Alpha 1-antitrypsin Pittsburgh Research Articles

1-antitrypsin Pittsburgh in a family with bleeding tendency

We describe a 16-year-old girl and her 41-year-old father who both had a bleeding tendency, dramatic prolongation of all standard clotting assays, undetectable levels of plasma protein C activity, and low or borderline levels of factors X, XI and XII. Plasma and serum electrophoresis revealed a minor peak following the main alpha(1) globulin peak, of which the proportion was increased. Platelet aggregation by thrombin (final concentration 1 U/mL) was absent in both patients, but this inhibition can be overcome by increasing the concentration of thrombin (4 U/mL). The molecular defect responsible for these coagulation abnormalities was identified by genomic sequencing. Both patients are heterozygous for alpha(1)-antitrypsin Met 358 to Arg (alpha(1)-antitrypsin Pittsburgh). Seven other members of this pedigree had normal coagulation tests and do not carry the same genetic mutation. This unique family with alpha1-antitrypsin Pittsburgh sheds some light on the study of this extremely rare mutation and its inheritance.

Purification and characterization of the candidate prohormone-processing enzyme SPC3 produced in a mouse L cell line.

SPC3 is a member of a growing family of mammalian subtilisin-like serine proteases which play a probable role in proprotein maturation. In this study we have prepared a mouse L cell line stably expressing rat SPC3 cDNA and characterized the recombinant SPC3 protein secreted into the medium. Three molecular forms of recombinant SPC3 were identified with molecular masses of 86, 75, and 64 kDa. NH2-terminal sequence analysis indicated that all three forms were cleaved following the sequence -Arg107-Arg-Lys-Arg110, indicating removal of an SPC3 prosequence. All three molecular forms showed a 3-4-kDa decrease in molecular mass following incubation with endoglycosidase F. Two SPC3 carboxyl-terminal-directed antisera recognized only the 86-kDa molecular form of recombinant SPC3, demonstrating that COOH-terminal truncation of SPC3 protein is responsible for the different molecular mass forms. Recombinant SPC3 had a pH optimum of 6.0 and was stimulated by calcium, with maximum activity at 10 mM. Recombinant SPC3 was inhibited most effectively by the thiol-reactive reagent p-hydroxymecuribenzoate and the heavy metal chelators EDTA and EGTA. Recombinant SPC3 was also inhibited by alpha 1-antitrypsin Pittsburgh as well as wild type alpha 1-antitrypsin and antithrombin III. The inhibitor specificities revealed using these high molecular mass serpins differ from those reported for other members of the subtilisin-like serine protease family and may be able to be exploited to distinguish between closely related members of this new enzyme superfamily. Studies of cleavage specificity using tri- and tetrapeptidyl coumarins that contained pairs of basic residues indicated that tetrapeptide substrates that contained an S4 Arg residue as part of an -Arg-X-Lys/Arg-Arg motif were the most effective synthetic peptide substrates. Recombinant SPC3 also cleaved human proalbumin following the Arg-Gly-Val-Phe-Arg-Arg prosequence. Circulating human proalbumin variants that contained a mutation at either of the basic amino acids adjacent to the cleavage site were not cleaved by recombinant SPC3. Recombinant SPC3 was also able to cleave after a single arginine residue in chicken proalbumin following the Arg-Asn-Leu-Gln-Arg-Phe-Ala-Arg prosequence. These results define the primary structure requirements for cleavage by recombinant SPC3 and remain consistent with a role for SPC3 in proprotein/prohormone maturation.

PACE/furin can process the vitamin K-dependent pro-factor IX precursor within the secretory pathway.

