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The effect of lyophilization from Tris buffer on purified α2M. Images of native PAGE (3–8% Tris-acetate) gels showing the migration of (A) reconstituted α2M that had been lyophilized from 20 mM Tris, 130 mM glycine, 80 mM trehalose, pH 8.0 and (B) α2M stored in solution at 4°C in 20 mM Tris, pH 8.0 for 2 months or following reconstitution after it had been lyophilized from 20 mM Tris, pH 8.0 and stored at -20°C for 7 days. As references, the positions of native and transformed α2M are also shown in (A). (C) Matched α2M samples in 20 mM phosphate, pH 7.4 or 20 mM Tris, pH 8.0 stored at 4°C for 2 months. Both samples contained 0.02% (w/v) sodium azide.
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Alpha-2-macroglobulin is an abundant secreted protein that is of particular interest because of its diverse ligand binding profile and multifunctional nature, which includes roles as a pro-tease inhibitor and as a molecular chaperone. The activities of alpha-2-macroglobulin are typically dependent on whether its conformation is native or transforme...
Citations
... The physiological threshold for Na + in the plasma of mammals and freshwater fish is $130-145 mmol kg À1 (Anderson et al., 1985;Han et al., 2016;Lahlou et al., 1969). Isolated examples indicate that when this threshold is exceeded, some plasma oligomers begin to dissociate, such as human α 2 -macroglobulin tetramers in a solution with 150 mmol kg À1 NaCl (in vitro) (Wyatt et al., 2015). Under CS, fish plasma Na + concentrations reach even higher levels ($205 mmol kg À1 ) (Lahlou et al., 1969), which can lead to changes in the organization of serum oligomers. ...
To investigate the stress response and physiological adaptations of goldfish (Carassius auratus) to critical salinity (CS) waters, we analyzed high‐density lipoprotein (HDL) stoichiometry, stress markers (cortisol, glucose), and plasma osmotic properties (Na⁺, osmolality, water content) using ichthyology, biochemistry, and proteomics approaches. After 21 days of exposure to CS, plasma concentrations of cortisol, glucose, and Na⁺ increased, indicating stress. Total plasma osmolality (Osmtotal) and osmolality generated by inorganic (Osminorg) and organic osmolytes (Osmorg) also increased, the latter by ~2%. We associated the increase of Osmorg with (1) increased metabolite concentration (glucose), (2) dissociation of HDL particles resulting in increased HDL number per unit plasma volume (~1.5–2‐fold) and (3) increased HDL osmotic activity. HDL remodeling may be the reason for the redistribution of bound and free water in plasma, which may contribute to water retention in plasma and, at the same time, to hemodynamic disturbances under CS conditions. The study's findings suggest that HDL remodeling is an important mechanism for maintaining osmotic homeostasis in fish, which is consistent with current capillary exchange models in vertebrates.
... Alpha 2 -M is found mostly in a native conformation in the bloodstream [21]. However, its cleavage by a large number of proteases results in a dramatic conformational change leading to the exposure of low-density lipoprotein receptor-related protein-1 (LRP1)-binding sites, thus reducing its plasma half-life to a few minutes by endosomal clearance in hepatocytes and macrophages [22,23]. ...
The brain-derived neurotrophic factor (BDNF) has been recently shown to have activating effects in isolated platelets. However, BDNF circulates in plasma and a mechanism to preclude constant activation of platelets appears necessary. Hence, we investigated the mechanism regulating BDNF bioavailability in blood. Protein-protein interactions were predicted by molecular docking and validated through immunoprecipitation. Platelet aggregation was assessed using light transmission aggregometry with washed platelets in response to classical agonists or BDNF, in the absence or presence of alpha-2-macroglobulin (α2M), and in platelet-rich plasma. BDNF signaling was assessed with phospho-blots. As little as 25% autologous plasma was sufficient to completely abolish platelet aggregation in response to BDNF. Docking predicted two forms of BDNF binding to native or activated α2M, parallel and perpendicular arrangements, and the model suggested that the BDNF-α2M complex cannot bind to the high-affinity BDNF receptor, tropomyosin receptor kinase B (TrkB). Experimentally, native and activated α2M formed stable complexes with BDNF preventing BDNF-induced TrkB activation and signal transduction. Both native and activated α2M inhibited BDNF induced-platelet aggregation in a concentration-dependent manner with comparable half-maximal inhibitory concentrations (IC50≈ 125-150 nM). Our study implicates α2M as a physiological regulator of BDNF bioavailability, and as inhibitor of BDNF-induced platelet activation in blood.
