C-terminal Arginine Residue Research Articles

Alternative Mechanism of Aspirin in Anti-Thrombotic Therapy: Inhibition of Thrombin Activatable Fibrinolysis Inhibitor

The use of aspirin is widely recommended for the prevention of heart attacks owing to its ability to inhibit platelet activation by irreversibly blocking cyclooxygenase 1. However, aspirin also affects the fibrinolytic and hemostatic pathways by mechanisms that are not well understood, causing severe hemorrhagic complications. Here, we investigated the ability of aspirin and aspirin metabolites to inhibit thrombin-activatable fibrinolysis inhibitor (TAFI), the major inhibitor of plasma fibrinolysis. TAFI is activated via proteolytic cleavage by the thrombin-thrombomodulin complex to TAFIa, a carboxypeptidase B-like enzyme. TAFIa modulates fibrinolysis by removing the C-terminal arginine and lysine residues from partially degraded fibrin, which in turn inhibits the binding of plasminogen to fibrin clots. Aspirin and its major metabolites, salicylic acid, gentisic acid, and salicyluric acid, inhibit TAFIa carboxypeptidase activity. Salicyluric acid effectively blocks activation of TAFI by thrombin-thrombomodulin; however, salicylates do not inhibit carboxypeptidase N or pancreatic carboxypeptidase B. Aspirin and other salicylates accelerated the dissolution of fibrin clots and reduced thrombus formation in an in vitro model of fibrinolysis. Inhibition of TAFI represents a novel hemostatic mechanism that contributes to aspirin’s therapy-associated antithrombotic activity and hemorrhagic complications.

Characterization of Cleavage Events in the Multifunctional Cilium Adhesin Mhp684 (P146) Reveals a Mechanism by Which Mycoplasma hyopneumoniae Regulates Surface Topography

ABSTRACT Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50P146, P40P146, and P85P146 that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence 672ATEF↓QQ677, consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1P146-F3P146 that mimic P50P146, P40P146, and P85P146 were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3P146 generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography.

Identification of isomeric 5-hydroxytryptophan- and oxindolylalanine-containing peptides by mass spectrometry

Cells continuously produce reactive oxidative species that can modify all cellular components. In proteins, for example, cysteine, methionine, tryptophan (Trp), and tyrosine residues are particularly prone to oxidation. Here, we report two new approaches to distinguish two isomeric oxidation products of Trp residues, i.e. 5-hydroxytryptophan (5-HTP) and oxindolylalanine (Oia) residues, in peptides. First, 2-nitrobenzenesulfenyl chloride, known to derivatize Trp residues in position 2 of the indole ring, was used to label 5-HTP residues. The mass shift of 152.98 m/z units allowed identifying 5-HTP- besides Trp-containing peptides by mass spectrometry, whereas Oia residues were not labeled. Second, fragmentation of the Oia- and 5-HTP-derived immonium ions at m/z 175.08 produced ions characteristic for each residue that allowed their identification even in the presence of y(1) ions at m/z 175.12 derived from peptides with C-terminal arginine residues. The pseudo MS(3) spectra acquired on a quadrupole time-of-flight hybrid mass spectrometer displayed two signals at m/z 130.05 and m/z 132.05 characteristic for Oia-containing peptides and a group of six signals (m/z 103.04, 120.04, 130.04, 133.03, 146.04, and 148.04) for 5-HTP-cointaining peptides. In both cases, the relative signal intensities appeared to be independent of the sequence providing a specific fingerprint of each oxidative modification.

Matrix-assisted laser desorption/ionisation mass spectrometric response factors of peptides generated using different proteolytic enzymes

Matrix-assisted laser desorption/ionisation (MALDI) mechanisms and the factors that influence the intensity of the ion signal in the mass spectrum remain imperfectly understood. In proteomics, it is often necessary to maximise the peptide response in the mass spectrum, especially for low abundant proteins or for proteolytic peptides of particular significance. We set out to determine which of the common proteolytic enzymes give rise to peptides with the best response factors under MALDI conditions. Standard proteins were enzymatically digested using four common proteases. We assessed relative response factors by coanalyzing the resulting digests. Thus, when tryptic peptides were added in equimolar quantities to their corresponding Asp-N, chymotrypsin and Glu-C digests, tryptic peptide signals were always predominant in the resulting MALDI mass spectra. Observable peaks attributable to non-tryptic peptides generally contained a terminal basic residue. It was proposed that a terminal basic residue has a disproportionate influence upon gas-phase basicity, and this hypothesis was supported by experiments with model isotopically labelled peptides. Experiments applying Cook's kinetic method showed that the peptide with a C-terminal arginine residue was more basic than the equivalent peptide with an N-terminal arginine, which was more basic than the peptide in which the arginine was mid-chain. Thus, the observation of the higher MALDI mass spectrometry response factors of tryptic peptides in comparison with peptides derived using other proteolytic enzymes corresponds with higher gas-phase basicities and may, along with other factors such as the complexity of the digest, influence the choice of enzyme in "bottom-up" proteomic experiments.

