Functional characterization of an abnormal factor XII molecule (F XII Bern)

An 18-year-old healthy woman was found to have cross-reacting material (CRM)-positive factor XII (F XII) deficiency, F XII clotting activity was less than 0.01 U/mL, whereas F XII antigen was 0.11 U/mL. An F XII inhibitor was excluded. To partially characterize the molecular defect of the abnormal F XII, immunologic and functional studies were performed on the proposita's plasma. The abnormal F XII was a single chain molecule with the same molecular weight (80 Kd) and the same isoelectric points (pl, 5.9 to 6.8) as normal F XII. Dextran sulfate activation of the proposita's plasma showed no proteolytic cleavage of F XII even after 120 minutes, whereas F XII in pooled normal plasma, diluted 1:10 with CRM-negative F XII-deficient plasma, was completely cleaved after 40 minutes. Adsorption to kaolin was identical for both abnormal and normal F XII. In the presence of dextran sulfate and exogenous plasma kallikrein, the abnormal F XII was cleaved with the same rate as normal F XII. However, kallikrein-cleaved abnormal F XII was not able to cleave factor XI and plasma prekallikrein, in contrast to activated normal F XII. Thus, these studies show that the functional defect of this abnormal F XII, denoted as F XII Bern, is due to the lack of protease activity of the kallikrein-cleaved molecule. Therefore, the structural defect is likely to be located in the light chain region of F XII, containing the enzymatic active site.     

Hb Baden: Structural and functional characterization

Hb Baden (β18Val→Met) is a rare variant hemoglobin that has never been functionally or clinically characterized. We describe a Hb Baden heterozygote who exhibits normal growth and development, as well as age‐ and gender‐appropriate hematological values. Surprisingly, in vitro analyses demonstrate that Hb Baden is relatively unstable and exhibits an abnormally high affinity for O₂. These properties are likely to affect the physiologies of individuals who inherit the β^Baden mutation in trans to a determinant for either a functionally relevant hemoglobinopathy or a mild thalassemia. The data also provide insights into the function of the A‐helix/AB‐segment of β globin, supporting a structural model in which this poorly understood region serves as a scaffold that fixes the positions of other helices that directly impact β‐globin function. Am. J. Hematol., 2010. © 2010 Wiley‐Liss, Inc.     

Structural and functional characterization of novel F7 mutations identified in Chinese factor VII-deficient patients

Hereditary factor VII (FVII) deficiency is a rare recessive bleeding disorder with an estimated prevalence of 1/500 000. We had investigated 50 unrelated Chinese patients with FVII deficiency and identified, in total, 25 mutations, including 18 missense mutations and 5 splicing mutations, on the F7 gene. The nucleotide transition c.1224T>G (p.His408Gln) in exon 9 constitutes a hotspot of mutation, with 19 patients harbouring this genetic variance. Few patients were homozygous or compound heterozygous for deleterious mutations, such as non-sense mutations, large insertion or deletions, indicating that complete deficiency of FVII may not be compatible with life. The eight novel mutations identified in the study, including one small deletion (p.Glu49GlyfsTer101), three type I missense mutations, p.Cys238Phe, p.Gly420Asp, p.Ala252Val and four type II missense mutations, p.Val336Met, p.Ser342Gly, p.Gly432Ser and p.Ile213Asn, were further analysed by in vitro expression and functional studies. The laboratory phenotype and structural analysis confirmed the functional consequence of p.Ile213Asn mutation involving cleavage and activation site. The molecular dynamic simulations and binding energy calculations along with functional probing of p.Gly432Ser mutation revealed the critical role of residue Gly432 in the binding between activated factor VII (factor VIIa) and tissue factor.     

Structural and functional characterization of the mouse von Willebrand factor receptor GPIb-IX with novel monoclonal antibodies

Five novel monoclonal antibodies (mAbs; p0p 1-5) were used to characterize the structural and functional properties and the in vivo expression of the murine GPIb-IX complex (von Willebrand factor receptor). The molecular weights of the subunits are similar to the human homologs: GPIbalpha (150 kd), GPIbbeta (25 kd), and GPIX (25 kd). Activation of platelets with thrombin or PMA predominantly induced shedding of glycocalicin (GC; 130 kd) but only low levels of receptor internalization. The GC concentration in normal mouse plasma was found to be at least 10 times higher than that described for human plasma (approximately 25 microg/mL versus 1-2 microg/mL). Two additional cleavage sites for unidentified platelet-derived proteases were found on GPIbalpha, as demonstrated by the generation of 3 N-terminal fragments during in vitro incubation of washed platelets (GC, 60 kd, 45 kd). Occupancy of GPIbalpha with p0p mAbs or F(ab)(2)-fragments resulted in aggregate formation in vitro and rapid irreversible thrombocytopenia in vivo, irrespective of the exact binding epitopes of the individual antibodies. GPIb-IX was not detectable immunohistochemically on endothelial cells in the major organs under normal or inflammatory conditions. The authors conclude that the mouse system might become an interesting model for studies on GPIb-IX function and regulation.     

