scholarly journals Human α2-macroglobulin is composed of multiple domains, as predicted by homology with complement component C3

2007 ◽  
Vol 407 (1) ◽  
pp. 23-30 ◽  
Author(s):  
Ninh Doan ◽  
Peter G. W. Gettins

Human α2M (α2-macroglobulin) and the complement components C3 and C4 are thiol ester-containing proteins that evolved from the same ancestral gene. The recent structure determination of human C3 has allowed a detailed prediction of the location of domains within human α2M to be made. We describe here the expression and characterization of three α2M domains predicted to be involved in the stabilization of the thiol ester in native α2M and in its activation upon bait region proteolysis. The three newly expressed domains are MG2 (macroglobulin domain 2), TED (thiol ester-containing domain) and CUB (complement protein subcomponents C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein 1) domain. Together with the previously characterized RBD (receptor-binding domain), they represent approx. 42% of the α2M polypeptide. Their expression as folded domains strongly supports the predicted domain organization of α2M. An X-ray crystal structure of MG2 shows it to have a fibronectin type-3 fold analogous to MG1–MG8 of C3. TED is, as predicted, an α-helical domain. CUB is a spliced domain composed of two stretches of polypeptide that flank TED in the primary structure. In intact C3 TED interacts with RBD, where it is in direct contact with the thiol ester, and with MG2 and CUB on opposite, flanking sides. In contrast, these α2M domains, as isolated species, show negligible interaction with one another, suggesting that the native conformation of α2M, and the consequent thiol ester-stabilizing domain–domain interactions, result from additional restraints imposed by the physical linkage of these domains or by additional domains in the protein.

2020 ◽  
Vol 295 (49) ◽  
pp. 16732-16742 ◽  
Author(s):  
Seandean Lykke Harwood ◽  
Nadia Sukusu Nielsen ◽  
Kathrine Tejlgård Jensen ◽  
Peter Kresten Nielsen ◽  
Ida B. Thøgersen ◽  
...  

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin–like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity–conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.


1998 ◽  
Vol 4 (S2) ◽  
pp. 986-987
Author(s):  
James K. Stoops ◽  
Steven J. Kolodziej ◽  
Usman Qazi ◽  
Norman J. Nolasco ◽  
Peter G.W. Gettins ◽  
...  

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.


1996 ◽  
Vol 318 (2) ◽  
pp. 539-545 ◽  
Author(s):  
Hanne GRØN ◽  
Ida B. THØGERSEN ◽  
Jan J. ENGHILD ◽  
Salvatore V PIZZO

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.


2021 ◽  
Vol 296 ◽  
pp. 100208
Author(s):  
Seandean Lykke Harwood ◽  
Nadia Sukusu Nielsen ◽  
Kathrine Tejlgård Jensen ◽  
Peter Kresten Nielsen ◽  
Ida B. Thøgersen ◽  
...  

2021 ◽  
pp. 100879
Author(s):  
Seandean Lykke Harwood ◽  
Nadia Sukusu Nielsen ◽  
Khang Diep ◽  
Kathrine Tejlgård Jensen ◽  
Peter Kresten Nielsen ◽  
...  

Blood ◽  
2019 ◽  
Vol 133 (17) ◽  
pp. 1909-1918 ◽  
Author(s):  
Jian Zhu ◽  
Joshua Muia ◽  
Garima Gupta ◽  
Lisa A. Westfield ◽  
Karen Vanhoorelbeke ◽  
...  

Abstract Human ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains, followed by 7 additional T domains and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. ADAMTS13 inhibits the growth of von Willebrand factor (VWF)–platelet aggregates by cleaving the cryptic Tyr1605-Met1606 bond in the VWF A2 domain. ADAMTS13 is regulated by substrate-induced allosteric activation; without shear stress, the distal T8-CUB domains markedly inhibit VWF cleavage, and binding of VWF domain D4 or selected monoclonal antibodies (MAbs) to distal ADAMTS13 domains relieves this autoinhibition. By small angle X-ray scattering (SAXS), ADAMTS13 adopts a hairpin-like conformation with distal T7-CUB domains close to the proximal MDTCS domains and a hinge point between T4 and T5. The hairpin projects like a handle away from the core MDTCS and T7-CUB complex and contains distal T domains that are dispensable for allosteric regulation. Truncated constructs that lack the T8-CUB domains are not autoinhibited and cannot be activated by VWF D4 but retain the hairpin fold. Allosteric activation by VWF D4 requires T7, T8, and the 58–amino acid residue linker between T8 and CUB1. Deletion of T3 to T6 produced the smallest construct (delT3-6) examined that could be activated by MAbs and VWF D4. Columba livia (pigeon) ADAMTS13 (pADAMTS13) resembles human delT3-6, retains normal activation by VWF D4, and has a SAXS envelope consistent with amputation of the hairpin containing the dispensable T domains of human ADAMTS13. Our findings suggest that human delT3-6 and pADAMTS13 approach a “minimal” structure for allosterically regulated ADAMTS13.


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