Discerning the Role of the Hydroxyproline Residue in the Structure of Conantokin Rl-B and Its Role in GluN2B Subunit-Selective Antagonistic Activity toward N-Methyl-d-Aspartate Receptors.

Conantokins (con) are short γ-carboxyglutamate (Gla)-containing polypeptides expressed by marine snails that function as antagonists of N-methyl-d-aspartate receptor (NMDAR) ion channels. The Gla residues govern structural conformations and antagonistic activities of the conantokins. In addition to Gla, some conantokins, e.g., conRl-B, also contain a hydroxyproline (HyP or O) residue, which in this case is centrally located in the peptide at position 10. Because conRl-B specifically inhibits ion channels of GluN2B subunit-containing heterotetrameric NMDARs, we evaluated the unusual role of HyP10 in this effect. To accomplish this goal, we examined synthetic variants of conRl-B in which HyP10 was either deleted (conRl-B[ΔO10]) or replaced with alanine (conRl-B[O10A]) or proline (conRl-B[O10P]). The solution structures of these variants were determined by nuclear magnetic resonance spectroscopy. Deletion of HyP10, or replacement of HyP10 with Ala10, attenuated the distortion in the central region of the apo-conRl-B helix and allowed Mg2+-complexed end-to-end α-helix formation. The inhibitory properties of these variants were assessed by measuring NMDA/Gly-stimulated intracellular Ca2+ influx in mice neurons. ConRl-B[O10P] retained its NMDAR ion channel inhibitory activity in wild-type (WT) neurons but lost its GluN2B specificity, whereas conRl-B[ΔO10] showed overall diminished inhibitory function. ConRl-B[O10A] showed attenuated inhibitory function but retained its GluN2B specificity. Thus, HyP10 plays a critical role in maintaining the structural integrity of conRl-B, which can be correlated with its GluN2B subunit-selective inhibition. Weakened inhibition by conRl-B was also observed in neurons lacking either the GluN2C or GluN2D subunit, compared to WT neurons. This suggests that GluN2C and GluN2D are also required for inhibition by conRl-B.

Quantifying vitamin K-dependent holoprotein compaction caused by differential γ-carboxylation using high-pressure size exclusion chromatography.

This study uses high-pressure size exclusion chromatography (HPSEC) to quantify divalent metal ion (X(2+))-induced compaction found in vitamin K-dependent (VKD) proteins. Multiple X(2+) binding sites formed by the presence of up to 12 γ-carboxyglutamic acid (Gla) residues are present in plasma-derived FIX (pd-FIX) and recombinant FIX (r-FIX). Analytical ultracentrifugation (AUC) was used to calibrate the Stokes radius (R) measured by HPSEC. A compaction of pd-FIX caused by the filling of Ca(2+) and Mg(2+) binding sites resulted in a 5 to 6% decrease in radius of hydration as observed by HPSEC. The filling of Ca(2+) sites resulted in greater compaction than for Mg(2+) alone where this effect was additive or greater when both ions were present at physiological levels. Less X(2+)-induced compaction was observed in r-FIX with lower Gla content populations, which enabled the separation of biologically active r-FIX species from inactive ones by HPSEC. HPSEC was sensitive to R changes of approximately 0.01nm that enabled the detection of FIX compaction that was likely cooperative in nature between lower avidity X(2+) sites of the Gla domain and higher avidity X(2+) sites of the epidermal growth factor 1 (EGF1)-like domain.

Noncanonical amino acids to improve the pH response of pHLIP insertion at tumor acidity.

The pH low insertion peptide (pHLIP) offers the potential to deliver drugs selectively to the cytoplasm of cancer cells based on tumor acidosis. The WT pHLIP inserts into membranes with a pH50 of 6.1, while most solid tumors have extracellular pH (pH(e)) of 6.5-7.0. To close this gap, a SAR study was carried out to search for pHLIP variants with improved pH response. Replacing Asp25 with α-aminoadipic acid (Aad) adjusts the pH50 to 6.74, matching average tumor acidity, and replacing Asp14 with γ-carboxyglutamic acid (Gla) increases the sharpness of pH response (transition over 0.5 instead of 1 pH unit). These effects are additive: the Asp14Gla/Asp25Aad double variant shows a pH50 of 6.79, with sharper transition than Asp25Aad. Furthermore, the advantage of the double variant over WT pHLIP in terms of cargo delivery was demonstrated in turn-on fluorescence assays and anti-proliferation studies (using paclitaxel as cargo) in A549 lung cancer cells at pH 6.6.

