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

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.

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

Journal of Biological Chemistry ◽

10.1016/j.jbc.2020.100208 ◽

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

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Synthesis of a Cys949Tyr α2-macroglobulin thiol ester mutant: co-transfection with wild-type α2-macroglobulin in an episomal expression system

Biochemical Journal ◽

10.1042/bj3120183 ◽

1995 ◽

Vol 312 (1) ◽

pp. 183-190 ◽

Cited By ~ 1

Author(s):

L Van Rompaey ◽

H Van den Berghe ◽

P Marynen

Keyword(s):

Receptor Binding ◽

Covalent Binding ◽

Binding Domain ◽

Expression Vectors ◽

Receptor Binding Domain ◽

Wild Type ◽

Thiol Ester ◽

Thiol Esters ◽

Α2 Macroglobulin ◽

K 562 Cells

A full-length alpha 2-macroglobulin (alpha 2M) cDNA was cloned into the episomal expression vectors pREP7 and pMEP4. Electroporation of the cell lines WI-L2-729HF2, U-937, K-562 and an Epstein-Barr virus-transformed cell line resulted in stable transfectants only with K-562 cells. Stable expression was obtained exclusively with pMEP4-alpha 2M and was driven from the inducible human metallothionein IIA promoter. Expression of the wild-type alpha 2M cDNA resulted in a recombinant protein (r alpha 2M) that could not be distinguished from plasma alpha 2M (p-alpha 2M): the transfected K-562 cells secreted tetrameric alpha 2M with intact internal thiol esters, a functional bait domain and a latent receptor-binding domain. r alpha 2M inhibited trypsin and elastase from cleaving a high-molecular-mass substrate. When the Cys-949 involved in the formation of the internal thiol ester was mutated to tyrosine (C949Y-r alpha 2M), a tetrameric alpha 2M was secreted, with the electrophoretic mobility of methylamine-treated p-alpha 2M (p-alpha 2M/MA) and with a functional receptor-binding domain. The C949Y-r alpha 2M did not possess proteinase-inhibiting capacity. Heterozygosity was mimicked by co-transfecting the K-562 cells with wild-type and mutant expression vectors. In this case, r alpha 2M was secreted with zero, one, two, three or four internal thiol esters. A comparison of the interaction of interleukin 1 beta and basic fibroblast growth factor with native p-alpha 2M, p-alpha 2M/MA and the mutant C949Y-r alpha 2M revealed that when assayed under nondenaturing conditions, no binding occurred to ‘slow’ p-alpha 2M whereas quantitatively similar binding was observed to ‘fast’ p-alpha 2M/MA and C949Y-r alpha 2M. Covalent binding, however, was essentially limited to p-alpha 2M/MA, suggesting the involvement of Cys-949 in the process. Covalent binding of insulin, on the contrary, was only observed when it was present during hydrolysis of the internal thiol esters of p-alpha 2M by trypsin treatment, and thus involves the activated Glx residue.

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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

Biochemical Journal ◽

10.1042/bj3180539 ◽

1996 ◽

Vol 318 (2) ◽

pp. 539-545 ◽

Cited By ~ 12

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.

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

Biochemical Journal ◽

10.1042/bj20070764 ◽

2007 ◽

Vol 407 (1) ◽

pp. 23-30 ◽

Cited By ~ 30

Author(s):

Ninh Doan ◽

Peter G. W. Gettins

Keyword(s):

Complement Protein ◽

Complement Components ◽

Ancestral Gene ◽

Thiol Ester ◽

Bait Region ◽

Morphogenetic Protein ◽

Complement Component C3 ◽

Α2 Macroglobulin ◽

Domain Interactions ◽

Type 3

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.

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Renal effects of the serine protease inhibitor aprotinin in healthy conscious mice

Acta Pharmacologica Sinica ◽

10.1038/s41401-021-00628-1 ◽

2021 ◽

Author(s):

Stefan Wörner ◽

Bernhard N. Bohnert ◽

Matthias Wörn ◽

Mengyun Xiao ◽

Andrea Janessa ◽

...

