Expression and purification of recombinant extracellular sulfatase HSulf-2 allows deciphering of enzyme sub-domain coordinated role for the binding and 6-O-desulfation of heparan sulfate.

Through their ability to edit 6-O-sulfation pattern of Heparan sulfate (HS) polysaccharides, Sulf extracellular endosulfatases have emerged as critical regulators of many biological processes, including tumor progression. However, study of Sulfs remains extremely intricate and progress in characterizing their functional and structural features has been hampered by limited access to recombinant enzyme. In this study, we unlock this critical bottleneck, by reporting an efficient expression and purification system of recombinant HSulf-2 in mammalian HEK293 cells. This novel source of enzyme enabled us to investigate the way the enzyme domain organization dictates its functional properties. By generating mutants, we confirmed previous studies that HSulf-2 catalytic (CAT) domain was sufficient to elicit arylsulfatase activity and that its hydrophilic (HD) domain was necessary for the enzyme 6-O-endosulfatase activity. However, we demonstrated for the first time that high-affinity binding of HS substrates occurred through the coordinated action of both domains, and we identified and characterized 2 novel HS binding sites within the CAT domain. Altogether, our findings contribute to better understand the molecular mechanism governing HSulf-2 substrate recognition and processing. Furthermore, access to purified recombinant protein opens new perspectives for the resolution of HSulf structure and molecular features, as well as for the development of Sulf-specific inhibitors.

A Two-component NADPH Oxidase (NOX)-like System in Bacteria Is Involved in the Electron Transfer Chain to the Methionine Sulfoxide Reductase MsrP.

MsrPQ is a newly identified methionine sulfoxide reductase system found in bacteria, which appears to be specifically involved in the repair of periplasmic proteins oxidized by hypochlorous acid. It involves two proteins: a periplasmic one, MsrP, previously named YedY, carrying out the Msr activity, and MsrQ, an integral b-type heme membrane-spanning protein, which acts as the specific electron donor to MsrP. MsrQ, previously named YedZ, was mainly characterized by bioinformatics as a member of the FRD superfamily of heme-containing membrane proteins, which include the NADPH oxidase proteins (NOX/DUOX). Here we report a detailed biochemical characterization of the MsrQ protein from Escherichia coli We optimized conditions for the overexpression and membrane solubilization of an MsrQ-GFP fusion and set up a purification scheme allowing the production of pure MsrQ. Combining UV-visible spectroscopy, heme quantification, and site-directed mutagenesis of histidine residues, we demonstrated that MsrQ is able to bind two b-type hemes through the histidine residues conserved between the MsrQ and NOX protein families. In addition, we identify the E. coli flavin reductase Fre, which is related to the dehydrogenase domain of eukaryotic NOX enzymes, as an efficient cytosolic electron donor to the MsrQ heme moieties. Cross-linking experiments as well as surface Plasmon resonance showed that Fre interacts with MsrQ to form a specific complex. Taken together, these data support the identification of the first prokaryotic two-component protein system related to the eukaryotic NOX family and involved in the reduction of periplasmic oxidized proteins.

The TET2 and TET3 aminopeptidases from Pyrococcus horikoshii form a hetero-subunit peptidasome with enhanced peptide destruction properties.

TET aminopeptidases assemble as large homo-dodecameric complexes. The reason why prokaryotic genomes often encode a diverse set of TET peptidases homologues remains unclear. In the archaeon Pyrococcus horikoshii, PhTET1, PhTET2 and PhTET3 homo-oligomeric particles have been proposed to work in concert to breakdown intracellular polypeptides. When coexpressed in Escherichia coli, the PhTET2 and PhTET3 proteins were found to assemble efficiently as heteromeric complexes. Biophysical analysis demonstrated that these particles possess the same quaternary structure as the homomeric TET dodecamers. The same hetero-oligomeric complexes were immunodetected in P. horikoshii cell extracts analysed by sucrose gradient fractionation and ion exchange chromatography. The biochemical activity of a purified hetero-oligomeric TET particle, assessed on chromogenic substrates and on a complex mixture of peptides, reveals that it displays higher efficiency than an equivalent combination of homo-oligomeric TET particles. Interestingly, phylogenetic analysis shows that PhTET2 and PhTET3 are paralogous proteins that arose from gene duplication in the ancestor of Thermococcales. Together, these results establish that the PhTET2 and PhTET3 proteins are two subunits of the same enzymatic complex aimed at the destruction of polypeptidic chains of very different composition. This is the first report for such a mechanism intended to improve multi-enzymatic complex efficiency among exopeptidases.