Over-Expression of PAI-1 and TAFI Predict Stroke: Two Faces of Ame Coin

Abstract 5249 TAFI (thrombin-activated fibrinolysis inhibitor) and PAI-1 play important roles in fibrinolysis, as they both reduce plasmin generation; PAI-1 a 50 KDa glycoprotein is a serine-proteinase inhibitor; TAFI is a 55 KDa plasma zymogen that is activated by either plasmin/thrombin or thrombin/thrombomodulin complex into an active carboxypeptidase, in particular, it is a basic zinc dependent metallo-carboxipeptidase specific for C-terminal lysine and arginine residue inhibiting degradation of the fibrin clot. We investigated the influence of PAI-1 and TAFI levels on stroke by determining their plasmatic antigen concentration by an electro-immunoassay. We found that PAI-1 levels are increased during the early stage of stroke and that it may also predict clot lysis resistance in patients treated with trombolysis (38.8 ng/ml +/− 14.6, p <0.0001) compared with control subjects (16.2 ng/ml +/−6.8, p <0.0001); TAFI levels were significantly higher in patient with stroke (184.8 nmol/l +/− 62.6 nmol/l), than in healthy control subjects (102.4 nmol/l +/− 36.4 nmol/l, p <0.0001). We have observed that a combination of both PAI-1 higher than 24.6 mg/ml (OR: 12.8. CI: 1.16 to 134.2), and TAFI higher than >180 nmol/l (OR.12.9 CI 1.42 to 116.6), had a sensibility of 92.2%, and a specificity of 98.4% in predicting stroke. The analysis of these results showed that TAFI antigen levels higher than 180nmo/L combined with PAI-1 levels higher than 24 mg/ml are a strong risk factor for clot lysis, stroke (for the evolution, severity, and outcome) and showed a potentially attractive target for fibrinolytic therapies. Disclosures: No relevant conflicts of interest to declare.

Negative Ion Fragmentation of Cysteic Acid Containing Peptides: Cysteic Acid as a Fixed Negative Charge

We present here a study of the collision induced dissociation (CID) of deprotonated cysteic acid containing peptides produced by MALDI. The effect of cysteic acid (C(ox)) position is interrogated by considering the positional isomers, C(ox)LVINVLSQG, LVINVLSQGC(ox), and LVINVC(ox)LSQG. Although considerable variation between the CID spectra is observed, the mechanistic picture that emerges involves charge retention at the deprotonated cysteic acid side chain. Fragmentation occurs in the proximity of the cysteic acid group by charge directed mechanisms as well as remote from this group to form ions, which may be rationalized by charge remote mechanisms. Additionally, the formation of the SO(3)(-•) ion is observed in all cases. Fragmentation of C(ox)LVINVLSQC(ox) provides both N- and C-terminal, y and b ions, respectively indicating that the negative charge may be retained at either of the cysteic acids; however, there is some evidence that charge retention at the C-terminal cysteic acid may be preferred. Fragmentation of tryptic type peptides containing a C-terminal arginine or lysine residue is considered through comparison of three peptides C(ox)LVINKLSQG, C(ox)LVINVLSQK, and C(ox)LVINVLSQR. Lastly, we rationalize the formation of b(n-1)+ H(2)O and a(n-1) ions through a mechanism involving rearrangement of the C-terminal residue to form a mixed anhydride intermediate.