Role of gamma carboxylated Glu47 in connexin 26 hemichannel regulation by extracellular Ca²âº: insight from a local quantum chemistry study.

Connexin hemichannels are regulated by several gating mechanisms, some of which depend critically on the extracellular Ca(2+) concentration ([Ca(2+)]e). It is well established that hemichannel activity is inhibited at normal (∼1 mM) [Ca(2+)]e, whereas lowering [Ca(2+)]e to micromolar levels fosters hemichannel opening. Atomic force microscopy imaging shows significant and reversible changes of pore diameter at the extracellular mouth of Cx26 hemichannels exposed to different [Ca(2+)]e, however, the underlying molecular mechanisms are not fully elucidated. Analysis of the crystal structure of connexin 26 (Cx26) gap junction channels, corroborated by molecular dynamics (MD) simulations, suggests that several negatively charged amino acids create a favorable environment for low-affinity Ca(2+) binding within the extracellular vestibule of the Cx26 hemichannel. In particular a highly conserved glutammic acid, found in position 47 in most connexins, is thought to undergo post translational gamma carboxylation (γGlu47), and is thus likely to play an important role in Ca(2+) coordination. γGlu47 may also form salt bridges with two conserved arginines (Arg75 and Arg184 in Cx26), which are considered important in stabilizing the structure of the extracellular region. Using a combination of quantum chemistry methods, we analyzed the interaction between γGlu47, Arg75 and Arg184 in a Cx26 hemichannel model both in the absence and in the presence of Ca(2+). We show that Ca(2+) imparts significant local structural changes and speculate that these modifications may alter the structure of the extracellular loops in Cx26, and may thus account for the mechanism of hemichannel closure in the presence of mM [Ca(2+)]e.

Factor VII and protein C are phosphatidic acid-binding proteins.

Seven proteins in the human blood clotting cascade bind, via their GLA (γ-carboxyglutamate-rich) domains, to membranes containing exposed phosphatidylserine (PS), although with membrane binding affinities that vary by 3 orders of magnitude. Here we employed nanodiscs of defined phospholipid composition to quantify the phospholipid binding specificities of these seven clotting proteins. All bound preferentially to nanobilayers in which PS headgroups contained l-serine versus d-serine. Surprisingly, however, nanobilayers containing phosphatidic acid (PA) bound substantially more of two of these proteins, factor VIIa and activated protein C, than did equivalent bilayers containing PS. Consistent with this finding, liposomes containing PA supported higher proteolytic activity by factor VIIa and activated protein C toward their natural substrates (factors X and Va, respectively) than did PS-containing liposomes. Moreover, treating activated human platelets with phospholipase D enhanced the rates of factor X activation by factor VIIa in the presence of soluble tissue factor. We hypothesize that factor VII and protein C bind preferentially to the monoester phosphate of PA because of its accessibility and higher negative charge compared with the diester phosphates of most other phospholipids. We further found that phosphatidylinositol 4-phosphate, which contains a monoester phosphate attached to its myo-inositol headgroup, also supported enhanced enzymatic activity of factor VIIa and activated protein C. We conclude that factor VII and protein C bind preferentially to monoester phosphates, which may have implications for the function of these proteases in vivo.

Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors.

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 µm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.

Activated protein C cofactor function of protein S: a novel role for a γ-carboxyglutamic acid residue.