Agar-supported cultivation of Halorubrum sp. SSR, and production of halocin C8 on the scale-up prototype Platotex.

Halorubrum sp. SSR was isolated from a solar saltern in Algeria. The strain exhibited a high antibiotic activity against the indicator strain Natronorubrum aibiense G23, and the bioactive compound showed thermal, acid and alkali stability. SSR was grown on agar-supported cultivation (AgSF) to compare yields and applicability with traditional submerged cultivation. AgSF scale-up was implemented taking benefit from the solid-state cultivation prototype Platotex. This technology leads to high amounts of the target Halocin and facilitate the downstream steps. The antibiotic compound was purified according to a fast efficient procedure including ion exchange chromatography followed by a fractionation on C18 Sep-Pack cartridge. The compound was identified as Halocin C8 according to N-terminal amino acid sequencing and high-resolution mass spectrometry.

[Ethics and solitary confinement in psychiatry]. / Ethique et isolement en psychiatrie.

The decision to treat a patient in solitary confinement in psychiatry does not follow any protocol and is not made on a case-by-case basis. Team deliberation opens discussion and enables the group as a whole to take responsibility for clarifying what is to be supported by the team and implemented by the carer during treatment. When presented with complex situations, uncertainty can be a force when it calls upon an ethical dilemma.

Experimental determination of organelle targeting-peptide cleavage sites using transient expression of green fluorescent protein translational fusions.

The majority of nuclear-encoded organellar proteins contain a cleavable presequence, which is necessary for protein targeting and import into the correct cellular compartment. Knowledge about targeting-peptide cleavage sites is essential for the structural and functional characterization of the mature organellar proteins as well as for a deeper understanding of the import process. Because of the low consensus and high variability of presequences, bioinformatics of targeting-peptide cleavage fails to predict the length of the targeting peptide with high confidence. Therefore, we have developed a rapid and robust method to experimentally determine the cleavage site of the transit peptide for proteins imported into mitochondria or plastids. The protein precursor with green fluorescent protein (GFP) fused to its C-terminus is transiently expressed in cells (for animal proteins) or protoplasts (for plant proteins), allowing translocation into organelles and removal of the transit peptide. After lysis, the matured protein is immunopurified using an anti-GFP antibody coupled to magnetic beads. The N-terminal amino sequence is then determined by Edman microsequencing or mass spectrometry. The method has been validated using proteins with known targeting-peptide sequences and is suitable for animal and plant organelle-targeted proteins.

Fibrillin-1 genetic deficiency leads to pathological ageing of arteries in mice.

Fibrillin-1, the major component of extracellular microfibrils that associate with insoluble elastin in elastic fibres, is mainly synthesized during development and postnatal growth and is believed to guide elastogenesis. Mutations in the fibrillin-1 gene cause Marfan syndrome, a multisystem disorder characterized by aortic aneurysms and dissections. The recent finding that early deficiency of elastin modifies vascular ageing has raised the possibility that fibrillin-1 deficiency could also contribute to late-onset pathology of vascular remodelling. To address this question, we examined cardiovascular function in 3-week-old, 6-month-old, and 24-month-old mice that are heterozygous for a hypomorphic structural mutation of fibrillin-1 (Fbn1{+/mgΔ} mice). Our results indicate that Fbn1{+/mgΔ} mice, particularly those that are 24 months old, are slightly more hypotensive than wild-type littermates. Additionally, aneurysm and aortic insufficiency were more frequently observed in ageing Fbn1{+/mgΔ}$ mice than in the wild-type counterparts. We also noted substantial fragmentation and decreased number of elastic lamellae in the aortic wall of Fbn1{+/mgΔ} mice, which were correlated with an increase in aortic stiffness, a decrease in vasoreactivity, altered expression of elastic fibre-related genes, including fibrillin-1 and elastin, and a decrease in the relative ratio between tissue elastin and collagen. Collectively, our findings suggest that the heterozygous mgΔ mutation accelerates some aspects of vascular ageing and eventually leads to aortic manifestations resembling those of Marfan syndrome. Importantly, our data also indicate that vascular abnormalities in Fbn1{+/mgΔ} mice are opposite to those induced by elastin haploinsufficiency during ageing that affect blood pressure, vascular dimensions, and number of elastic lamellae.

PrP N-terminal domain triggers PrP(Sc)-like aggregation of Dpl.