Keyword(s):

Protease Inhibitor ◽

Serine Protease ◽

Kidney Injury ◽

Serine Protease Inhibitor ◽

Tubular Function ◽

Proteolytic Activation ◽

Salt Diet ◽

Low Salt ◽

Proximal Tubular ◽

Proximal Tubular Function

AbstractTreatment with aprotinin, a broad-spectrum serine protease inhibitor with a molecular weight of 6512 Da, was associated with acute kidney injury, which was one of the reasons for withdrawal from the market in 2007. Inhibition of renal serine proteases regulating the epithelial sodium channel ENaC could be a possible mechanism. Herein, we studied the effect of aprotinin in wild-type 129S1/SvImJ mice on sodium handling, tubular function, and integrity under a control and low-salt diet. Mice were studied in metabolic cages, and aprotinin was delivered by subcutaneously implanted sustained release pellets (2 mg/day over 10 days). Mean urinary aprotinin concentration ranged between 642 ± 135 (day 2) and 127 ± 16 (day 8) µg/mL . Aprotinin caused impaired sodium preservation under a low-salt diet while stimulating excessive hyperaldosteronism and unexpectedly, proteolytic activation of ENaC. Aprotinin inhibited proximal tubular function leading to glucosuria and proteinuria. Plasma urea and cystatin C concentration increased significantly under aprotinin treatment. Kidney tissues from aprotinin-treated mice showed accumulation of intracellular aprotinin and expression of the kidney injury molecule 1 (KIM-1). In electron microscopy, electron-dense deposits were observed. There was no evidence for kidney injury in mice treated with a lower aprotinin dose (0.5 mg/day). In conclusion, high doses of aprotinin exert nephrotoxic effects by accumulation in the tubular system of healthy mice, leading to inhibition of proximal tubular function and counterregulatory stimulation of ENaC-mediated sodium transport.

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α2-Macroglobulin and Related Thiol Ester Plasma Proteins

The Plasma Proteins ◽

10.1016/b978-0-12-568405-7.50010-2 ◽

1987 ◽

pp. 191-291 ◽

Cited By ~ 76

Author(s):

Lars Sottrup-Jensen

Keyword(s):

Plasma Proteins ◽

Thiol Ester ◽

Α2 Macroglobulin

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Structure and Properties of Oxidized Chitosan Grafted Cashmere Fiber by Amide Covalent Modification

Molecules ◽

10.3390/molecules25173812 ◽

2020 ◽

Vol 25 (17) ◽

pp. 3812

Author(s):

Jifeng Li ◽

Ting Fang ◽

Wenjing Yan ◽

Fei Zhang ◽

Yunhui Xu ◽

...

Keyword(s):

Magic Angle Spinning ◽

Covalent Modification ◽

Primary Amines ◽

Carboxyl Groups ◽

Hydroxyl Groups ◽

Magic Angle ◽

Structure And Properties ◽

Carboxyl Content ◽

Moisture Regain ◽

Degree Structure

In this study, oxidized chitosan grafted cashmere fibers (OCGCFs) were obtained by crosslinking the oxidized chitosan onto cashmere fibers by amide covalent modification. A novel method was developed for the selective oxidation of the C6 primary hydroxyls into carboxyl groups for chitosan. The effect of oxidization reaction parameters of HNO3/H3PO4–NaNO2 mediated oxidation system on the oxidation degree, structure, and properties of chitosan were investigated. The chemical structure of the oxidized chitosan was characterized by solid-state cross-polarization magic angle spinning carbon-13 Nuclear Magnetic Resonance (CP/MAS 13C-NMR), Fourier transform infrared spectroscopy (FT-IR), and its morphology was investigated by scanning electron microscopy (SEM). Subsequently, the effect of the oxidized chitosan grafting on OCGCF was examined, and the physical properties, moisture regain, and antibacterial activity of OCGCFs were also evaluated. The results showed that oxidation of chitosan mostly occurred at the C6 primary hydroxyl groups. Moreover, an oxidized chitosan with 43.5–56.8% carboxyl content was realized by ranging the oxidation time from 30 to 180 min. The resulting OCGCF had a C–N amido bond, formed as a result of the reaction between the primary amines in the cashmere fibers and the carboxyl groups in the oxidized chitosan through the amide reaction. The OCGCF exhibited good moisture regain and remarkable bacteriostasis against both Staphylococcus aureus and Escherichia coli bacteria with its durability.

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