Protein S has an important anticoagulant function by acting as a cofactor for activated protein C (APC). We recently reported that the EGF1 domain residue Asp95 is critical for APC cofactor function. In the present study, we examined whether additional interaction sites within the Gla domain of protein S might contribute to its APC cofactor function. We examined 4 residues, composing the previously reported "Face1" (N33S/P35T/E36A/Y39V) variant, as single point substitutions. Of these protein S variants, protein S E36A was found to be almost completely inactive using calibrated automated thrombography. In factor Va inactivation assays, protein S E36A had 89% reduced cofactor activity compared with wild-type protein S and was almost completely inactive in factor VIIIa inactivation; phospholipid binding was, however, normal. Glu36 lies outside the ω-loop that mediates Ca(2+)-dependent phospholipid binding. Using mass spectrometry, it was nevertheless confirmed that Glu36 is γ-carboxylated. Our finding that Gla36 is important for APC cofactor function, but not for phospholipid binding, defines a novel function (other than Ca(2+) coordination/phospholipid binding) for a Gla residue in vitamin K-dependent proteins. It also suggests that residues within the Gla and EGF1 domains of protein S act cooperatively for its APC cofactor function.

Using the same organocatalyst for asymmetric synthesis of both enantiomers of glutamic acid-derived Ni(II) complexes via 1,4-additions of achiral glycine and dehydroalanine Schiff base Ni(II) complexes.

(S)- and (R)-BIMBOL were efficient PT catalysts of asymmetric Michael addition of prochiral Ni-PBP-Gly (1) to acrylic esters and malonic esters to Ni-PBP-Δ-Ala (2) correspondingly. The salient feature of the catalysis is opposite configurations of Glu prepared via the two paths with BIMBOL of the same configuration and a perspective novel catalytic procedure for the synthesis of Gla derivatives.

Effect of anticoagulation with citrate versus heparin on the adsorption of coagulation factors to blood purification resins with different charge.

In liver failure, hydrophobic toxins accumulate in the blood circulation. To support hepatic function, extracorporeal blood purification systems have been developed, in which both cationic and neutral adsorbents are used to remove albumin-bound metabolites from blood. An issue of these systems is the additional removal of coagulation factors containing negatively charged γ-carboxyglutamate (Gla) domains, which, in physiological conditions, are shielded by calcium ions. We hypothesized that complexation of calcium ions by citrate leads to exposure of negative Gla domains, resulting in their binding to the positively charged adsorbents. The data presented here confirm that the binding of coagulation factors containing Gla domains to positively charged polymers is enhanced in the presence of citrate as compared to heparin. This effect increased with increasing charge density of the polymer and has important implications for the clinical application of positively charged polymers.

Carboxylator: incorporating solvent-accessible surface area for identifying protein carboxylation sites.

In proteins, glutamate (Glu) residues are transformed into γ-carboxyglutamate (Gla) residues in a process called carboxylation. The process of protein carboxylation catalyzed by γ-glutamyl carboxylase is deemed to be important due to its involvement in biological processes such as blood clotting cascade and bone growth. There is an increasing interest within the scientific community to identify protein carboxylation sites. However, experimental identification of carboxylation sites via mass spectrometry-based methods is observed to be expensive, time-consuming, and labor-intensive. Thus, we were motivated to design a computational method for identifying protein carboxylation sites. This work aims to investigate the protein carboxylation by considering the composition of amino acids that surround modification sites. With the implication of a modified residue prefers to be accessible on the surface of a protein, the solvent-accessible surface area (ASA) around carboxylation sites is also investigated. Radial basis function network is then employed to build a predictive model using various features for identifying carboxylation sites. Based on a five-fold cross-validation evaluation, a predictive model trained using the combined features of amino acid sequence (AA20D), amino acid composition, and ASA, yields the highest accuracy at 0.874. Furthermore, an independent test done involving data not included in the cross-validation process indicates that in silico identification is a feasible means of preliminary analysis. Additionally, the predictive method presented in this work is implemented as Carboxylator ( http://csb.cse.yzu.edu.tw/Carboxylator/ ), a web-based tool for identifying carboxylated proteins with modification sites in order to help users in investigating γ-glutamyl carboxylation.

Membrane-dependent interaction of factor Xa and prothrombin with factor Va in the prothrombinase complex.

