The prevalence of proteolytic antibodies against factor VIII in hemophilia A.

BACKGROUND: Factor VIII inhibitors are IgG alloantibodies that arise during replacement therapy in 25 to 50 percent of patients with severe hemophilia A. The hydrolysis of factor VIII by anti--factor VIII antibodies has been proposed as a mechanism of inactivation of factor VIII. METHODS: We purified IgG from patients with severe hemophilia A. The proteolytic activity of the antibodies was assessed by incubating the IgG with biotinylated human factor VIII and analyzing patterns of factor VIII cleavage by sodium dodecyl sulfate--polyacrylamide-gel electrophoresis and immunoblotting. The controls were normal human IgG and IgG purified from plasma of patients with hemophilia who did not have inhibitory antibodies. RESULTS: Significant proteolytic activity was detected in IgG from 13 of 24 inhibitor-positive patients. No hydrolytic activity was detected in control antibodies of IgG from patients without inhibitors. The rate of hydrolysis of factor VIII by purified IgG correlated positively with the factor VIII--neutralizing activity of IgG in plasma (r2=0.67, P=0.029). Principal-component analysis of migration profiles of digestion fragments demonstrated the heterogeneity of the catalytic potential of factor VIII inhibitors among patients. CONCLUSIONS: Proteolysis is a mechanism by which IgG antibodies against factor VIII can inactivate factor VIII.

Why do p-nitro-substituted aryl azides provide unintended dark reactions with proteins?

Aryl azide-mediated photo cross-linking has been widely used to obtain structural features in biological systems, even though the reactive species generated upon photolysis in aqueous solution have not been well characterized. We have established a mechanistic framework for the formation of adducts between photoactivated 5-azido-2-nitrobenzoyl reagents and protein functional groups. Photolysis of the aryl azide tethered to biotin via an amide linkage yields a cross-link with streptavidin. The ability of the pre-irradiated reagent to form a similar cross-link indicates that it is the long-lived reactive intermediate that contributes to the cross-link formation. The reactive intermediate forms an adduct with tryptophan. The sequence of the labeled peptide is found to be GlyTrp(*)ThrValAlaTrp(*)LysAsn, corresponding to residues 74-81 of the streptavidin sequence, where Trp(*) designates the modified Trp-75 and Trp-79. A peak at m/z 1455.1 corresponding to the calculated [M(peptide)+aryl nitrene+2O](+) molecular ion value has been observed for the labeled peptide. Product structure identification experiments support the assignment that the long-lived reactive intermediate is a p-nitro-N-arylhydroxylamine, which undergoes a number of transformations in aqueous solution leading to nitroso derivatives. A plausible mechanism of the interaction between tryptophan and nitroso compound is discussed.

IgG from patients with systemic sclerosis bind to DNA antitopoisomerase 1 in normal human fibroblasts extracts.

By using a semi-quantitative immunoblotting technique, we have analyzed serum immunoglobulin G (IgG) reactivities of patients with limited cutaneous systemic sclerosis and anticentromere antibodies, patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies, patients with diffuse systemic sclerosis without antitopoisomerase 1 or anticentromere antibodies and age- and gender-matched healthy controls with normal human skin fibroblasts and HEp-2 cells antigens. Serum IgG reactivities of patients with diffuse systemic sclerosis and antitopoisomerase 1 antibodies differed significantly from those of healthy controls or systemic sclerosis patients in other groups for reactivity with fibroblast proteins. IgG from patients with antitopoisomerase 1 antibodies bound to a 90 kDa fibroblast band and to a 100 kDa protein band in a HEp-2 cell protein extract. These two bands were further identified as DNA topoisomerase 1. Our results indicate that IgG from patients with diffuse systemic sclerosis bind DNA topoisomerase 1 in normal human fibroblasts extracts.

Immunoblotting on HEp-2 cells increases the detection of antitopoisomerase 1 antibodies in patients with systemic sclerosis.