... Repeated freezing and thawing cycles of purified hα2M induce sample heterogeneity. It has been reported that, once purified, native hα 2 M is more susceptible to freeze and thaw 39 . To assess this in the complex context of plasma, we analysed the biophysical and functional properties of pure native hα 2 M after several fast (Fig. 3a) or slow (Fig. 3c,d) freeze-and-thaw cycles (FTs). ...
... In agreement with 39 , fast freezing in liquid nitrogen in up to three FTs apparently did not significantly impair the protein and high-molecular-weight species, which are indicative of conformational changes as shown in Fig. 1 of 39 , appeared only after the third cycle (Fig. 3a). Contrary to 39 , however, we did not detect induced species, which we attribute to the presence of sodium azide in these published studies. This reagent is a chemical preservative and potent nucleophile 55 , which can attack the thioester bond and induce rearrangement in a similar fashion to MA. ...
Human α2-macroglobulin (hα2M) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hα2M undergoes stepwise transitions from its native, expanded, highly flexible, active conformation to an induced, compact, triggered conformation. As a consequence, the peptidase is entrapped by an irreversible Venus flytrap mechanism. Given the importance of hα2M, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hα2M purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hα2M is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hα2M. We therefore used a combination of native and sodium dodecylsulfate polyacrylamide gel electrophoresis, affinity and ion-exchange chromatography, multi-angle laser light scattering after size-exclusion chromatography, free cysteine quantification, and peptidase inhibition assays with endopeptidases of two catalytic classes and three protein substrates, to characterize the biochemical and biophysical properties of hα2M purified ad hoc either from fresh plasma or frozen fresh plasma after thawing. We found no differences in the molecular or functional properties of the preparations, indicating that protective components in plasma maintain native hα2M in a functionally competent state despite freezing.
... Both forms bind to a number of cytokines and growth factors at different degrees and with different efficiencies to regulate their activities. Compared with the native one, iA2M has been found involved in more functions, such as activating the signaling cascades in the target cell that facilitates neutrophil migration, improving the cell division of macrophages, and promoting bacterial phagocytosis and antigen presentation by macrophages (Asplin et al., 2001;Borth and Luger, 1989;LaMarre et al., 1991;Liu et al., 2001;Panyutich and Ganz, 1991;Peslova et al., 2009;Rehman et al., 2013;Vandooren and Itoh, 2021;Wyatt et al., 2015). Moreover, iA2M inhibits amyloid formation, and hypochlorite-induced A2M dimer with potent chaperone activity helps remove the damaged proteins through lipoprotein receptors (Rehman et al., 2013;Vandooren and Itoh, 2021). ...
... Human native A2M was isolated from blood plasma as previously described (Chuang et al., 2014;Wyatt et al., 2015). Human blood from healthy consenting donors was centrifuged at 1000×g and 4°C for 30 min to obtain pellet cells. ...
Human alpha-2-macroglobulin is a well-known inhibitor of a broad spectrum of proteases and plays important roles in immunity, inflammation, and infections. Here, we report the cryo-EM structures of human alpha-2-macroglobulin in its native state, induced state transformed by its authentic substrate, human trypsin, and serial intermediate states between the native and fully induced states. These structures exhibit distinct conformations, which reveal the dynamic transformation of alpha-2-macro-globulin that acts as a protease inhibitor. The results shed light on the molecular mechanism of alpha-2-macroglobulin in entrapping substrates.