A conserved arginine residue in the terminal protein domain of hepatitis B virus polymerase is critical for RNA pre-genome encapsidation

Hepadnaviruses, including human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV), replicate their DNA genome through reverse transcription. Although hepadnaviral polymerase (Pol) is distantly related to retroviral reverse transcriptases, some of its features are distinct. In particular, in addition to the reverse transcriptase and RNase H domains, which are commonly encoded by retroviral reverse transcriptases, the N-terminally extended terminal protein (TP) domain confers unique features such as protein-priming capability. Importantly, the TP domain is also essential for encapsidation of the viral RNA pre-genome. To gain further insight into the TP domain, this study used clustered charged residue-to-alanine mutagenesis of HBV Pol. Of the 20 charged residues examined, only one arginine (R105) was critical for RNA encapsidation. This result contrasts with previous findings for DHBV Pol regarding the critical residue of the TP domain required for RNA binding. Firstly, R128 of DHBV Pol, which corresponds to R105 of HBV Pol, was reportedly tolerable to alanine substitution for RNA binding. Secondly, the C-terminal arginine residue of the DHBV Pol TP domain (R183) was shown to be critical for RNA binding, whereas alanine substitution of the corresponding arginine residue of the HBV Pol TP domain (R160) remained able to support RNA encapsidation. Together, these data highlight the divergence between avian and mammalian hepadnaviral Pols with respect to an arginine residue critical for RNA encapsidation.

The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: Insights into their use as tools for the removal of affinity tags

Carboxypeptidases may serve as tools for removal of C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline, suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint.

SrGAP2 Arginine Methylation Regulates Cell Migration and Cell Spreading through Promoting Dimerization

The Slit-Robo GTPase-activating proteins (srGAPs) are critical for neuronal migration through inactivation of Rho GTPases Cdc42, Rac1, and RhoA. Here we report that srGAP2 physically interacts with protein arginine methyltransferase 5 (PRMT5). srGAP2 localizes to the cytoplasm and plasma membrane protrusion. srGAP2 knockdown reduces cell adhesion spreading and increases cell migration, but has no effect on cell proliferation. PRMT5 binds to the N terminus of srGAP2 (225-538 aa) and methylates its C-terminal arginine residue Arg-927. The methylation mutant srGAP2-R927A fails to rescue the cell spreading rate, is unable to localize to the plasma membrane leading edge, and perturbs srGAP2 homodimer formation mediated by the F-BAR domain. These results suggest that srGAP2 arginine methylation plays important roles in cell spreading and cell migration through influencing membrane protrusion.

Conversion of arginine to ornithine for improving the fragmentation pattern of peptides labeled with the N-terminal tris(2,4,6-trimethoxyphenyl)phosphonium group in tandem mass spectrometry

We propose a method of converting arginine to ornithine residues by controlled hydrazinolysis, in order to facilitate the sequencing of peptides by tandem mass spectrometry (MS/MS). Whereas the presence of C-terminal arginine residue occurring in a tryptic peptide is generally desirable in MS and MS/MS analyses, that of arginine in the middle of a peptide often adversely affects the appearance of fragment peaks. This problem still arises in peptides, which are acylated with a reagent containing the tris(2,4,6-trimethoxyphenyl)phosphonium (TMPP) group at the N-termini. Using four model peptides, angiotensin III (RVYIHPF), a mucin-related peptide (APDTRPAPG), α-bag cell peptide (1–9) (APRLRFYSL), and [des-Pro2] bradykinin (RPGFSPFR), we optimized the protocol of hydrazinolysis to remove the guanidino group of arginine. Owing to this derivatization, we obtained much simpler MS/MS spectra composing mainly a-type ions characteristic of peptides tagged with the TMPP group. Formylation of the δ-amino group of ornithine further enhanced the efficacy of the derivatization, which would be applicable to the sequencing of non-tryptic peptides.

The Kinetics of Activated Thrombin-Activatable Fibrinolysis Inhibitor with Respect to Plasminogen Binding Site Removal From Fibrin Degradation Products.

