Stephen O. Brennan

Mutation of antitrypsin to antithrombin: α1-antitrypsin Pittsburgh (358 Met→Arg), a fatal bleeding disorder

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant proteins being an acute-phase reactant, the level of which increased several-fold after trauma.

Fibrinogen Brescia : Hepatic Endoplasmic Reticulum Storage and Hypofibrinogenemia Because of a γ284 Gly→Arg Mutation

The proposita suffered from liver cirrhosis and biopsy showed type 1 membrane-bound fiberglass inclusions. The hepatic inclusion bodies were weakly periodic acid-Schiff diastase-positive, and on immunoperoxidase staining reacted specifically with anti-fibrinogen antisera. Coagulation investigations revealed low functional and antigenic fibrinogen together with a prolonged thrombin time of 37 seconds (normal, 17 to 22 seconds) suggestive of a hypodysfibrinogenemia. DNA sequencing of all three fibrinogen genes showed a single heterozygous mutation of GGG (Gly)-->CGG (Arg) at codon 284 of the gamma-chain gene. However, examination of purified fibrinogen chains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase high-performance liquid chromatography, ion-exchange high-performance liquid chromatography, and isoelectric focusing, failed to show any evidence of the mutant gamma(Br) chain in plasma fibrinogen. This finding was substantiated by electrospray ionization mass spectrometry, which showed only a normal gamma (and Bbeta) chain mass, but a large increase in the portion of their disialo isoforms. We speculate that misfolding of the variant protein causes hepatic retention and the subsequent hypofibrinogenemia, and that the functional defect (dysfibrinogenemia) results from hypersialylation of otherwise normal Bbeta and gamma chains consequent to the liver cirrhosis. These conclusions were supported by studies on six other family members with hypofibrinogenemia, and essentially normal clotting times, who were heterozygous for the gamma284 Gly-->Arg mutation.

Physiological variant of antithrombin-III lacks carbohydrate sidechain at Asn 135

Both normal antithrombin‐III (AT‐IIIα) and the high heparin affinity form (AT‐IIIβ) were isolated from pooled human plasma. AT‐IIIβ had a lower negative charge and lower molecular mass than AT‐IIIα. Sialidase and endo‐F digestion indicated that the inherent difference resided in the oligosaccharide component of the molecule. CNBr fragmentation showed there was an oligosaccharide sidechain missing between residues 104 and 251, subdigestion with trypsin indicated that Asn 135 was not glycosylated in AT‐IIIβ. Chromatography of total tryptic digests on concanavalin A‐Sepharose confirmed that the high heparin affinity form of antithrombin lacked an oligosaccharide moiety at Asn 135.

Mutation of antitrypsin to antithrombin. alpha 1-antitrypsin Pittsburgh (358 Met leads to Arg), a fatal bleeding disorder.

Our previous studies predicted a functional relationship between the plasma proteins alpha 1-antitrypsin and antithrombin III. To elucidate this relationship we investigated the plasma of a 14-year-old boy who had died from an episodic bleeding disorder. A variant alpha 1-antitrypsin was identified in which the methionine at position 358 had been replaced by an arginine. This had converted the alpha 1-antitrypsin from its normal function as an inhibitor of elastase to that of an inhibitor of thrombin. This finding indicates that the reactive center of alpha 1-antitrypsin is methionine 358, which acts as a bait for elastase, just as the normal reactive center of antithrombin III is arginine 393, which acts as a bait for thrombin. The independence of the new thrombin inhibitor from heparin control explains the bleeding disorder; it also indicates that heparin normally acts directly on antithrombin III, revealing its inherent inhibitory activity. The episodic nature of the bleeding was a consequence of the mutant proteins being an acute-phase reactant, the level of which increased several-fold after trauma.

Characterization of non-covalent oligomers of proteins treated with hypochlorous acid

Hypochlorous acid (HOCl) is a potent oxidant produced by myeloperoxidase that causes aggregation of many proteins. Treatment of apohaemoglobin and apomyoglobin with HOCl produced a regular series of oligomer bands when the proteins were separated by SDS/PAGE under reducing conditions. Aggregation was detectable at a HOCl/protein molar ratio of 0.5:1 and was maximal at ratios of 10:1-20:1. Dimers formed within 1 min of adding HOCl, and further aggregation occurred over the next 30 min. No convincing evidence for covalent cross-linking was obtained by amino acid analysis, peptide analysis or electrospray ionization-MS of HOCl-modified apomyoglobin. The latter showed an increase in mass consistent with conversion of the two methionine residues into sulphoxides. A 5-fold excess of HOCl generated approximately three chloramines on the apomyoglobin. These underwent slow decay. Protein carbonyls were formed and were almost entirely located only on the polymer bands. Conversion of positively into negatively charged groups on the protein by succinylation caused preformed aggregates to dissociate. Treatment of apomyoglobin with taurine chloramine generated methionine sulphoxides but few protein carbonyls, and did not result in aggregation. We conclude that aggregation was due to strong, non-covalent interactions between protein chains. We propose that formation of protein carbonyls and possibly chloramines, along with methionine oxidation, alters protein folding to expose hydrophobic areas on neighbouring molecules that associate to form dimers and higher-molecular-mass aggregates. This process could lead to the formation of aggregated proteins at sites of myeloperoxidase activity and contribute to inflammatory tissue injury.