Production and characterization of a murine monoclonal antibody against a heavy chain of Hageman factor (factor XII)

A murine hybridoma cell line that produces a monoclonal antibody to human Hageman factor (HF, factor XII) is described. The antibody (P 5-2- 1) consists of mouse IgG2b heavy chains and lambda light chains, selectively neutralizes HF procoagulant activity, and prevents the proteolytic cleavage of HF during contact activation in plasma. When HF is exposed to P 5-2-1 before the absorption of HF to kaolin, HF procoagulant activity is markedly inhibited. In contrast, P 5-2-1 does not interfere with HF activity after the adsorption of HF to kaolin. P 5-2-1 does not inactivate the prekallikrein-activating activity of 28,000-mol wt HF fragments (HFf). P 5-2-1 binds exclusively to the 40,000-mol wt portion of a heavy chain of HF and inhibits the adsorption of HF to negatively charged surfaces. P 5-2-1 immobilized on Sepharose can be used to deplete HF from normal human plasma. This immunoaffinity-depleted plasma is indistinguishable from congenital HF- deficient plasma and can be used as the substrate for HF procoagulant activity assay.     

Structural and functional differences between decay-accelerating factor and red cell acetylcholinesterase

The abnormal erythrocytes in paroxysmal nocturnal hemoglobinuria, both PNH II (the moderately abnormal cells) and PNH III (the markedly abnormal cells), lack both acetylcholinesterase (AChE) activity and decay-accelerating factor (DAF) activity. Both of these activities are found on glycoprotein molecules with a molecular weight of about 70 Kd. To demonstrate that these two activities are in fact on different proteins, we have shown that binding to normal red cells of antibody to DAF does not inhibit the subsequent binding of monoclonal antibody to AChE nor AChE activity. Inhibition of DAF activity by polyclonal antibody increases the susceptibility of normal erythrocytes to lysis by complement but inhibition of AChE activity by antibody does not. The rate of decay of the C3 convertase complex of the classical pathway of complement activation was inhibited by DAF added in the fluid phase but not by AChE. When DAF was exhaustively immunoprecipitated from a solution of the erythrocyte membrane proteins, AChE remained and vice versa. These studies indicate that acetylcholinesterase and decay-accelerating factor are two different proteins, both of which are lacking on PNH II and PNH III erythrocytes.     

Structural and functional characterization of the pore-forming domain of pinholin S2168

Pinholin S²¹68 triggers the lytic cycle of bacteriophage φ21 in infected Escherichia coli. Activated transmembrane dimers oligomerize into small holes and uncouple the proton gradient. Transmembrane domain 1 (TMD1) regulates this activity, while TMD2 is postulated to form the actual “pinholes.” Focusing on the TMD2 fragment, we used synchrotron radiation-based circular dichroism to confirm its α-helical conformation and transmembrane alignment. Solid-state ¹⁵N-NMR in oriented DMPC bilayers yielded a helix tilt angle of τ = 14°, a high order parameter (S_mol = 0.9), and revealed the azimuthal angle. The resulting rotational orientation places an extended glycine zipper motif (G₄₀xxxS₄₄xxxG₄₈) together with a patch of H-bonding residues (T₅₁, T₅₄, N₅₅) sideways along TMD2, available for helix–helix interactions. Using fluorescence vesicle leakage assays, we demonstrate that TMD2 forms stable holes with an estimated diameter of 2 nm, as long as the glycine zipper motif remains intact. Based on our experimental data, we suggest structural models for the oligomeric pinhole (right-handed heptameric TMD2 bundle), for the active dimer (right-handed Gly-zipped TMD2/TMD2 dimer), and for the full-length pinholin protein before being triggered (Gly-zipped TMD2/TMD1-TMD1/TMD2 dimer in a line).