Abstract 3185Poster Board III-122TAFI (thrombin activatable fibrinolysis inhibitor, or carboxypeptidase U) is a plasma zymogen that can be activated by thrombin, thrombin-thrombomodulin or plasmin. When activated, TAFIa cleaves C-terminal lysine and arginine residues from plasmin modified fibrin (Fn'). Fn' as a cofactor increases the rate of plasminogen activation by 3-fold over intact fibrin and 3000-fold compared to in the absence of fibrin. Upon extensive treatment with TAFIa, the cofactor activity of TAFIa modified fibrin decreases by approximately 97%. Determining the kinetics of TAFIa will give insight into how much TAFIa is required to efficiently inhibit plasminogen activation and fibrinolysis. The kinetics of TAFIa on its primary physiological substrate were measured by exploiting the binding of plasminogen to fibrin degradation products (FDPs). Fluorescently labeled plasminogen (5IAF-Pg) was equilibrated with FDPs labeled with a quencher, QSY C5-maleimide (QSY-FDP). When 5IAF-Pg is bound to QSY-FDP a baseline fluorescence reading is obtained. When treated with TAFIa, plasminogen binding sites are removed from the QSY-FDP and the fluorescence increases. A model was used to convert the rate of fluorescence increase into the rate of Plasminogen binding site removal. The model includes two distinct binding sites on QSY-FDPs (C-terminal and internal lysines), only one of which is susceptible to removal by TAFIa (C-terminal lysine). 5IAF-Glu-Pg (fluorescent native plasminogen) binds to QSY-FDP with a Kd of 176nM and when QSY-FDP are treated with TAFIa the Kd increases to 1.06μM. It appears that 5IAF-Glu-Pg has the ability to weakly bind TAFIa-treated QSY-FDP, however, the capacity is greatly reduced. Similar binding constants were obtained for 5IAF-Lys-Pg (fluorescent plasmin-cleaved plasminogen) (Kd=92nM; Kd (+TAFIa)=1.55μM). The increase in Kd upon treatment of the QSY-FDP with TAFIa is similar to that observed with 5IAF-Glu-Pg, however, the capacity of the FDPs to bind 5IAF-Lys-Pg is relatively unchanged. The calculated rate of 5IAF-Glu-Pg binding site removal by TAFIa was determined at various QSY-FDP concentrations (0-2 μM). The data are hyperbolic in nature and when fit using the Michaelis-Menten model the kcat and Km of plasminogen binding site removal were 2.34 s-1 and 142.6nM, respectively, implying a catalytic efficiency of 16.41 μM-1s-1. The rate is sensitive to the TAFIa concentration with all TAFIa concentrations (50, 75 and 100pM) yielding similar kinetic parameters. The data described here suggest that TAFIa is very efficient in removing plasminogen binding sites. The catalytic efficiency of TAFIa toward QSY-FDP is 60-fold higher than reported for bradykinin, which was previously the best known substrate of TAFIa. This increased catalytic efficiency is due to a much lower Km (0.146 μM compared to 70.6 μM). These data are reflective of plasminogen site removal and not every C-terminal lysine or arginine cleaved by TAFIa is expected to be involved in plasminogen binding. Therefore, the catalytic efficiency of TAFIa reported here (16.41 μM-1s-1) is likely a lower limit for the true value. DisclosuresNo relevant conflicts of interest to declare.

C-end rule peptides mediate neuropilin-1-dependent cell, vascular, and tissue penetration

Screening of phage libraries expressing random peptides for binding to prostate cancer cells primarily yielded peptides that had a C-terminal arginine (or rarely lysine) residue, usually in a consensus context R/KXXR/K. Phage expressing these sequences and synthetic nanoparticles coated with them bound to and were internalized into cells. The C-terminal arginine (or lysine) was essential to the activity; adding another amino acid, or even blocking the free carboxyl group of this arginine residue by amidation, eliminated the binding and internalizing activity. An internal R/KXXR/K can be exposed and switched on by a cleavage by a protease. The strict requirement for C-terminal exposure of the motif prompted us to term the phenomenon the C-end rule (CendR). Affinity chromatography showed that the CendR peptides bind to neuropilin-1 (NRP-1) on the target cells. NRP-1 is a cell-surface receptor that plays an essential role in angiogenesis, regulation of vascular permeability, and the development of the nervous system. VEGF-A165 and other ligands of NRP-1 possess a C-terminal CendR sequence that interacts with the b1 domain of NRP-1 and causes cellular internalization and vascular leakage. Our CendR peptides have similar effects, particularly when made multivalent through coupling to a particle. We also noted a unique and important activity of these peptides: penetration and transportation through tissues. The peptides were able to take payloads up to the nanoparticle size scale deep into extravascular tissue. Our observations have implications in drug delivery and penetration of tissue barriers and tumors.

Thrombin-activatable fibrinolysis inhibitor and its relation with inflammation in rheumatoid arthritis

Rheumatoid arthritis (RA) is associated with an excessive risk of cardiovascular morbidity and mortality, and inflammation appears to be the missing link explaining this markedly elevated risk.1 Inflammation is a...