Because all three protein components of prothrombinase, factors (f) Xa and Va and prothrombin, bind to negatively charged membrane phospholipids, the exact role of the membrane in the prothrombinase reaction has not been fully understood. In this study, we prepared deletion derivatives of fXa and prothrombin in which both the Gla and first EGF-like domains of the protease (E2-fXa) as well as the Gla and both kringle domains of the substrate (prethrombin-2) had been deleted. The fVa-mediated catalytic activity of E2-fXa toward prethrombin-2 was analyzed in both the absence and presence of phospholipids composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS). PCPS markedly accelerated the initial rate of prethrombin-2 activation by E2-fXa, with the cofactor exhibiting saturation only in the presence of phospholipids (apparent K(d) of approximately 60 nM). Competitive kinetic studies in the presence of the two exosite-1-specific ligands Tyr(63)-sulfated hirudin(54-65) and TM456 suggested that while both peptides are highly effective inhibitors of the fVa-mediated activation of prethrombin-2 by E2-fXa in the absence of PCPS, they are ineffective competitors in the presence of phospholipids. Since neither E2-fXa nor prethrombin-2 can interact with membranes, these results suggest that interaction of fVa with PCPS improves the affinity of the activation complex for proexosite-1 of the substrate. Direct binding studies employing OG(488)-EGR-labeled fXa and E2-fXa revealed that the interaction of the Gla domain of fXa with PCPS also induces conformational changes in the protease to facilitate its high-affinity interaction with fVa.

Metal-binding affinity and selectivity of nonstandard natural amino acid residues from DFT/CDM calculations.

Unnatural amino acid residues are increasingly being used in metalloprotein design and engineering to expand the repertoire of protein structures/folds and functions. However, natural but nonstandard amino acid residues (not in the basic set of 20) possessing metal-ligating groups such as selenocysteine (Sec), pyrrolysine (Pyl), and gamma-carboxyglutamic acid (Gla) have attracted little attention, and their potential as metal-binding entities in metalloprotein engineering has not been assessed. In particular, the metal-binding affinity/selectivity of these three rare residues remains unclear. Herein, the metal-binding affinity/selectivity of the Gla, Pyl, and Sec side chains have been systematically studied using a combined density functional theory and continuum dielectric method. The calculations reveal an advantage of using these noncanonical protein building blocks instead of the standard 20 amino acid residues. Gla2-, Pyl0, and Sec- have greater potential in trapping the metal cation than their standard amino acid counterparts. They prefer binding to Zn2+ rather than to Mg2+ or Ca2+ in a protein cavity due to the better electron-accepting ability and lower coordination number preference of Zn2+, as compared to Mg2+ and Ca2+. Between Ca2+ and Mg2+, Gla2- prefers Ca2+, whereas Pyl0 and Sec- poorly discriminate between the two metal cations. The results herein suggest that Gla2-, Pyl0, and Sec- could be employed as very efficient metal-binding entities in engineering metalloproteins with preprogrammed properties.

The PT1-Ca2+ Gla domain binds to a membrane through two dipalmitoylphosphatidylserines. A computational study.

Binding of vitamin K-dependent proteins to cell membranes containing phosphatidylserine (PS) via gamma-carboxyglutamic acid (Gla) domains is one of the essential steps in the blood coagulation pathway. During activation of the coagulation cascade, prothrombin is converted to thrombin by prothrombinase, a complex consisting of serine protease FXa and cofactor FVa, anchored to anionic phospholipids on the surface of activated platelets in the presence of calcium ions. To investigate the binding of the Gla domain of prothrombin fragment 1 (PT1) to anionic lipids in the presence of Ca2+, we have conducted MD simulations of the protein with one and two dipalmitoylphosphatidylserines (DPPS) in a dipalmitoylphosphatidylcholine (DPPC) bilayer membrane. The results show a well-defined phosphatidylserine binding site, which agrees generally with crystallographic studies [Huang, M., et al. (2003) Nat. Struct. Biol. 10, 751-756]. However, in the presence of the lipid membrane, some of the interactions observed in the crystal structure adjust during the simulations possibly because in our system the PT1-Ca2+ complex is embedded in a DPPC lipid membrane. Our simulations confirm the existence of a second phospholipid headgroup binding site on the opposite face of the PT1-Ca2+ complex as suggested by MacDonald et al. [(1997) Biochemistry 36, 5120-5127]. The serine headgroup in the second site binds through a Gla domain-bound calcium ion Ca1, Gla30, and Lys11. On the basis of free energy simulations, we estimate the energy of binding of the PT1-Ca2+ complex to a single DPPS to be around -11.5 kcal/mol. The estimated free energy of binding of a DPPS lipid to the second binding site is around -8.8 kcal/mol and is in part caused by the nature of the second site and in part by entropic effects.