Upon infecting a host, bacteriophages release their newly produced offspring into the environment by lysis of the host cell. The infection cycle of double-stranded DNA bacteriophages that infect gram-negative bacteria is regulated by small viral membrane proteins, so-called holins (1–3). These holins accumulate in an inactive form in the hosts’ cytoplasmic membrane during phage morphogenesis (3). At an allele-specific time, the holins start to form big membrane lesions. Through these holes the muralytic endolysins escape from the cytoplasm and start to degrade the peptidoglycane layer (4, 5). Host lysis is then completed by the Rz–Rz1 spanin complex that disintegrates the outer membrane (6). In addition to this canonical holin endolysin system, a second class of holins, referred to as pinholins, has been described (3, 7). This class is represented by pinholin S²¹68 of lambdoid phage φ21 (7, 8). In contrast to canonical holins, for example those encoded by phage λ, the holes formed by pinholins are much smaller (“pinholes”) and do not allow the passage of proteins (7, 9).The pinholin S²¹68 protein is encoded by gene S²¹ of the lysis cassette, which possesses a dual start motif to allow expression of an antipinholin as well. This antipinholin protein has three additional N-terminal amino acids (M₁-K₂-S₃) and serves as a specific inhibitor of pinholin (Fig. 1A) (8, 10). The (anti)pinholin protein structure consists of two transmembrane domains (TMDs) and an unstructured C terminus (8). It is postulated that TMD1 possesses regulatory functions, while TMD2 is responsible for the actual pinhole formation (8, 11–13). During phage morphogenesis, (anti)pinholin initially accumulates as inactive heterodimers in the cytoplasmic membrane. For pinhole formation, TMD1 has to flip out of the membrane to allow TMD2–TMD2 interactions that build up the pinhole (Fig. 1B) (8, 14). The presence of antipinholin reduces the pinholin activity, because the additional charge on the N terminus delays the flipping of TMD1 (8, 11). Previous cross-linking and modeling studies by Pang et al. (9) have suggested that the pinholes are composed of seven pinholin S²¹68 monomers. These heptamers are supposed to form small holes with an inner diameter of around 1.5 nm, which lead to a collapse of the proton gradient across the membrane (9).Open in a separate windowFig. 1.Pinholin S²¹68. (A) Primary structure of the (anti)pinholin. The dual start motif of gene S²¹ of phage φ21 allows the expression of both antipinholin S²¹71 and pinholin S²¹68. The postulated TMDs are indicated, and our synthetic TMD2 fragment (W₃₆-E₆₂) is underlined. (B) In its inactive state, both helices of pinholin S²¹68 are supposed to be aligned in a transmembrane state (Left). Above a certain threshold concentration, TMD1 is supposed to flip out of the membrane into a surface bound state (Right) (14). This triggering allows further self-assembly through TMD2–TMD2 interactions and leads to the formation of a small pinhole pore by a putative heptamer (9, 13).The machinery also contains associated endolysins carrying an N-terminal secretory signal, called the signal-anchor release (SAR) domain. In their membrane-tethered state, the SAR-endolysins are enzymatically inactive, which prevents premature lysis (15, 16). This inactive state is highly dependent on the presence of an intact membrane potential (7, 17). After disruption of this potential by the formation of pinholes, the SAR-endolysins are released from the membrane, and become refolded and activated during this process (7, 16).Drew et al. (18) demonstrated recently that the wild-type protein is predominately α-helical (∼83%) in DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine; di-14:0-PC) vesicles. Ahammad et al. (14) from the same group used electron paramagnetic resonance (EPR) experiments on synthetic reconstituted pinholin to show that TMD1 is partly externalized from the lipid, whereas TMD2 is stably membrane inserted. In the present study, we aimed to determine the detailed membrane orientation of the postulated pore-forming TMD2 fragment and examine its putative role as the basic homo-/hetero-/oligomeric assembly unit. We used synchrotron radiation-based circular dichroism spectroscopy (SRCD) for quantitative secondary structure determination, and oriented CD (SROCD) for qualitative orientational analysis. Accurate parameters on the helix alignment were obtained by solid-state NMR spectroscopy (ssNMR) on nonperturbing selective ¹⁵N-isotope labels, which yielded a detailed picture of the TMD2 as it is positioned in the membrane. Using a vesicle leakage assay based on ANTS (8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt)/DPX (p-xylene-bis-pyridinium bromide), we proved that TMD2 is indeed the pore-forming domain and could estimate the size of the pinhole from the leakage of fluorescein-isothiocyanate-dextrans (FITC-dextrans, FDs). Based on these experimental data, we have constructed putative models for the pinholin in all three stages of its functional cycle.     