The Roles of Selected Arginine and Lysine Residues of TAFI (Pro-CPU) in Its Activation to TAFIa by the Thrombin-Thrombomodulin Complex

Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7-2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1+/-0.6 min, and that of wild type was 8.4+/-0.3 min at 37 degrees C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

Methyl Group Migration During the Fragmentation of Singly Charged Ions of Trimethyllysine-Containing Peptides: Precaution of Using MS/MS of Singly Charged Ions for Interrogating Peptide Methylation

Core histones are susceptible to a range of post-translational modifications (PTMs), including acetylation, phosphorylation, methylation, and ubiquitination, which play important roles in the epigenetic control of gene expression. Here, we observed an unusual discrepancy between MALDI-MS/MS and ESI-MS/MS on the methylation of trimethyllysine-containing peptides with residues 9-17 from human histone H3 and residues 73-83 from yeast histone H3. It turned out that the discrepancy could be attributed to an unusual methyl group migration from the side chain of trimethyllysine to the C-terminal arginine residue during peptide fragmentation, and this methyl group transfer only occurred for singly charged ions, but not for doubly charged ions. The methyl group transfer argument received its support from the results on the studies of the fragmentation of the ESI- or MALDI-produced singly charged ions of several synthetic trimethyllysine-bearing peptides. The results presented in this study highlighted that caution should be exerted while MS/MS of singly charged ions is employed to interrogate the PTMs of trimethyllysine-containing peptides.

Carboxypeptidase Z (CPZ) Links Thyroid Hormone and Wnt Signaling Pathways in Growth Plate Chondrocytes

Carboxypeptidase Z (CPZ) removes carboxyl-terminal basic amino acid residues, particularly arginine residues, from proteins. CPZ contains a cysteine-rich domain (CRD) similar to the CRD found in the frizzled family of Wnt receptors. We have previously shown that thyroid hormone regulates terminal differentiation of growth plate chondrocytes through activation of Wnt-4 expression and Wnt/β-catenin signaling. The Wnt-4 protein contains a C-terminal arginine residue and binds to CPZ through the CRD. The objective of this study was to determine whether CPZ modulates Wnt/β-catenin signaling and terminal differentiation of growth plate chondrocytes. Our results show that CPZ and Wnt-4 mRNA are co-expressed throughout growth plate cartilage. In primary pellet cultures of rat growth plate chondrocytes, thyroid hormone increases both Wnt-4 and CPZ expression, as well as CPZ enzymatic activity. Knockdown of either Wnt-4 or CPZ mRNA levels using an RNA interference technique or blocking CPZ enzymatic activity with the carboxypeptidase inhibitor GEMSA reduces the thyroid hormone effect on both alkaline phosphatase activity and Col10a1 mRNA expression. Adenoviral overexpression of CPZ activates Wnt/β-catenin signaling and promotes the terminal differentiation of growth plate cells. Overexpression of CPZ in growth plate chondrocytes also removes the C-terminal arginine residue from a synthetic peptide consisting of the carboxyl-terminal 16 amino acids of the Wnt-4 protein. Removal of the C-terminal arginine residue of Wnt-4 by site-directed mutagenesis enhances the positive effect of Wnt-4 on terminal differentiation. These data indicate that thyroid hormone may regulate terminal differentiation of growth plate chondrocytes in part by modulating Wnt signaling pathways through the induction of CPZ and subsequent CPZ-enhanced activation of Wnt-4.

Receptor Subtype-specific Docking of Asp6.59 with C-terminal Arginine Residues in Y Receptor Ligands

Y receptors (YRs) are G protein-coupled receptors whose Y(1)R, Y(2)R, and Y(5)R subtypes preferentially bind neuropeptide Y (NPY) and peptide YY, whereas mammalian Y(4)Rs show a higher affinity for pancreatic polypeptide (PP). Comparison of YR orthologs and paralogs revealed Asp(6.59) to be fully conserved throughout all of the YRs reported so far. By replacing this conserved aspartic acid residue with alanine, asparagine, glutamate, and arginine, we now show that this residue plays a crucial role in binding and signal transduction of NPY/PP at all YRs. Sensitivity to distinct replacements is, however, receptor subtype-specific. Next, we performed a complementary mutagenesis approach to identify the contact site of the ligand. Surprisingly, this conserved residue interacts with two different ligand arginine residues by ionic interactions; although in Y(2)R and Y(5)R, Arg(33) is the binding partner of Asp(6.59), in Y(1)R and Y(4)R, Arg(35) of human PP and NPY interacts with Asp(6.59). Furthermore, Arg(25) of PP and NPY is involved in ligand binding only at Y(2)R and Y(5)R. This suggests significant differences in the docking of YR ligands between Y(1/4)R and Y(2/5)R and provides new insights into the molecular binding mode of peptide agonists at GPCRs. Furthermore, the proposed model of a subtype-specific binding mode is in agreement with the evolution of YRs.