Factor IX is synthesized as a precursor polypeptide which requires proteolytic cleavage of the propeptide for functional activity. Expression of factor IX at high levels in Chinese hamster ovary (CHO) cells results in the secretion of a mixture of profactor IX and mature factor IX. We have studied whether the processing of profactor IX may be mediated by the recently discovered subtilisin-like serine proteases PACE/furin and/or PACE4. Co-transfection of a PACE expression vector with a profactor IX expression vector resulted in the secretion of fully processed factor IX. In contrast, co-transfection of a PACE4 expression vector with a profactor IX expression vector did not increase processing of profactor IX to the mature form. A factor IX Arg-to-Thr mutation at the P1 position (residue 39) destroyed the ability for PACE to process profactor IX. Amino-terminal sequence analysis demonstrated that processing mediated by PACE occurred at the authentic site within profactor IX. The specificity of profactor IX processing by PACE was also evaluated by transfection of a vector encoding the serine protease inhibitor alpha 1-antitrypsin. Expression of wild-type alpha 1-antitrypsin, which does not inhibit PACE, did not influence processing of profactor IX mediated by co-expression of PACE. In contrast, the alpha 1-antitrypsin Pittsburgh mutant, which inhibits PACE, inhibited profactor IX processing activity mediated by transfected PACE as well as the endogenous CHO cell propeptide processing enzyme. Pulse-chase labeling indicated that PACE processed profactor IX late within the secretory pathway, although a secreted soluble mutant PACE was also capable of processing profactor IX in the conditioned medium. The results implicate PACE as a candidate for the enzyme that processes profactor IX in vivo.

Modulators of coagulation. A critical appraisal of their role in sepsis.

Widespread intravascular coagulation is common in patients with sepsis. Coagulation abnormalities may result from exposure to endotoxin, from tumor necrosis factor alpha or interleukin 1 release, or from the actions of a more specific mediator, such as vascular permeability factor. The result is marked activation of the contact and coagulation systems; simultaneously, there is decreased fibrinolysis and depressed levels of the inhibitors of the contact and coagulation systems. Multiple agents are being studied to correct these abnormalities. Antithrombin III holds promise because it inhibits a number of factors important in contact and coagulation activation, not just thrombin. Plasminogen activators may prove helpful in increasing fibrinolysis during sepsis; because they have been associated with rebound thrombin generation, however, plasminogen activators may be most effective if used in conjunction with hirudin or a synthetic hirudin analogue. Bradykinin may offset hypotension in sepsis. Protein C may inhibit thrombin formation and also complex with plasminogen activator inhibitor 1, thereby promoting fibrinolysis. Other agents that may prove effective include alpha 1-antitrypsin Pittsburgh, C1-esterase inhibitor, monoclonal antibodies to contact factors, soybean trypsin inhibitors, thrombomodulin, prostaglandin I2, and aprotinin. There are no data to support the use of heparin or fibronectin, except in limited circumstances.

Saccharomyces cerevisiae strains that overexpress heterologous proteins.

We describe a system that facilitates the selection of host mutants that overproduce a range of secreted and internally produced heterologous proteins in Saccharomyces cerevisiae. These mutants were initially selected for their ability to oversecrete recombinant human albumin (rHA), as detected by a direct visual assay that relies upon antibody precipitation in solid media. Yeast strains that were able to synthesize and secrete increased levels of rHA also produced elevated levels of internally expressed proteins including alpha 1-antitrypsin Pittsburgh variant and plasminogen activator inhibitor type 2.

Substitution of arginine for Leu444 in the reactive site of heparin cofactor II enhances the rate of thrombin inhibition.