Transmissible spongiform encephalopathies are fatal neurodegenerative disorders thought to be transmitted by self-perpetuating conformational conversion of a neuronal membrane glycoprotein (PrP(C), for "cellular prion protein") into an abnormal state (PrP(Sc), for "scrapie prion protein"). Doppel (Dpl) is a protein that shares significant biochemical and structural homology with PrP(C). In contrast to its homologue PrP(C), Dpl is unable to participate in prion disease progression or to achieve an abnormal PrP(Sc)-like state. We have constructed a chimeric mouse protein, composed of the N-terminal domain of PrP(C) (residues 23-125) and the C-terminal part of Dpl (residues 58-157). This chimeric protein displays PrP-like biochemical and structural features; when incubated in presence of NaCl, the alpha-helical monomer forms soluble beta-sheet-rich oligomers which acquire partial resistance to pepsin proteolysis in vitro, as do PrP oligomers. Moreover, the presence of aggregates akin to protofibrils is observed in soluble oligomeric species by electron microscopy.

Human iron regulatory protein 2 is easily cleaved in its specific domain: consequences for the haem binding properties of the protein.

Mammalian IRPs (iron regulatory proteins), IRP1 and IRP2, are cytosolic RNA-binding proteins that post-transcriptionally control the mRNA of proteins involved in storage, transport, and utilization of iron. In iron-replete cells, IRP2 undergoes degradation by the ubiquitin/proteasome pathway. Binding of haem to a 73aa-Domain (73-amino-acid domain) that is unique in IRP2 has been previously proposed as the initial iron-sensing mechanism. It is shown here that recombinant IRP2 and the 73aa-Domain are sensitive to proteolysis at the same site. NMR results suggest that the isolated 73aa-Domain is not structured. Iron-independent cleavage of IRP2 within the 73aa-Domain also occurs in lung cancer (H1299) cells. Haem interacts with a cysteine residue only in truncated forms of the 73aa-Domain, as shown by a series of complementary physicochemical approaches, including NMR, EPR and UV-visible absorption spectroscopy. In contrast, the cofactor is not ligated by the same residue in the full-length peptide or intact IRP2, although non-specific interaction occurs between these molecular forms and haem. Therefore it is unlikely that the iron-dependent degradation of IRP2 is mediated by haem binding to the intact 73aa-Domain, since the sequence resembling an HRM (haem-regulatory motif) in the 73aa-Domain does not provide an axial ligand of the cofactor unless this domain is cleaved.

Folding and turnover of human iron regulatory protein 1 depend on its subcellular localization.

Aconitases are iron-sulfur hydrolyases catalysing the interconversion of citrate and isocitrate in a wide variety of organisms. Eukaryotic aconitases have been assigned additional roles, as in the case of the metazoan dual activity cytosolic aconitase-iron regulatory protein 1 (IRP1). This human protein was produced in yeast mitochondria to probe IRP1 folding in this organelle where iron-sulfur synthesis originates. The behaviour of human IRP1 was compared with that of genuine mitochondrial (yeast or human) aconitases. All enzymes were functional in yeast mitochondria, but IRP1 was found to form dense particles as detected by electron microscopy. MS analysis of purified inclusion bodies evidenced the presence of human IRP1 and alpha-ketoglutarate dehydrogenase complex component 1 (KGD1), one of the subunits of alpha-ketoglutarate dehydrogenase. KGD1 triggered formation of the mitochondrial aggregates, because the latter were absent in a KGD1(-) mutant, but it did not efficiently do so in the cytosol. Despite the iron-binding capacity of IRP1 and the readily synthesis of iron-sulfur clusters in mitochondria, the dense particles were not iron-rich, as indicated by elemental analysis of purified mitochondria. The data show that proper folding of dual activity IRP1-cytosolic aconitase is deficient in mitochondria, in contrast to genuine mitochondrial aconitases. Furthermore, efficient clearance of the aggregated IRP1-KGD1 complex does not occur in the organelle, which emphasizes the role of molecular interactions in determining the fate of IRP1. Thus, proper folding of human IRP1 strongly depends on its cellular environment, in contrast to other members of the aconitase family.

Chronic Treatment with Minoxidil Induces Elastic Fiber Neosynthesis and Functional Improvement in the Aorta of Aged Mice.