Thrombin-activable Fibrinolysis Inhibitor (TAFI) Zymogen Is an Active Carboxypeptidase

Thrombin-activable fibrinolysis inhibitor (TAFI) is a carboxypeptidase found in human plasma, presumably as an inactive zymogen. The current dogma is that proteolytic activation by thrombin/thrombomodulin generates the active enzyme (TAFIa), which down-regulates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. In this study, we have shown that the zymogen exhibits continuous and stable carboxypeptidase activity against large peptide substrates, and we suggest that the activity down-regulates fibrinolysis in vivo.

The human cathepsin H gene encodes two novel minor histocompatibility antigen epitopes restricted by HLA-A*3101 and -A*3303

Minor histocompatibility antigens (mHags) play crucial roles in the induction of graft versus host disease (GVHD) and/or graft versus leukaemia (GVL) effects following human leucocyte antigen (HLA)-identical haematopoietic stem cell transplantation (HSCT). Using HLA-A*3101- and -A*3303-restricted cytotoxic T lymphocyte (CTL) clones generated from different post-HSCT recipients, we identified two novel mHag epitopes encoded by the leader sequence of cathepsin H (CTSH) isoform a. The nonameric sequence ATLPLLCAR was defined as an HLA-A*3101-restricted epitope (CTSH(R)/A31), while a decameric peptide featuring a one N-terminal amino acid extension, WATLPLLCAR, was presented by HLA-A*3303 (CTSH(R)/A33). The immunogenicity of both epitopes was totally dependent on the polymorphic C-terminal arginine residue and substitution with glycine completely abolished binding to the corresponding HLA molecules. Thus, the immunogenicity of this mHag is exerted by differential HLA binding capacity. CTSH is relatively ubiquitously expressed at protein levels, thus it may be involved in GVHD and anti-leukaemic/tumour responses. Interestingly, however, CTL clones predominantly lysed targets of haematopoietic cell origin, which could not be explained in terms of the immunoproteasome system. Although the mechanisms involved in the differential susceptibility remain to be determined, these data suggest that CTSH-encoded mHags could be targets for GVL effects.

Fragmentation of doubly-protonated peptide ion populations labeled by H/D exchange with CD 3OD

Doubly-protonated bradykinin (RPPGFSPFR) and an angiotensin III analogue (RVYIFPF) were subjected to hydrogen/deuterium (H/D) exchange with CD 3OD in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. A bimodal distribution of deuterium incorporation was present for bradykinin after H/D exchange for 90 s at a CD 3OD pressure of 4 × 10 −7 Torr, indicating the existence of at least two distinct populations. Bradykinin ion populations corresponding to 0–2 and 5–11 deuteriums (i.e., D 0, D 1, D 2, D 5, D 6, D 7, D 8, D 9, D 10, and D 11) were each monoisotopically selected and fragmented via sustained off-resonance irradiation (SORI) collision-induced dissociation (CID). The D 0–D 2 ion populations, which correspond to the slower exchanging population, consistently require lower SORI amplitude to achieve a similar precursor ion survival yield as the faster-reacting (D 5–D 11) populations. These results demonstrate that conformation/protonation motif has an effect on fragmentation efficiency for bradykinin. Also, the partitioning of the deuterium atoms into fragment ions suggests that the C-terminal arginine residue exchanges more rapidly than the N-terminal arginine. Total deuterium incorporation in the b 1/y 8 and b 2/y 7 ion pairs matches very closely the theoretical values for all ion populations studied, indicating that the ions of a complementary pair are likely formed during the same fragmentation event, or that no scrambling occurs upon SORI. Deuterium incorporation into the y 1/a 8 pseudo-ion pair does not closely match the expected theoretical values. The other peptide, doubly-protonated RVYIFPF, has a trimodal distribution of deuterium incorporation upon H/D exchange with CD 3OD at a pressure of 1 × 10 −7 Torr for 600 s, indicating at least three distinct ion populations. After 90 s of H/D exchange where at least two distinct populations are detected, the D 0–D 7 ion populations were monoisotopically selected and fragmented via SORI-CID over a range of SORI amplitudes. The precursor ion survival yield as a function of SORI amplitude falls into two distinct behaviors corresponding to slower- and faster-reacting ion populations. The slower-reacting population requires larger SORI amplitudes to achieve the same precursor ion survival yield as the faster exchanging population. Total deuterium incorporation into the y 2/b 5 ion pairs matches closely the theoretical values over all ion populations and SORI amplitudes studied. This result indicates the y 2 and b 5 ions are likely formed by the same mechanism over the SORI amplitudes studied.