Two clusters of charged residues located in the electropositive face of the von Willebrand factor A1 domain are essential for heparin binding

The VWF A1 domain seems to possess two heparin binding regions (residues 565-587 and 633-648) displaying positively charged amino acids, but the overall polyanion-A1 domain interaction scheme remains essentially elusive. To probe this molecular reaction as well as the role of electrostatic forces in VWF-heparin interaction, we performed mutagenesis and molecular modeling experiments. Fifteen mutated rVWFs were expressed [R571A, K572A, R573A, K585A, R571A/K572A/R573A, R578A/R579A, R578A/R579A/K585A, R571A/K572A/R573A/R578A/R579A/K585A (6A), K642G, K643G, K644G, K645G, K642G/K645G, K643G/K644G, and K642G/K643G/K644G/K645G (4G)]. Experimental results indicate that the multimeric structure of the mutants was similar to that of wild-type (WT) rVWF and that all rVWFs displayed normal binding to four conformation-dependent mAbs directed against the A1 domain. Three variants displayed significant reductions in the level of heparin binding. The 6A variant showed 39.2 +/- 1.3% of the WT rVWF level (p < 0.005), while mutants K643G/K644G and 4G showed 63.6 +/- 3.2 and 53.3 +/- 5% of the WT rVWF level, respectively (p < 0.005). Computational investigations showed that one face of the A1 domain is strongly electropositive, indicating that electrostatic forces should be essential in steering heparin onto its binding site. In agreement with our experimental data, the most striking alterations of the electrostatic potential contours were seen for mutants 4G, K643G/K644G, and 6A. Our data suggest that two clusters, one at positions 571-573, 578, 579, and 585 and the other at positions 642-645, act in concert for the recognition of heparin, forming a single extended binding surface across the electropositive face of the VWF A1 domain. A structural model of the VWF A1 domain-heparin complex is proposed, taking into account both experimental and computer modeling data.     

Short inverse complementary amino acid sequences generate protein complexity

Inversions of short genomic sequences play a central role in the generation of protein complexity. More than half of the 1300 motifs registered in ProSite have protein inverse complementary sequences (princoms) among proteins registered in SwissProt. The observed number of princoms occurrences exceeds by far the expected number (p < 10(-10)). Princoms often endow their host proteins with a whole new range of biochemical and physiological capabilities, including the possibility of intramolecular and intermolecular disulfide bond formation. These results support the idea that, like the duplications, the inversions of small genomic fragments have been a fundamental mechanism for shaping genomes.     

The number of vertebrate repeats can be regulated at yeast telomeres by Rap1-independent mechanisms

The number of telomeric DNA repeats at chromosome ends is maintained around a mean value by a dynamic balance between elongation and shortening. In particular, proteins binding along the duplex part of telomeric DNA set the number of repeats by progressively limiting telomere growth. The paradigm of this counting mechanism is the Rap1 protein in Saccharomyces cerevisiae. We demonstrate here that a Rap1-independent mechanism regulates the number of yeast telomeric repeats (TG(1-3)) and of vertebrate repeats (T(2)AG(3)) when TEL1, a yeast ortholog of the human gene encoding the ATM kinase, is inactivated. In addition, we show that a T(2)AG(3)-only telomere can be formed and maintained in humanized yeast cells carrying a template mutation of the gene encoding the telomerase RNA, which leads to the synthesis of vertebrate instead of yeast repeats. Genetic and biochemical evidences indicate that this telomere is regulated in a Rap1-independent manner, both in TEL1 and in tel1Delta humanized yeast cells. Altogether, these findings shed light on multiple repeat-counting mechanisms, which may share critical features between lower and higher eukaryotes.     

Arabinogalactan-proteins in Cichorium somatic embryogenesis: effect of β-glucosyl Yariv reagent and epitope localisation during embryo development