Normal arterial aging processes involve vascular cell dysfunction associated with wall stiffening, the latter being due to progressive elastin and elastic fiber degradation, and elastin and collagen cross-linking by advanced glycation end products (AGEs). These processes progressively lead to cardiovascular dysfunction during aging. Elastin is only synthesized during late gestation and childhood, and further degradation occurring throughout adulthood cannot be physiologically compensated by replacement of altered material. However, the ATP-dependent K+ channel opener minoxidil has been shown to stimulate elastin expression in vitro and in vivo in the aorta of young adult rats. Therefore, we have studied the effect of a 10-week chronic oral treatment with minoxidil (120 mg/L in drinking water) on the aortic structure and function in aged 24-month-old mice. Minoxidil treatment increased tropoelastin, fibulin-5, and lysyl-oxidase messenger RNA levels, reinduced a moderate expression of elastin, and lowered the levels of AGE-related molecules. This was accompanied by the formation of newly synthesized elastic fibers, which had diverse orientations in the wall. A decrease in the glycation capacity of aortic elastin was also produced by minoxidil treatment. The ascending aorta also underwent a minoxidil-induced increase in diameter and decrease in wall thickness, which partly reversed the age-associated thickening and returned the wall thickness value and strain-stress relation closer to those of younger adult animals. In conclusion, our results suggest that minoxidil presents an interesting potential for arterial remodeling in an antiaging perspective, even when treating already aged animals.

Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography.

Human plasma paraoxonase (PON1) is calcium-dependent enzyme that hydrolyses esters, including organophosphates and lactones, and exhibits anti-atherogenic properties. Human phosphate binding protein (HPBP) was discovered as contaminant during crystallization trials of PON1. This observation and uncertainties for the real activities of PON1 led us to re-evaluate the purity of PON1 preparations. We developed a hydroxyapatite chromatography for the separation of both HDL-associated proteins. We confirmed that: (1) HPBP is strongly associated to PON1 in HDL, and generally both proteins are co-purified; (2) standard purification protocols of PON1 lead to impure enzyme; (3) hydroxyapatite chromatography allows the simultaneous purification of PON1 and HPBP.

Proteomic and profile analysis of the proteins laced with aragonite and vaterite in the freshwater mussel Hyriopsis cumingii shell biominerals.

Hyriopsis cumingii (Lea, Unionidae), a freshwater bivalve species widely distributed in China and commercially exploited for freshwater pearl production, was chosen as the reference model to investigate the protein signature in the organic scaffold matching calcium carbonate crystallization mode. This study takes advantage of different calcium carbonate habits production by the organism: aragonite in shell and pearl and vaterite in alternative pearl formation. Amino acid global composition and proteomics analysis have been undertaken to study the amino acid imbalance with respect to biominerals and microstructures. Forty peptides sequences were obtained by proteomics, of which ten are shared by all the different samples, nine are laced with aragonite; another nine with vaterite and twelve are related to pearls. Bioinformatics analysis allowed the peptides to be matched to the deduced protein sequences from EST databases and allowed functional assignment (e.g. scaffolding, strain strength, chitin binding or carbonic anhydrase function) to the proteins found in the different materials. Such panel of motifs tailored in vaterite and aragonite habits produced in a freshwater mollusk gives food for thought about organic control of the biomineralization processes.

What is the difference in organic matrix of aragonite vs. vaterite polymorph in natural shell and pearl? Study of the pearl-forming freshwater bivalve mollusc Hyriopsis cumingii.

Aragonite pearl, vaterite pearl and shell nacre of the freshwater mollusc Hyriopsis cumingii (Zhejiang province, China) were chosen to analyze microstructure and organic composition in the different habits of calcium carbonate. SEM and TEM were used to reveal the microstructure and mineralogical phase. We found that tablets in vaterite exhibited more irregular texture and were packaged with more organic matrices than in aragonite forms. Then a peculiar method was introduced to extract water soluble matrix (WSM), acid soluble matrix (ASM) and acid insoluble matrix (AIM) from the three samples, and biochemical analysis of these organic matrixes involved in crystal formation and polymorph selection was carried out. High performance liquid chromatography (HPLC) confirms the hydrophobic pattern of the organic matrix intermingled with mineral, the opposite of the early mobilizable water soluble fraction. Amino acid composition confirms hydrophobic residues as major components of all the extracts, but it reveals an imbalance in acidic residues rates in WSM vs. ASM and in aragonite vs. vaterite. Electrophoresis gives evidence for signatures in proteins with a 140 kDa material specific for aragonite in WSM. Conversely all ASM extracts reveal the presence of about 55 kDa components, including a discrete band in vaterite extract.

The structural basis for the integrity of adenovirus Ad3 dodecahedron.