In order to improve the detection of antitopoisomerase 1 antibodies in a cohort of 111 systemic sclerosis patients, we have performed immunoblots on protein extracts of HEp-2 cells. Using indirect immunofluorescence and ELISA, 27 patients (24.3%) had antitopoisomerase 1 antibodies, 32 (28.8%) had anticentromere antibodies, 31 (27.9%) had antinuclear antibodies with none of these two antibodies and 21 (18.9%) had no antinuclear antibody. IgG from 24/27 (88.9%) patients with antitopoisomerase 1 antibodies bound to 2 protein bands of 63 and 100 kDa identified as topoisomerase 1 by N-terminal sequencing. Antitopoisomerase 1 antibodies were detected in 9 (8.1%) patients who had no antitopoisomerase 1 antibody as determined by ELISA. Patients with antitopoisomerase 1 antibodies had an almost similar phenotype without distinction between ELISA or immunoblot approaches. Our results provide evidence for the use of a combination of ELISA and immunoblot approaches for the detection of antitopoisomerase 1 antibodies.

Anti-endothelial cell antibodies from patients with limited cutaneous systemic sclerosis bind to centromeric protein B (CENP-B).

By using a quantitative immunoblotting technique on protein extracts of human macrovascular and microvascular endothelial cells, we have analyzed the self-reactive repertoires of IgG from 20 patients with limited cutaneous SSc, 40 patients with diffuse SSc and 60 age- and sex-matched healthy controls. Serum IgG from 15/20 patients with limited cutaneous SSc and anti-centromere antibodies bound to at least one of the two 75- and 85-kDa protein bands in the different endothelial cell extracts, whereas IgG from healthy controls or patients with diffuse SSc did not. N-terminal sequencing of the 75- and 85-kDa bands identified CENP-B as the sole antigen in both bands. Moreover, IgG from all of the SSc patients who recognized the 75- and/or 85-kDa bands bound to a full-length recombinant CENP-B protein as assessed by ELISA, whereas IgG from other SSc patients did not. The main target of anti-endothelial cell antibodies in patients with limited cutaneous SSc is the nuclear and ubiquitous protein CENP-B.

Catalytic IgG from patients with hemophilia A inactivate therapeutic factor VIII.

Factor VIII (FVIII) inhibitors are anti-FVIII IgG that arise in up to 50% of the patients with hemophilia A, upon therapeutic administration of exogenous FVIII. Factor VIII inhibitors neutralize the activity of the administered FVIII by sterically hindering its interaction with molecules of the coagulation cascade, or by forming immune complexes with FVIII and accelerating its clearance from the circulation. We have shown previously that a subset of anti-factor VIII IgG hydrolyzes FVIII. FVIII-hydrolyzing IgG are detected in over 50% of inhibitor-positive patients with severe hemophilia A, and are not found in inhibitor-negative patients. Although human proficient catalytic Abs have been described in a number of inflammatory and autoimmune disorders, their pathological relevance remains elusive. We demonstrate here that the kinetics of FVIII degradation by FVIII-hydrolyzing IgG are compatible with a pathogenic role for IgG catalysts. We also report that FVIII-hydrolyzing IgG from each patient exhibit multiple cleavage sites on FVIII and that, while the specificity of cleavage varies from one patient to another, catalytic IgG preferentially hydrolyze peptide bonds containing basic amino acids.

Analysis of autoantibody repertoires in small- and medium-sized vessels vasculitides. Evidence for specific perturbations in polyarteritis nodosa, microscopic polyangiitis, Churg-Strauss syndrome and Wegener's granulomatosis.

