scholarly journals Limulus α2-macroglobulin. First evidence in an invertebrate for a protein containing an internal thiol ester bond

1987 ◽  
Vol 248 (3) ◽  
pp. 703-707 ◽  
Author(s):  
P B Armstrong ◽  
J P Quigley
Keyword(s):  
Proteinase Inhibitor ◽  
Thermal Denaturation ◽  
Horseshoe Crab ◽  
Proteinase Inhibitors ◽  
Binding Activity ◽  
Ester Bond ◽  
Thiol Groups ◽  
Thiol Ester ◽  
Complement Proteins ◽  
Α2 Macroglobulin

Intra-chain thiol ester bonds are present in a limited number of proteins. The thiol ester class of proteins includes vertebrate alpha 2-macroglobulin and the complement proteins C3 and C4. We report here the first instance of a thiol ester protein from an invertebrate, the alpha 2-macroglobulin proteinase-inhibitor homologue present in the plasma of the American horseshoe crab Limulus polyphemus. Our evidence is of three kinds: (1) the proteinase-binding activity of Limulus alpha 2-macroglobulin is inactivated by the low-molecular-mass primary amine methylamine; (2) the native protein is subject to autolytic fragmentation during mild thermal denaturation, yielding fragments of approx. 125 kDa and 55 kDa, whereas the methylamine-treated protein is stable under these conditions of thermal treatment; (3) new thiol groups are generated rapidly during reaction of the protein with trypsin. The demonstration of the thiol ester bond in a protein from an ancient invertebrate provides evolutionary evidence for the importance of this bond in the function of plasma forms of the alpha 2-macroglobulin-like proteinase inhibitors.

On the Structure-Function Relationships of the Unique Proteinase Inhibitor Human α2 Macroglobulin

1998 ◽  
Vol 4 (S2) ◽  
pp. 986-987
Author(s):  
James K. Stoops ◽  
Steven J. Kolodziej ◽  
Usman Qazi ◽  
Norman J. Nolasco ◽  
Peter G.W. Gettins ◽  
...  
Keyword(s):  
Structural Change ◽  
Complement Activation ◽  
Proteinase Inhibitor ◽  
Vital Role ◽  
Ester Bond ◽  
Cleavage Sites ◽  
Thiol Ester ◽  
Bait Region ◽  
Α2 Macroglobulin ◽  
Structural And Functional Properties

Human α2 macroglobulin (α2M) has structural and functional properties that contribute to its uniqueness as proteinase inhibitor. It is the largest known (Mr=720,000) and the only natural proteinase inhibitor which has a broad range of reactivity and for which the reaction is irreversible. It has a vital role in the clearance of proteinases from the circulation and in regulating their activity in fibrinolysis, coagulation and complement activation.An α2M molecule can entrap two proteinase molecules such as chymotrypsin and trypsin and can therefore be considered to contain two functional domains. Each subunit in the homotetramer has a bait region with cleavage sites for nearly all known endoproteinases and an internal thiol ester bond. A proteinase cleaves the two bait regions within both functional units leading to an activation and cleavage of the thiol ester bonds. Consequently, α2M undergoes a major structural change resulting in the irreversible entrapment of the proteinase.

scholarly journals Structural and functional analysis of the spontaneous re-formation of the thiol ester bond in human α2-macroglobulin, rat α1-inhibitor-3 and chemically modified derivatives

1996 ◽  
Vol 318 (2) ◽  
pp. 539-545 ◽  
Author(s):  
Hanne GRØN ◽  
Ida B. THØGERSEN ◽  
Jan J. ENGHILD ◽  
Salvatore V PIZZO
Keyword(s):  
Room Temperature ◽  
Elevated Temperatures ◽  
Proteinase Inhibitors ◽  
Gel Filtration ◽  
Complement Components ◽  
Thiol Ester ◽  
Chemically Modified ◽  
Inhibitory Capacity ◽  
Α2 Macroglobulin ◽  
The Complement System

The α-macroglobulins are proteinase inhibitors that form part of a superfamily along with components of the complement system. Internal β-cysteinyl–γ-glutamyl thiol ester bonds are an important structural feature of most α-macroglobulins and several complement components. We have studied the reversibility of thiol ester cleavage caused by NH3 or CH3NH2 in tetrameric human α2-macroglobulin (α2M) and monomeric rat α1-inhibitor-3 (α1 I3). When employing NH3 as the nucleophile, the thiol ester in α1I3 re-formed spontaneously at room temperature after gel filtration to remove excess nucleophile, and an active proteinase inhibitor was regained. When CH3NH2 was employed as the nucleophile, thiol ester reversibility was more energy-demanding. With either nucleophile, α2M once inactivated did not regain proteinase-inhibitory capacity at room temperature. At elevated temperatures, however, the reaction between α2M and NH3 or CH3NH2 was reversible and the inhibitory capacity could be recovered. Modification of the cysteinyl groups from the thiol ester prevented its re-formation but did not prevent the heat-induced retrieval of inhibitory capacity, suggesting that conformational features rather than the thiol ester are essential for α2M to function as an inhibitor. As demonstrated by non-denaturing PAGE, the conformation of native α2M is restored when the proteinase-inhibitory capacity is recovered.