During the viral life cycle adenoviruses produce excess capsid proteins. Human adenovirus serotype 3 (Ad3) synthesizes predominantly an excess of free pentons, the complexes of pentameric penton base and trimeric fiber proteins, which are responsible for virus penetration. In infected cells Ad3 pentons spontaneously assemble into dodecahedral virus-like nano-particles containing twelve pentons. They also form in insect cells during expression in the baculovirus system. Similarly, in the absence of fiber protein dodecahedric particles built of 12 penton base pentamers can be produced. Both kinds of dodecahedra show remarkable efficiency of intracellular penetration and can be engineered to deliver several millions of foreign cargo molecules to a single target cell. For this reason, they are of great interest as a delivery vector. In order to successfully manipulate this potential vector for drug and/or gene delivery, an understanding of the molecular basis of vector assembly and integrity is critical. Crystallographic data in conjunction with site-directed mutagenesis and biochemical analysis provide a model for the molecular determinants of dodecamer particle assembly and the requirements for stability. The 3.8 Å crystal structure of Ad3 penton base dodecamer (Dd) shows that the dodecahedric structure is stabilized by strand-swapping between neighboring penton base molecules. Such N-terminal strand-swapping does not occur for Dd of Ad2, a serotype which does not form Dd under physiological conditions. This unique stabilization of the Ad3 dodecamer is controlled by residues 59-61 located at the site of strand switching, the residues involved in putative salt bridges between pentamers and by the disordered N-terminus (residues 1-47), as confirmed by site directed mutagenesis and biochemical analysis of mutant and wild type protein. We also provide evidence that the distal N-terminal residues are externally exposed and available for attaching cargo.

Adenovirus dodecahedron, as a drug delivery vector.

BACKGROUND: Bleomycin (BLM) is an anticancer antibiotic used in many cancer regimens. Its utility is limited by systemic toxicity and dose-dependent pneumonitis able to progress to lung fibrosis. The latter can affect up to nearly 50% of the total patient population, out of which 3% will die. We propose to improve BLM delivery by tethering it to an efficient delivery vector. Adenovirus (Ad) dodecahedron base (DB) is a particulate vector composed of 12 copies of a pentameric viral protein responsible for virus penetration. The vector efficiently penetrates the plasma membrane, is liberated in the cytoplasm and has a propensity to concentrate around the nucleus; up to 300000 particles can be observed in one cell in vitro. PRINCIPAL FINDINGS: Dodecahedron (Dd) structure is preserved at up to about 50 degrees C at pH 7-8 and during dialysis, freezing and drying in the speed-vac in the presence of 150 mM ammonium sulfate, as well as during lyophilization in the presence of cryoprotectants. The vector is also stable in human serum for 2 h at 37 degrees C. We prepared a Dd-BLM conjugate which upon penetration induced death of transformed cells. Similarly to free bleomycin, Dd-BLM caused dsDNA breaks. Significantly, effective cytotoxic concentration of BLM delivered with Dd was 100 times lower than that of free bleomycin. CONCLUSIONS/SIGNIFICANCE: Stability studies show that Dds can be conveniently stored and transported, and can potentially be used for therapeutic purposes under various climates. Successful BLM delivery by Ad Dds demonstrates that the use of virus like particle (VLP) results in significantly improved drug bioavailability. These experiments open new vistas for delivery of non-permeant labile drugs.

The HIV-1 envelope glycoprotein gp120 features four heparan sulfate binding domains, including the co-receptor binding site.

It is well established that the human immunodeficiency virus-1 envelope glycoprotein surface unit, gp120, binds to cell-associated heparan sulfate (HS). Virus infectivity is increased by such interaction, and a variety of soluble polyanions efficiently neutralize immunodeficiency virus-1 in vitro. This interaction has been mainly attributed to the gp120 V3 loop. However, although evidence suggested that this particular domain does not fully recapitulate the binding activity of the protein, the ability of HS to bind to other regions of gp120 has not been completely addressed, and the exact localizations of the polysaccharide binding sites are not known. To investigate in more detail the structural basis of the HS-gp120 interaction, we used a mapping strategy and compared the heparin binding activity of wild type and mutant gp120 using surface plasmon resonance-based binding assays. Four heparin binding domains (1-4) were identified in the V2 and V3 loops, in the C-terminal domain, and within the CD4-induced bridging sheet. Interestingly, three of them were found in domains of the protein that undergo structural changes upon binding to CD4 and are involved in co-receptor recognition. In particular, Arg(419), Lys(421), and Lys(432), which directly interact with the co-receptor, are targeted by heparin. This study provides a complete account of the gp120 residues involved in heparin binding and identified several binding surfaces that constitute potential target for viral entry inhibition.

