three temperatures in membranes of 7 of the 65 double mutants constructed, i.e., in the aS207C/cI55C, aN214C/cA62C, aN214C/cM65C, aI221C/cG69C, aI223C/cL72C, aL224C/cY73C, and aI225C/cY73C double mutant proteins. The pattern of cross-linking aligns the helices in a parallel fashion over a span of 19 residues with the aN214C residue lying close to the cA62C and cM65C residues in the middle of the membrane. Lesser a–c dimer formation was observed in nine other double mutants after treatment at 20°C in a pattern generally supporting that indicated by the seven landmark residues cited above. Cross-link formation was not observed between helix-1 of subunit c and helix-4 of subunit a in 19 additional combinations of doubly Cys-substituted proteins. These results provide direct chemical evidence that helix-2 of subunit c and helix-4 of subunit a pack close enough to each other in the membrane to interact during function. The proximity of helices supports the possibility of an interaction between Arg210 in helix-4 of subunit a and Asp61 in helix-2 of subunit c during proton translocation, as has been suggested previously.

Btk dosage determines sensitivity to B cell antigen receptor cross-linking

Mutations in Btk result in the B cell immunodeficiencies X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. Btk is a critical component of signaling pathways regulating B cell development and function. We used a genetic approach to determine whether Btk is also limiting for these processes. One allele of a murine Btk transgene expressed a dosage of Btk (25% of endogenous levels in splenic B cells) sufficient to restore normal numbers of phenotypically mature conventional B cells in xid mice. 2,4,6-trinitrophenyl–Ficoll response, anti-IgM-induced proliferation, B1 cell development, and serum IgM and IgG3 levels remained significantly impaired in these animals. B cells from Btk −/− transgenic mice also responded poorly to anti-IgM, indicating that the xid mutation does not create a dominant negative form of Btk. Response to 2,4,6-trinitrophenyl–Ficoll and B cell receptor cross-linking were increased 3- to 4-fold in xid mice homozygous for the transgene. These results demonstrate that Btk is a limiting component of B cell antigen receptor signaling pathways and suggest that B cell development and response to antigen may require different levels of Btk activity.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.

Chemistry for the analysis of protein–protein interactions: Rapid and efficient cross-linking triggered by long wavelength light

Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the de novo design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents.

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm1 : Proteolytic Removal by Thermolysin and in Vitro Cross-Linking of Granule-Associated Polypeptides

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix.

Detecting the conformational change of transmembrane signaling in a bacterial chemoreceptor by measuring effects on disulfide cross-linking in vivo.

Transmembrane signaling by bacterial chemoreceptors is thought to involve relative movement among the four transmembrane helices of the homodimer. We assayed that movement by measuring effects of ligand occupancy on rates of oxidative cross-linking between cysteines introduced into neighboring helices of the transmembrane domain of chemoreceptor Trg from Escherichia coli. Measurements were done on chemoreceptors in their native environment, intact cells that were motile and chemotactically responsive. Receptor occupancy did not appear to cause drastic rearrangement of the four-helix structure since, among 67 cysteine pairs tested, the same 19 exhibited oxidative cross-linking in the presence or absence of saturating chemoattractant. However, occupancy did cause subtle changes that were detected as effects on rates of cross-linking. Among the seven disulfides appropriate for measurements of initial rates of formation, ligand occupancy had significant and different effects on all three cross-links that connected the two helices within a subunit but had minimal effects on the four that spanned the packing interface between subunits. This constitutes direct evidence that the conformational change of transmembrane signaling involves significant movement within a subunit and minimal movement between subunits, a pattern deduced from several previous studies and now documented directly. Among possible modes of movement between the two helices of a subunit, axial sliding of one helix relative to the other was the conformational change that best accounted for the observed effects on cross-linking.

Fas (CD95) expression and death-mediating function are induced by CD4 cross-linking on CD4+ T cells.

The CD4 receptor contributes to T-cell activation by coligating major histocompatibility complex class II on antigen presenting cells with the T-cell receptor (TCR)/CD3 complex, and triggering a cascade of signaling events including tyrosine phosphorylation of intracellular proteins. Paradoxically, CD3 cross-linking prior to TCR stimulation results in apoptotic cell death, as does injection of anti-CD4 antibodies in vivo of CD4 ligation by HIV glycoprotein (gp) 120. In this report we investigate the mechanism by which CD4 cross-linking induces cell death. We have found that CD4 cross-linking results in a small but rapid increase in levels of cell surface Fas, a member of the tumor necrosis factor receptor family implicated in apoptotic death and maintenance of immune homeostasis. Importantly, CD4 cross-linking triggered the ability of Fas to function as a death molecule. Subsequent to CD4 cross-linking, CD4+ splenocytes cultured overnight became sensitive to Fas-mediated death. Death was Fas-dependent, as demonstrated by cell survival in the absence of plate-bound anti-Fas antibody, and by the lack of CD4-induced death in cells from Fas-defective lymphoproliferative (lpr) mice. We demonstrate here that CD4 regulates the ability of Fas to induce cell death in Cd4+ T cells.

Signature p53 mutation at DNA cross-linking sites in 8-methoxypsoralen and ultraviolet A (PUVA)-induced murine skin cancers.

A combination of psoralen and ultraviolet A radiation (PUVA) is widely used in the treatment of psoriasis. However, PUVA treatment increases the risk of developing skin cancer in psoriasis patients and induces skin cancer in mice. Since the DNA damage induced by PUVA is quite different from that induced by UV, we investigated whether PUVA-induced mouse skin cancers display carcinogen-specific mutations in the p53 tumor suppressor gene. The results indicated that 10 of 13 (77%) PUVA-induced skin tumors contained missense mutations predominantly at exons 6 and 7. In contrast, tumor-adjacent, PUVA-exposed skin from tumor-bearing animals did not exhibit p53 mutation in exons 4-8. Interestingly, about 40% of all mutations in PUVA-induced skin tumors occurred at 5'-TA sites, and an equal number of mutations occurred at one base flanking 5'TA or 5'-TAT sites. Since PUVA induces DNA cross-links exclusively at these sites and since UV "signature" mutations were rarely detected in PUVA-induced skin cancers, we can conclude that PUVA acts as a carcinogen by inducing unique PUVA signature mutations in p53. This finding may have implications for identifying the etiology of skin cancer in psoriasis patients who have undergone PUVA therapy.