scholarly journals Binding of α2-macroglobulin and limulin: regulation of the plasma haemolytic system of the American horseshoe crab, Limulus

2000 ◽  
Vol 347 (3) ◽  
pp. 679-685 ◽  
Author(s):  
Snehasikta SWARNAKAR ◽  
Rengasamy ASOKAN ◽  
James P. QUIGLEY ◽  
Peter B. ARMSTRONG
Keyword(s):  
Horseshoe Crab ◽  
Solid Phase ◽  
Haemolytic Activity ◽  
Thiol Ester ◽  
The Third ◽  
Α2 Macroglobulin ◽  
Sialic Acid Binding ◽  
Small Primary ◽  
Solid Phase Assay ◽  
Binding Lectin

The mediator of haemolysis in the plasma of the horseshoe crab, Limulus polyphemus, is limulin, a sialic acid-binding lectin. The haemolytic activity of limulin is inhibited by thiol ester-reacted forms of Limulus α2-macroglobulin, the third-most abundant protein of the plasma. Limulus α2-macroglobulin that has experienced cleavage of its internal thiol ester bond, consequent to reaction with proteases, or with the small primary amine, methylamine, reduces the haemolytic activity of limulin when present at molar excesses that approximate the relative concentrations of these two proteins in the plasma. Native, unreacted Limulus α2-macroglobulin has no effect on the haemolytic activity of limulin. Limulin binds thiol ester-reacted forms of Limulus α2-macroglobulin both in a solid-phase assay and in solution with an avidity 10-25 times higher than native, unreacted Limulus α2-macroglobulin. Protease-reacted α2-macroglobulin functions as a marker for the presence of foreign proteases in the blood of Limulus, and thus of pathogenic organisms that release proteases as facilitators of invasion and pathogenicity. Modulation of the haemolytic system represents a novel function for α2-macroglobulin.

Role of Endogenous Proteinase Inhibitors in the Regulation of the Blood Clotting System of the Horseshoe Crab, Limulus Polyphemus

1984 ◽  
Vol 52 (02) ◽  
pp. 117-120 ◽  
Author(s):  
Peter B Armstrong ◽  
Jack Levin ◽  
James P Quigley
Keyword(s):  
Blood Clotting ◽  
Horseshoe Crab ◽  
Proteinase Inhibitors ◽  
Low Molecular Weight ◽  
Clotting System ◽  
Heat Stable ◽  
Α2 Macroglobulin ◽  
Inhibitory Activities ◽  
Acid Labile

SummaryBlood clotting in Limulus is dependent on the activity of a proteinase which converts the zymogen, coagulogen, into a form that undergoes polymerization to form the clot. The abilities of a series of recently discovered endogenous proteinase inhibitors to inhibit this enzyme and thereby serve as potential regulators of its activity were explored. The blood plasma of Limulus contains a single inhibitor that is functionally and structurally homologous to vertebrate α2 macroglobulin. During exocytosis, the blood cells (amebocytes) release a series of inhibitors, including small quantities of the α2 macroglobulin homologue; a low molecular weight, acid-and heat-stable inhibitor; and an acid-labile activity. Of the three inhibitory activities, only the cell-released, acidlabile inhibitor is capable of inhibiting the clotting enzyme.

scholarly journals α1-Proteinase Inhibitor, α1-Antichymotrypsin, and α2-Macroglobulin Are the Antiapoptotic Factors of Vascular Smooth Muscle Cells

2000 ◽  
Vol 276 (15) ◽  
pp. 11798-11803 ◽  
Author(s):  
Yuji Ikari ◽  
Eileen Mulvihill ◽  
Stephen M. Schwartz
Keyword(s):  
Extracellular Matrix ◽  
Smooth Muscle ◽  
Vascular Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Survival ◽  
Vascular Smooth Muscle Cells ◽  
Proteinase Inhibitor ◽  
Proteinase Inhibitors ◽  
Muscle Cells ◽  
Α2 Macroglobulin

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors α1-proteinase inhibitor, α1-antichymotrypsin, and α2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.

scholarly journals Correction: α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester

2021 ◽  
Vol 296 ◽  
pp. 100208
Author(s):  
Seandean Lykke Harwood ◽  
Nadia Sukusu Nielsen ◽  
Kathrine Tejlgård Jensen ◽  
Peter Kresten Nielsen ◽  
Ida B. Thøgersen ◽  
...  
Keyword(s):  
Hydroxyl Groups ◽  
Thiol Ester ◽  
Α2 Macroglobulin

scholarly journals Reduction of the beta-Cys-gamma-Glu thiol ester bond of human C3 with sodium borohydride.

1983 ◽  
Vol 258 (22) ◽  
pp. 13580-13586 ◽  
Author(s):  
M L Thomas ◽  
F F Davidson ◽  
B F Tack
Keyword(s):  
Sodium Borohydride ◽  
Ester Bond ◽  
Thiol Ester
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