Active and exo-site inhibition of human factor Xa: structure of des-Gla factor Xa inhibited by NAP5, a potent nematode anticoagulant protein from Ancylostoma caninum.

Hookworms are hematophagous nematodes capable of growth, development and subsistence in living host systems such as humans and other mammals. Approximately one billion, or one in six, people worldwide are infected by hookworms causing gastrointestinal blood loss and iron deficiency anemia. The hematophagous hookworm Ancylostoma caninum produces a family of small, disulfide-linked protein anticoagulants (75-84 amino acid residues). One of these nematode anticoagulant proteins, NAP5, inhibits the amidolytic activity of factor Xa (fXa) with K(i)=43 pM, and is the most potent natural fXa inhibitor identified thus far. The crystal structure of NAP5 bound at the active site of gamma-carboxyglutamic acid domainless factor Xa (des-fXa) has been determined at 3.1 A resolution, which indicates that Asp189 (fXa, S1 subsite) binds to Arg40 (NAP5, P1 site) in a mode similar to that of the BPTI/trypsin interaction. However, the hydroxyl group of Ser39 of NAP5 additionally forms a hydrogen bond (2.5 A) with His57 NE2 of the catalytic triad, replacing the hydrogen bond of Ser195 OG to the latter in the native structure, resulting in an interaction that has not been observed before. Furthermore, the C-terminal extension of NAP5 surprisingly interacts with the fXa exosite of a symmetry-equivalent molecule forming a short intermolecular beta-strand as observed in the structure of the NAPc2/fXa complex. This indicates that NAP5 can bind to fXa at the active site, or the exosite, and to fX at the exosite. However, unlike NAPc2, NAP5 does not inhibit fVIIa of the fVIIa/TF complex.

Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks.

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPM gammaDPLgammaIIRI-nh2, (gamma, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.

Conantokin-P, an unusual conantokin with a long disulfide loop.

The conantokins are a family of Conus venom peptides (17-27AA) that are N-methyl-d-aspartate (NMDA) receptor antagonists. Conantokins lack disulfide bridges (six out of seven previously characterized peptides are linear), but contain multiple residues of gamma-carboxyglutamate. These post-translationally modified amino acids confer the largely helical structure of conantokins by coordinating divalent metal ions. Here, we report that a group of fish-hunting cone snails, Conus purpurascens and Conus ermineus, express a distinctive branch of the conantokin family in their venom ducts. Two novel conantokins, conantokin-P (Con-P) and conantokin-E (Con-E) are 24AA long and contain five gamma-carboxyglutamate residues. These two peptides are characterized by a long disulfide loop (12 amino acids including two Gla residues between the Cys residues). The oxidative folding studies of Con-P revealed that the formation of the disulfide bond proceeded significantly faster in the presence of Ca(++) ions. Circular dichroism suggested that Con-P is less helical than other previously characterized conantokins. Con-P blocks NMDA receptors containing NR2B subunit with submicromolar potency. Furthermore, the subtype-selectivity for different NR2 subunits differs from that of the previously characterized conantokins. Our results suggest that different branches of the phylogenetic tree of cone snails have evolved distinct groups of conantokins, each with its own unique biochemical features.

Snake-venom-derived Factor IX-binding protein specifically blocks the gamma-carboxyglutamic acid-rich-domain-mediated membrane binding of human Factors IX and X.

A potent anticoagulant protein, IX-bp (Factor IX binding protein), has been isolated from the venom of Trimeresurus flavoviridis (habu snake) and is known to bind specifically to the Gla (gamma-carboxyglutamic acid-rich) domain of Factor IX. To evaluate the molecular basis for its anticoagulation activity, we assessed its interactions with various clotting factors. We found that the anticoagulation activity is primarily due to binding to the Gla domains of Factors IX and X, thus preventing these factors from recognizing phosphatidylserine on the plasma membrane. The present study suggests that ligands that bind to the Gla domains of Factors IX and X may have the potential to become novel anticoagulants.