In order to identify new antibody reactivities, we have used a quantitative immunoblotting technique on extracts of normal human tissues to analyze the repertoires of serum IgM, serum IgG and purified IgG autoantibodies of patients with systemic vasculitides. Patients fulfilled the American College of Rheumatology and Chapel Hill criteria for the diagnosis of polyarteritis nodosa (PAN) (n=8), PAN related to hepatitis B virus (HBV) infection (n=5), Wegener's granulomatosis (WG) (n=6), microscopic polyangiitis (MPA) (n=18) or Churg-Strauss syndrome (CSS) (n=8). Sera from patients with chronic HBV infection without PAN (n=5) and age- and gender-matched healthy individuals (n=45) were used as controls. In the lung extract, IgM from 12/18 MPA patients reacted with high intensity with a 50 kDa band and serum IgG from 3/8 CSS patients bound to a 70 kDa protein band. In the artery extract, serum IgG from 6/18 MPA patients bound to an 85 kDa antigen, whereas purified IgG from all WG patients tested bound to a 28 kDa protein band and IgM from CSS patients bound to 2 main antigens of 38 and 60 kDa. These results provide evidence for the specificity of autoantibody repertoires from patients with PAN, WG, CSS and MPA.

When contemporary aminoacyl-tRNA synthetases invent their cognate amino acid metabolism.

Faithful protein synthesis relies on a family of essential enzymes called aminoacyl-tRNA synthetases, assembled in a piecewise fashion. Analysis of the completed archaeal genomes reveals that all archaea that possess asparaginyl-tRNA synthetase (AsnRS) also display a second ORF encoding an AsnRS truncated from its anticodon binding-domain (AsnRS2). We show herein that Pyrococcus abyssi AsnRS2, in contrast to AsnRS, does not sustain asparaginyl-tRNAAsn synthesis but is instead capable of converting aspartic acid into asparagine. Functional analysis and complementation of an Escherichia coli asparagine auxotrophic strain show that AsnRS2 constitutes the archaeal homologue of the bacterial ammonia-dependent asparagine synthetase A (AS-A), therefore named archaeal asparagine synthetase A (AS-AR). Primary sequence- and 3D-based phylogeny shows that an archaeal AspRS ancestor originated AS-AR, which was subsequently transferred into bacteria by lateral gene transfer in which it underwent structural changes producing AS-A. This study provides evidence that a contemporary aminoacyl-tRNA synthetase can be recruited to sustain amino acid metabolism.

Purification of turkey pancreatic phospholipase A2.

Turkey pancreatic phospholipase (TPP) has been purified from delipidated pancreases. The purification included ammonium sulfate fractionation, acidic (pH 5) treatment, followed by sequencial column chromatographies on DEAE-cellulose, Sephadex G-75, and reverse phase high pressure liquid chromatography. The purified enzyme was found to be a monomeric protein with molecular mass of 14 kDa. The optimal activity was measured at pH 8 and 37 degrees C using egg yolk emulsion as substrate. Our results show that the enzyme (TPP) was not stable for 1 h at 60 degrees C, and that bile salt and Ca2+ were required for the expression of the purified enzyme. The sequence of the N-terminal amino acids of the purified enzyme shows a very close similarity between TPP and all other known pancreatic phospholipases.

IgG reactivity with a 100-kDa tissue and endothelial cell antigen identified as topoisomerase 1 distinguishes between limited and diffuse systemic sclerosis patients.

We have analyzed antibody (Ab) reactivities of patients with limited systemic sclerosis (SSc) and anti-centromere Ab, patients with diffuse SSc and anti-topoisomerase 1 (anti-topo 1) Ab, patients with diffuse SSc without anti-topo 1 or anti-centromere Ab and age- and gender-matched healthy controls with normal human tissue and endothelial cell (EC) antigens. IgG reactivities with tissue antigens differed significantly between patients with anti-topo 1 Ab and patients with anti-centromere Ab. One 100-kDa band identified as topoisomerase 1 in macrovascular and microvascular EC extracts was recognized by IgG from patients with anti-topo 1 Ab and 50% of patients without specific Ab. IgG from patients with limited SSc and anti-centromere Ab, but not those of other patients or controls specifically recognized a 80-kDa band only in microvascular EC. Our results indicate that Ab from patients with limited or diffuse SSc with or without anti-topo 1 Ab exhibit specific and mutually exclusive reactivity patterns.