The two major oligomeric forms of human mannan-binding lectin: chemical characterization, carbohydrate-binding properties, and interaction with MBL-associated serine proteases.

Mannan-binding lectin (MBL) is an oligomeric C-type lectin assembled from homotrimeric structural units that binds to neutral carbohydrates on microbial surfaces. It forms individual complexes with MBL-associated serine proteases (MASP)-1, -2, -3 and a truncated form of MASP-2 (MAp19) and triggers the lectin pathway of complement through MASP-2 activation. To characterize the oligomerization state of the two major MBL forms present in human serum, both proteins were analyzed by mass spectrometry. Mass values of 228,098 +/- 170 Da (MBL-I) and 304,899 +/- 229 Da (MBL-II) were determined for the native proteins, whereas reduction of both species yielded a single chain with an average mass of 25,340 +/- 18 Da. This demonstrates that MBL-I and -II contain 9 and 12 disulfide-linked chains, respectively, and therefore are trimers and tetramers of the structural unit. As shown by surface plasmon resonance spectroscopy, trimeric and tetrameric MBL bound to immobilized mannose-BSA and N-acetylglucosamine-BSA with comparable K(D) values (2.2 and 0.55 nM and 1.2 and 0.96 nM, respectively). However, tetrameric MBL exhibited significantly higher maximal binding capacity and lower dissociation rate constants for both carbohydrates. In contrast, no significant difference was detected for binding of the recombinant MASPs or MAp19 to immobilized trimeric or tetrameric MBL. As shown by gel filtration, both MBL species formed 1:2 complexes with MASP-3 or MAp19. These results provide the first precise analysis of the major human MBL oligomers. The oligomerization state of MBL has a direct effect on its carbohydrate-binding properties, but no influence on the interaction with the MASPs.

Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii.

Pyrococcus horikoshii open reading frame PH1527 encodes a 39014 Da protein that shares about 30% identity with endoglucanases and members of the M42 peptidase family. Analytical ultracentrifugation and electron microscopy studies showed that the purified recombinant protein forms stable, large dodecameric complexes with a tetrahedral shape similar to the one described for DAP, a deblocking aminopeptidase that was characterized in the same organism. The two related proteins were named PhTET1 (for DAP) and PhTET2 (for PH1527). The substrate specificity and the mode of action of the PhTET2 complex were studied in detail and compared to those of PhTET1 and other assigned M42 peptidases. When assayed with short chromogenic peptides, PhTET2 was found to be an aminopeptidase, with a clear preference for leucine as the N-terminal amino acid. However, the enzyme can cleave moderately long polypeptide substrates of various compositions in a fairly unspecific manner. The hydrolytic mechanism was found to be nonprocessive. The enzyme has neither carboxypeptidase nor endoproteolytic activities, and it is devoid of N-terminal deblocking activity. PhTET2 was inhibited in the presence of EDTA and bestatin, and cobalt was found to be an activating metal. The PhTET2 protein is a highly thermostable enzyme that displays optimal activity around 100 degrees C over a broad pH array.

A novel strategy for defining critical amino acid residues involved in protein/glycosaminoglycan interactions.

The binding of proteins to glycosaminoglycans (GAGs) is the prerequisite for a large number of cellular processes and regulatory events and is associated to many pathologies. However, progress in the understanding of these mechanisms has been hampered by the lack of simple and comprehensive analytical tools for the identification of the structural attributes involved in protein/saccharide interaction. Characterization of GAG binding motifs on proteins has so far relied on site-directed mutagenesis studies, protein sequence mapping using synthetic peptides, molecular modeling, or structural analysis. Here, we report the development of a novel approach for identifying protein residues involved in the binding to heparin, the archetypal member of the GAG family. This method, which uses native proteins, is based on the formation of cross-linked complexes of the protein of interest with heparin beads, the proteolytic digestion of these complexes, and the subsequent identification of the heparin binding containing peptides by N terminus sequencing. Analysis of the CC chemokine regulated on activation, normal T-cell expressed, and secreted (RANTES), the envelope glycoprotein gC from pseudorabies virus and the laminin-5 alpha 3LG4/5 domain validated the techniques and provided novel information on the heparin binding motifs present within these proteins. Our results highlighted this method as a fast and valuable alternative to existing approaches. Application of this technique should greatly contribute to facilitate the structural study of protein/GAG interactions and the understanding of their biological functions.