Preferential localization of recombinant factor VIIa to platelets activated with a combination of thrombin and a glycoprotein VI receptor agonist.

BACKGROUND: Activation of platelets with a combination of collagen and thrombin generates a subpopulation of highly procoagulant 'coated' platelets characterized by high surface expression of fibrinogen and other procoagulant proteins. OBJECTIVES: To analyze the interaction of recombinant factor VIIa (rFVIIa) with coated platelets. METHODS AND RESULTS: rFVIIa localized to the coated platelets in flow cytometry experiments, while minimal rFVIIa was found on platelets activated with adenosine diphosphate, thrombin or via glycoprotein VI individually, and essentially no rFVIIa was found on non-stimulated platelets. Removal of the gamma-carboxyglutamic acid (Gla) domain of rFVIIa, and addition of EDTA, annexin V or excess prothrombin inhibited rFVIIa localization to the coated platelets, indicating that the interaction was mediated by the calcium-dependent conformation of the Gla domain and platelet exposure of negatively charged phospholipids. A reduced level of platelet fibrinogen exposure was observed at hemophilia A-like conditions in a model system of cell-based coagulation, indicating that coated platelet formation in hemophilia may be diminished. Addition of rFVIIa dose-dependently enhanced thrombin generation and partly restored platelet fibrinogen exposure. CONCLUSIONS: The data suggest that rFVIIa localized preferentially on platelets activated with dual agonists, thereby ensuring enhanced thrombin generation localized at the site of injury where both collagen and tissue factor are exposed, the latter ensuring the formation of thrombin necessary for coated platelet formation.

Novel gamma-carboxyglutamic acid-containing peptides from the venom of Conus textile.

The cone snail is the only invertebrate system in which the vitamin K-dependent carboxylase (or gamma-carboxylase) and its product gamma-carboxyglutamic acid (Gla) have been identified. It remains the sole source of structural information of invertebrate gamma-carboxylase substrates. Four novel Gla-containing peptides were purified from the venom of Conus textile and characterized using biochemical methods and mass spectrometry. The peptides Gla(1)-TxVI, Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI each have six Cys residues and belong to the O-superfamily of conotoxins. All four conopeptides contain 4-trans-hydroxyproline and the unusual amino acid 6-l-bromotryptophan. Gla(2)-TxVI/A and Gla(2)-TxVI/B are isoforms with an amidated C-terminus that differ at positions +1 and +13. Three isoforms of Gla(3)-TxVI were observed that differ at position +7: Gla(3)-TxVI, Glu7-Gla(3)-TxVI and Asp7-Gla(3)-TxVI. The cDNAs encoding the precursors of the four peptides were cloned. The predicted signal sequences (amino acids -46 to -27) were nearly identical and highly hydrophobic. The predicted propeptide region (-20 to -1) that contains the gamma-carboxylation recognition site (gamma-CRS) is very similar in Gla(2)-TxVI/A, Gla(2)-TxVI/B and Gla(3)-TxVI, but is more divergent for Gla(1)-TxVI. Kinetic studies utilizing the Conusgamma-carboxylase and synthetic peptide substrates localized the gamma-CRS of Gla(1)-TxVI to the region -14 to -1 of the polypeptide precursor: the Km was reduced from 1.8 mm for Gla (1)-TxVI lacking a propeptide to 24 microm when a 14-residue propeptide was attached to the substrate. Similarly, addition of an 18-residue propeptide to Gla(2)-TxVI/B reduced the Km value tenfold.

From gamma-carboxy-glutamate to protein C.

The story I shall recount started in 1969, when I was given the opportunity to join the Department of Clinical Chemistry at the University Hospital in Malmö. I had just finished medical school at the university in the neighboring town of Lund. Parallel to pursuing my medical studies I had spent some time in the Department of Biochemistry. I did not know much about biochemistry, but it was enough for me to realize that I wanted to do laboratory research rather than developing a clinical career. I was happy to accept an offer to start working in the laboratory, particularly as the head of the department, Professor Carl-Bertil Laurell, had an excellent reputation. As it turned out, I came to spend almost all of my professional life in the laboratory.