 Direct somatic embryogenesis was induced in root tissues of the Cichorium hybrid `474' (C. intybus L. var. sativum×C. endivia L. var. latifolia). Addition of β-d-glucosyl Yariv reagent (βGlcY), a synthetic phenylglycoside that specifically binds arabinogalactan-proteins (AGPs), to the culture medium blocked somatic embryogenesis in a concentration-dependent manner with complete inhibition of induction occurring at 250 μM βGlcY. The AGP-unreactive α-d-galactosyl Yariv reagent had no biological activity in this system. Upon transfer of 250 μM βGlcY-treated roots to control conditions, somatic embryogenesis was recovered with a time course similar to that of control roots. The βGlcY penetrated roots and bound abundantly to developing somatic embryos, to the root epidermis and the stele. Immunofluorescence and immunogold labelling using monoclonal antibodies (JIM13, JIM16 and LM2) revealed that AGPs were localised in the outer cell walls peripheral cells of the globular embryo. A spatio-temporal expression of AGPs appeared to be associated with differentiation events in the somatic embryo during the transition from the globular stage to the torpedo stage. To verify βGlcY specificity, molecules that bound βGlcY were extracted from treated conditioned medium and identified as AGPs by using the same monoclonal antibodies. In addition, AGPs were found to be abundantly present in the medium during embryogenic culture. All of these results establish the implication of AGPs in embryo development, and their putative role in somatic embryogenesis is discussed. Received: 26 August 1999 / Accepted: 28 January 2000     

New SUCLG1 patients expanding the phenotypic spectrum of this rare cause of mild methylmalonic aciduria

Deficiencies in two subunits of the succinyl-coenzyme A synthetase (SCS) have been involved in patients with encephalomyopathy and mild methylmalonic aciduria (MMA). In this study, we described three new SUCLG1 patients and performed a meta-analysis of the literature. Our report enlarges the phenotypic spectrum of SUCLG1 mutations and confirms that a characteristic metabolic profile (presence of MMA and C4-DC carnitine in urines) and basal ganglia MRI lesions are the hallmarks of SCS defects. As mitochondrial DNA depletion in muscle is not a constant finding in SUCLG1 patients, this may suggest that diagnosis should not be based on it, but also that alternative physiopathological mechanisms may be considered to explain the combined respiratory chain deficiency observed in SCS patients.     

Chips from chips: application to the study of antibody responses to methylated proteins

Peptide microarrays are useful tools for the characterization of humoral responses against peptide antigens. The study of post-translational modifications requires the printing of appropriately modified peptides, whose synthesis can be time-consuming and expensive. We describe here a method named "chips from chips", which allows probing the presence of antibodies directed toward modified peptide antigens starting from unmodified peptide microarrays. The chip from chip concept is based on the modification of peptide microspots by simple chemical reactions. The starting peptide chip (parent chip) is covered by the reagent solution, thereby allowing the modification of specific residues to occur, resulting in the production of a modified peptide chip (daughter chip). Both parent and daughter chips can then be used for interaction studies. The method is illustrated using reductive methylation for converting lysines into dimethyllysines. The rate of methylation was studied using specific antibodies and fluorescence detection, or surface-assisted laser desorption ionization mass spectrometry. This later technique showed unambiguously the efficient methylation of the peptide probes. The method was then used to study the humoral response against the Mycobacterium tuberculosis heparin-binding hemagglutinin, a methylated surface-associated virulence factor and powerful diagnostic and protective antigen.     

Variability in the bioluminescence response of the dinoflagellate Pyrocystis lunula

Light emission in dinoflagellates is induced by water motions. But although it is known that mechanical stimulations of these organisms trigger the bioluminescent response, the exact mechanism that involves some cell membrane excitations by fluid motions is not yet fully understood and is still controversial. We show in this experimental study that the accelerated shear flow, created by abrupt rotations of one or both co-axial cylinders of a Couette shearing chamber excites the light emission from cultured dinoflagellates Pyrocystis lunula. Following our first results published earlier that state that pure laminar shear does not excite the main bioluminescent response in dinoflagellates, our present experiments show that both shear and acceleration in the flow are needed to trigger the bioluminescent response. Besides, the probability to stimulate this bioluminescent response under acceleration and shear is deduced from the response curves. This response follows a Gaussian distribution that traduces a heterogeneity in individual cell thresholds for the stimulation of bioluminescence in a dinoflagellate population. All these results will have a repercussion in the possible applications of dinoflagellate bioluminescence in flow visualizations and measurements. Moreover, this study opens a new way in studying mechanically-induced stimulus thresholds at the cell level.     

Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia

Globozoospermia is a rare (incidence <0.1% in male infertile patients) form of teratozoospermia, mainly characterized by round-headed spermatozoa that lack an acrosome. It originates from a disturbed spermiogenesis, which is expected to be induced by a genetic factor. Several family cases and recessive mouse models with the same phenotype support this expectation. In this study, we present a consanguineous family with three affected brothers, in whom we have identified a homozygous mutation in the spermatogenesis-specific gene SPATA16. This is the first example of a nonsyndromic male infertility condition in humans caused by an autosomal gene defect, and it could also mean that the identification of other partners like SPATA16 could elucidate acrosome formation.