Activation of factor XII by dextran sulfate: the basis for an assay of factor XII

A system was developed for studying the activation of factor XII (Hageman factor) in the presence of dextran sulfate (DS). Salient features of the system included low ionic strength (0.08), low concentration of factor XII (approximately 1/10,000 that in normal plasma), and an excess of exogenous prekallikrein (PK). In this system, factor XII was rapidly converted to the 80,000 molecular weight (mol wt) form of factor XIIa (alpha-factor-XIIa). Once formed, the factor XIIa converted PK to kallikrein at a rate that was proportional to the amount of factor XII originally present in the incubation mixture. This system was used to construct a simple sensitive assay for factor XII in plasma and other biologic samples. The kallikrein produced was measured spectrophotometrically with the chromogenic substrate (H-D-Pro-Phe-Arg- p-nitroanilide (S-2302). This assay was shown to be independent of the high molecular weight kininogen and the PK content of the sample being analyzed. The measurements obtained were consistent with fundamental enzymologic principles and, if desired, could be processed with a simple calculator program to achieve linear standard curves. When applied to the quantitation of factor XII in plasma, the assay yielded values in close agreement with those determined by coagulant assay or by radial immunodiffusion.     

Structural and functional characterization of the nitrite channel NirC from Salmonella typhimurium

Nitrite (NO₂⁻) is a central intermediate in the nitrogen metabolism of microorganisms and plants, and is used as a cytotoxin by macrophages as part of the innate immune response. The bacterial membrane protein NirC acts as a specific channel to facilitate the transport of nitrite anions across lipid bilayers for cytoplasmic detoxification. Despite NirC’s importance in nitrogen metabolism and in the pathogenicity of enteric bacteria, available biochemical data are scarce. Here we present a functional and structural characterization of NirC from Salmonella typhimurium by lipid bilayer electrophysiology and X-ray crystallography. NirC is a pentameric member of the formate/nitrite transporter family of membrane proteins that operates as a channel with high conductance. Single-channel measurements reveal fast and slow gating events but, in contrast to the related FocA formate channel, no pH-dependent gating. A 2.4Å crystal structure of NirC at pH 5 shows similarity to FocA and aquaporins, but lacks the structural asymmetry observed in the formate channel at similarly low pH. Resolved water molecules in the protomers suggest a transport mechanism that also permits a facultative NO₂⁻/H⁺ symport.     

Activated factor XII in rheumatoid arthritis

Rheumatoid arthritis (RA) is associated with premature mortality, with approximately 50% of deaths being due to cardiovascular disease. It has been shown that the increased incidence of cardiovascular disease is independent of traditional risk factors. Previous studies have shown an increased risk of coronary heart disease with increased levels of activated factor XII (FXIIa). The aim of this study was to investigate levels of FXIIa in patients with RA. We studied 32 patients with RA and 30 age- and sex-matched control subjects. We found FXIIa levels significantly increased in the patient group, with 56% of the patients and 6.7% of controls having levels greater than or equal to 2 ng/ml. A previous study has shown that individuals with levels of 2 ng/ml or more have an increased risk of coronary heart disease. Measurement of FXIIa could perhaps help to identify an 'at risk' group of patients, allowing early intervention therapy.     

Combined dys-form of homozygous factor XI deficiency and heterozygous factor XII deficiency

A 12-year-old girl with lifelong hemorrhagic episodes was found to have both a dys-form of homozygous factor XI deficiency and heterozygous factor XII deficiency. The heredity of the coagulation defects was confirmed by family studies. Severe bleeding after dental surgery occurred in spite of replacement therapy and local measures including fibrin glue. Our findings suggest that the risk of bleeding in patients with homozygous factor XI deficiency must not be underestimated and that the most effective measure is the transfusion of sufficient amounts of fresh frozen plasma until at least the 5th postoperative day.     

Behçet's syndrome and factor XII deficiency

Summary Several mechanisms have been proposed to explain thrombotic tendency in Behçet's syndrome. We report the case of a 43-year old woman presenting retinal-vein thrombosis, factor XII deficiency and Behçet's syndrome. This kind of association has thus far never been reported. Factor XII deficiency is known to possibly induce various types of thrombosis and might explain the prevalence of ocular symptoms in our patient.