Heparin cofactor II (HCII), a member of the "serpin" family of serine protease inhibitors, is a 65,600-Da plasma glycoprotein that inhibits thrombin and chymotrypsin. The rate of thrombin inhibition is stimulated approximately 1000-fold by heparin or dermatan sulfate. Thrombin and chymotrypsin cleave the Leu444-Ser445 bond (designated P1-P'1) in the reactive site of HCII, forming a stable equimolar complex in which the protease is inactive. In this study, we have determined the effects of substituting an arginine for Leu444 in recombinant HCII (rHCII). The rHCII was expressed in Escherichia coli and partially purified by heparin-Sepharose chromatography. Apparent second-order rate constants (k2) for inhibition of thrombin, coagulation factor Xa, kallikrein, plasmin, and chymotrypsin by rHCII were determined using appropriate chromogenic substrates. In the absence of a glycosaminoglycan, rHCII(Leu444----Arg) inhibited thrombin at a 98-fold higher rate (k2 = 6.2 x 10(6) M-1 min-1) than native rHCII (k2 = 6.3 x 10(4) M-1 min-1). Dermatan sulfate accelerated thrombin inhibition by both forms of rHCII, but the maximum rate constant in the presence of dermatan sulfate was only 2-fold higher for rHCII(Leu444----Arg) (k2 = 5.3 x 10(8) M-1 min-1) than for native rHCII (k2 = 2.2 x 10(8) M-1 min-1). Heparin was less effective than dermatan sulfate in stimulating both forms of rHCII. Factor Xa, kallikrein, and plasmin were inhibited more rapidly and chymotrypsin more slowly by rHCII(Leu444----Arg) than by native rHCII. These effects are qualitatively similar to those observed with the natural mutant alpha 1-antitrypsin Pittsburgh (Met358----Arg at the P1 position) and strengthen the hypothesis that the P1 residue is a major determinant of protease specificity in the serpins. Furthermore, the rapid rate of inhibition of thrombin by rHCII(Leu444----Arg) in the absence of heparin or dermatan sulfate suggests that this variant may be useful as a therapeutic agent.

Proalbumin to Albumin Conversion by a Proinsulin Processing Endopeptidase of Insulin Secretory Granules

A lysate of purified insulin secretory granules, which contains two types of proinsulin processing activity (type 1, Arg-Arg-directed and type II, Lys-Arg-directed (Davidson, H.W., Rhodes, C.J., and Hutton, J. C. (1988) Nature 333, 93-96), was found to process proalbumin by specific proteolytic cleavage of the COOH-terminal side of the Arg-2-Arg-1 sequence. The subcellular distribution of proalbumin processing activity in insulinoma tissue paralleled that for proinsulin conversion and occurred principally in a secretory granule fraction. Cleavage appeared to result from the Arg-Arg-directed type 1 proinsulin processing endo-peptidase. It was Ca2+-dependent (K0.5 activation = 1.0-1.5 mM Ca2+), unaffected by group-specific inhibitors of serine, cysteinyl, or aspartyl proteinases, and had an acidic pH optimum (5.5). Active-site inhibitor studies showed this activity had a preference for dibasic over monobasic amino acid sequences and indicated that the sequence of the dibasic site was an important determinant of the susceptibility of the substrate to cleavage. The activity did not process the proalbumin Christchurch mutant (Arg-2-Arg-1 to Arg-2-Gln-1). It was inhibited by the variant alpha 1-antitrypsin Pittsburgh (Met358 to Arg358; K0.5 = 100 nM) but not by other related proteins normally co-secreted with albumin from hepatocytes, namely alpha 1-antitrypsin M, alpha 2-macroglobulin, or antithrombin III. The insulin secretory granule proalbumin processing activity was indistinguishable from a proalbumin endopeptidase reported in rat liver membranes and similar to the yeast KEX-2 protease. These findings suggest that a highly conserved set of proprotein endopeptidases exists, which are specific for a dibasic sequence but broadly specific for proprotein substrates. Such enzymic activities appear to be active within both the constitutive and regulated pathways of secretion. Intraorganellar Ca2+ and pH appear to play a key role in regulating their activities.

Identification of a calcium-dependent microsomal proteinase responsible for monobasic cleavage of chicken proalbumin

The location and nature of the endoproteolytic activity involved in processing of proproteins has been studied in chicken liver microsomes. A membrane-bound, calcium-dependent proteinase was found to cleave chicken proalbumin with a monobasic cleavage site approx. 10-times faster than human proalbumin, which has a dibasic cleavage site. The mutant (human) proalbumin Christchurch (Arg(-1)----Gln), with a potential monobasic site, was not processed. The enzyme, which had a pH optimum of between 5.0 and 7.0, was not inhibited by serine or aspartyl proteinase inhibitors but was affected by some inhibitors of cysteine proteinases. The convertase was specifically inhibited by the reactive centre variant alpha 1-antitrypsin Pittsburgh, but not by normal alpha 1-antitrypsin.