Characterization of the inactive fragment resulting from limited proteolysis of human alpha1-proteinase inhibitor by Crotalus adamanteus proteinase II.

Kress, Lawrence F.; Kurecki, T; Chan, S.K.; Laskowski, M.

doi:10.1016/s0021-9258(18)50597-4

Cited by 69 publications

(6 citation statements)

References 27 publications

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“…The primary cleavage sites generated by SV8 proteinase (Potempa et al, 1986) and CA2 (Kress et al, 1979) are shown in Figure 1. We confirmed both these sites as primary cleavages, and no secondary cleavages were detected.…”

Section: Resultsmentioning

confidence: 99%

Conformation of the reactive site loop of .alpha.1-proteinase inhibitor probed by limited proteolysis

Mast¹,

Enghild²,

Salvesen³

1992

Biochemistry

205

176

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Elucidation of the reactive site loop (RSL) structure of serpins is essential for understanding their inhibitory mechanism. Maintenance of the RSL structure is likely to depend on its interactions with a dominant unit of secondary structure known as the A-sheet. We investigated these interactions by subjecting alpha 1-proteinase inhibitor to limited proteolysis using several enzymes. The P1-P10 region of the RSL was extremely sensitive to proteolysis, indicating that residues P3'-P13 are exposed in the virgin inhibitor. Following cleavage eight or nine residues upstream from the reactive site, the protein noncovalently polymerized, sometimes forming circles. Polymerization resulted from insertion of the P1-P8 or P1-P9 region of one molecule into the A-sheet of an adjacent proteolytically modified molecule. The site of cleavage within the RSL had a distinct effect on the conformational stability of the protein, such that stability increased as more amino acids insert into the A-sheet. We conclude that the A-sheet of virgin alpha 1-proteinase inhibitor resembles that of ovalbumin, except that it contains a bulge where two or three RSL residues are inserted. Insertion of seven or eight RSL residues, allowed by proteolytic cleavage of the RSL, causes expansion of the sheet. It is likely that the RSL of alpha 1-proteinase inhibitor and several serpins exhibits significantly more mobility than is common among other protein inhibitors of serine proteinases.

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Section: Resultsmentioning

confidence: 99%

Conformation of the reactive site loop of .alpha.1-proteinase inhibitor probed by limited proteolysis

Mast¹,

Enghild²,

Salvesen³

1992

Biochemistry

205

176

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“…a in brackets cleavage site numbering according to 418 amino acid sequence length of A1AT including precursor molecule of 24 amino acids 60 using one-letter-code for amino acids. CAAP a in vivo (human) in vitro human tissue/ cells in vitro assays using purified enzymes References Enzyme name Enzyme host b C45 (G 373 A) Not detected in plasma or serum of healthy or septic individuals Current study Glycyl endopeptidase Carica papaya 29 , 61 C44 (A 374 M) Plasma & serum (no difference between healthy and septic individuals) Current study Placenta 43 , 44 BALF (higher concentrations in premature infants with bronchopulmonary dysplasia) 37 MMP-11 human endogenous 6 Adamalysin II Crotalus adamanteus 29 , 62 C43 (M 375 F) Not detected in plasma or serum of healthy or septic individuals Current study Thermolysin Bacillus thermoproteolyticus 61 C42 (F 376 L) Plasma & serum (higher concentrations in sepsis cohort than healthy individuals) …”

Section: Discussionmentioning

confidence: 99%

Multiplex quantification of C-terminal alpha-1-antitrypsin peptides provides a novel approach for characterizing systemic inflammation

Bigalke

Sponholz

Schnabel

et al. 2022

Sci Rep

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C-terminal peptides (CAAPs) of the highly abundant serine protease alpha-1-antitrypsin (A1AT) have been identified at various lengths in several human materials and have been proposed to serve as putative biomarkers for a variety of diseases. CAAPs are enzymatically formed and these enzymatic activities are often associated with excessive immune responses (e.g. sepsis, allergies). However, most of those CAAPs have been either detected using in vitro incubation experiments or in human materials which are not easily accessible. To gain a comprehensive understanding about the occurrence and function of CAAPs in health and disease, a LC–MS/MS method for the simultaneous detection of nine CAAPs was developed and validated for human plasma (EDTA and lithium-heparin) and serum. Using this newly developed method, we were able to detect and quantify five CAAPs in healthy individuals thereby providing an initial proof for the presence of C36, C37, C40 and C44 in human blood. Concentrations of four CAAPs in a clinical test cohort of patients suffering from sepsis were significantly higher compared to healthy controls. These results reveal that in addition to C42 other fragments of A1AT seem to play a crucial role during systemic infections. The proposed workflow is simple, rapid and robust; thus this method could be used as diagnostic tool in routine clinical chemistry as well as for research applications for elucidating the diagnostic potential of CAAPs in numerous diseases. To this end, we also provide an overview about the current state of knowledge for CAAPs identified in vitro and in vivo.

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“…On the other hand, it seems probable that /3-fibrinogenases of T. mucrosquamatus and T. gramineus may be resistant to inhibition in plasma. Kress and colleagues have recently shown that metalloendoproteases from C. adamanteus venom, called proteinases I and II, catalytically inactivated human a^I (Kress et al, 1979;Kurecki et al, 1978) and AT III (Kress & Catanese, 1981a) by a limited proteolytic cleavage. The inactivation of a^I by C. adamanteus proteinase II is probably not a physiological process, since these studies have been performed with purified inhibitors, wherein a2M, a most probable inhibitor of the protease, was absent.…”

Section: Discussionmentioning

confidence: 99%

Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

Pandya¹,

Budzynski²

1984

Biochemistry

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Crotalus atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.

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Characterization of the inactive fragment resulting from limited proteolysis of human alpha1-proteinase inhibitor by Crotalus adamanteus proteinase II.

Cited by 69 publications

References 27 publications

Conformation of the reactive site loop of .alpha.1-proteinase inhibitor probed by limited proteolysis

Conformation of the reactive site loop of .alpha.1-proteinase inhibitor probed by limited proteolysis

Multiplex quantification of C-terminal alpha-1-antitrypsin peptides provides a novel approach for characterizing systemic inflammation

Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom

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