Human α1-antitrypsin: carbohydrate attachment and sequence homology

Human err-antitrypsin (or-AT) has 3 carbohydrate sidechains [ 11. The attachment point of one of these sidechains has been determined in conjunction with the amino acid sequence of the C-terminal third of the molecule [2]. We now provide further sequence data which define the attachment points of the other 2 carbohydrate sidechains. These data also show the extent of the sequence homology with antithrombinIII and ovalbumin, and provide support for a single reactive centre situated near the C-terminus. tryptic digestion in 2 M guanidine hydrochloride in 0.1 M NI&HCOa buffer, pH 8.0 at 37’C for 15 h with an enzyme to substrate ratio of 1: 10.

Calcium-dependent KEX2-like protease found in hepatic secretory vesicles converts proalbumin to albumin.

The yeast KEX2 protease is the only enzyme that has a proven role in the activation of polypeptide hormones through cleavage at pairs of basic residues. The enzyme that fulfils this role in higher eukaryotes has yet to be unequivocally identified. In this investigation, a KEX2‐like calcium‐dependent protease has been identified in rat hepatic microsomes. The enzyme is membrane‐bound, has a pH optimum of 5–6 and converts proalbumin to albumin. More importantly, like the KEX2 protease, it meets two other exacting criteria defined by specific mutations in humans. Namely, it does not process proalbumin Christchurch (−1 Arg→Gln) which lacks one of the requisite basic residues and, whilst not itself a serine protease, it is inhibited by the reactive center variant, α1‐antitrypsin Pittsburgh (358 Met→Arg) but not by normal α1‐antitrypsin.

New carbohydrate site in mutant antithrombin (7 Ile+Asn) with decreased heparin affinity

A mutant antithrombin was isolated from the plasma of a patient with pulmonary embolism. The new protein, which accounted for 55% of the antithrombin, had decreased heparin affinity and contained two components when analysed on the basis of either charge or molecular mass. Sialidase and endo‐β‐N‐acetylglucosaminidase F treatment suggested that this heterogeneity was due to a partial glycosylation occurring at a new carbohydrate attachment sequence. Peptide mapping by reverse‐phase HPLC showed that the abnormality involved the N‐terminal tryptic peptide. Sequence analysis demonstrated that the underlying mutation was 7 Ile→Asn which introduces a new Asn‐Cys‐Thr glycosylation sequence. This new oligosaccharide attachment site occupies the base of the proposed heparin‐binding site, and the finding explains the consequent decrease in heparin affinity.

A deep intronic mutation in FGB creates a consensus exonic splicing enhancer motif that results in afibrinogenemia caused by aberrant mRNA splicing, which can be corrected in vitro with antisense oligonucleotide treatment

We previously described a novel homozygous point mutation (FGB c.115–600A>G) located deep within intron 1 of the fibrinogen beta gene (FGB), as a likely cause of afibrinogenemia. While this was the only mutation detected, its pathological mechanism was unclear. Here we show the mutation causes the inclusion of a 50‐bp cryptic exon by creating a consensus heptad motif recognized by the spliceosome recruiting protein pre‐mRNA splicing factor (SF2)/arginine/serine‐rich alternative splicing factor (ASF) splicing factor 2/alternative splicing factor (SF2/ASF). Translation of the aberrant mRNA would result in truncation of the Bβ chain, preventing fibrinogen synthesis. Selective introduction of a second mutation into the enhancer motif abolished the SF2/ASF binding motif and re‐established normal pre‐mRNA splicing. Subsequent introduction of antisense phosphorodiamidate morpholino oligonucleotides (PMOs) into transfected cells containing the mutant construct blocked the protein‐RNA interaction and successfully restored normal splicing (∼50% at 2 µM and ∼90% at 10 µM). The molecular characterization of this case has revealed a unique disease mechanism, shown the importance of screening for deep intronic mutations, and provided evidence that antisense gene therapy is potentially practical for the treatment of diseases caused by this class of mutation. Hum Mutat 0, 1–8, 2008.

The molecular mechanisms of congenital hypofibrinogenaemia.

Congenital hypofibrinogenaemia is characterized by abnormally low levels of fibrinogen and is usually caused by heterozygous mutations in the fibrinogen chain genes (α, β and γ). However, it does not usually result in a clinically significant condition unless inherited in a homozygous or compound heterozygous state, where it results in a severe bleeding disorder, afibrinogenaemia. Various protein and expression studies have improved our understanding of how mutations causing hypo- and afibrinogenaemia affect secretion of the mature fibrinogen molecule from the hepatocyte. Some mutations can perturb chain assembly as in the γ153 Cys → Arg case, while others such as the Bβ Leu → Arg and the Bβ414 Gly → Ser mutations allow intracellular hexamer assembly but inhibit protein secretion. An interesting group of mutations, such as γ284 Gly → Arg and γ375 Arg → Trp, not only cause hypofibrinogenaemia but are also associated with liver disease. The nonexpression of these variant chains in plasma fibrinogen is due to retention in the endoplasmic reticulum, which in turn leads to hypofibrinogenaemia.