... 10.491262 doi: bioRxiv preprint A2M bind a number of cytokines, growth factors to different degrees and with different efficiency to regulate their activities. Interestingly, induced A2M was found to perform more functions like activating the signaling cascades in target cell that facilitates neutrophil migration, improves cell division of macrophages, promotes bacterial phagocytosis and antigen presentation by macrophages (1, [3][4][5][6][7][8][9][10]. Moreover, induced A2M was found to inhibit amyloid formation and hypochlorite-induced A2M dimer with potent chaperone activity helps to remove the damaged proteins through lipoprotein receptors (1, 10). ...
Human alpha-2-macroglobulin is a well-known proteases inhibitor against a broad spectrum of proteases. It also plays important roles in immunity, inflammation, and infections. Here, we report cryo-EM structures of human alpha-2-macroglobulin of the native state, the transformed state induced by its authentic substrate, human trypsin, and serial intermediate states between the native and the fully induced state. These structures exhibit distinct conformations, which reveal a dynamic transformation process of alpha-2-macroglobulin acting as a protease inhibitor. The results shed light on the molecular mechanism of alpha-2-macroglobulin entrapping substrates, and help to understand how alpha-2-macroglobulin possesses variant physiological functions.
... Nevertheless, the possibility of their decay is indirectly indicated by data on the decay of other human plasma proteins under the infl uence of an increased level of Na + . Thus, in vitro, at the concentration of Na + in the solution of only 150 mmol/kg, the decomposition of the tetrameric human plasma of alpha-2-macroglobulin into dimers (in ~ 5% of the tetramer pool) was registered and the breakdown was accompanied by a change in the functional profi le of the protein (43). In this example, the concentration of Na + in the solution (150 mmol/kg) does not exceed its titer in the physiological solution used in clinical practice (154 mEq/L) (13). ...
Aim:
To evaluate the effect of hypernatremia on the organization of blood plasma high density lipoproteins (HDL) in goldfish; to compare the state of hypernatremia in fish and humans; to assess the possible risks and consequences of the effect of hypernatremia on human plasma lipoproteins.
Methods:
The fish were acclimated for 20 days at a critical salinity of 11.5 g/L; after that the salt water was gradually "desalinated". The concentration of Na+ and the content of total water were determined in tissues, cells, and body fluids. The HDL organization was assessed by the number of apolipoprotein molecules per particle. The methods of flame spectrophotometry, electrophoresis and MALDI were used.
Results:
In fresh water, the state of normonatremia was maintained in the fish body; at critical water salinity, the state of hypernatremia. Against the background of hypernatremia, the initial signs of muscle and erythrocyte dehydration appeared in fish, the total water content in the plasma did not change, and HDL disintegrated into small particles, which, upon restoration of normonatremia, were combined into the original large forms.
Conclusion:
In goldfish at the state of normonatremia, large forms of HDL are stable while at the state of hypernatremia, the small forms of HDL are stable. Under conditions of a hypertonic environment and plasma hypernatremia, the breakdown of HDL prevents the loss of water from the fish organism and reduces the threat of their dehydration. Human hypernatremia is characterized by plasma sodium levels comparable to that in goldfish, however accompanied by life-threatening metabolic changes. The results of this study may be useful for assessing the risks of HDL breakdown at hypernatremia and for the development of protocols for the treatment of pathological conditions in humans (Fig. 4, Ref. 45).
... Also, if one uses polyclonal antibodies to A2M, conformational changes in the latter may facilitate interactions with clones which recognize the modified A2M structure. Moreover, it has been established that A2M structure is acutely sensitive to freezing and lyophilization, which is a crucial factor with lyophilized A2M standards [60]. This is relevant because the commercial ELISA kit used here (Ref K6610A, Immundiagnostik AG, Germany), according to manufacturer's instructions, features an A2M standard that, after reconstitution, is stored at -20°C before use in analysis. ...
One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified proteolytic activity. Trypsin first hydrolyzes two bonds in A2M's ‘bait region’, and the peptide ⁷⁰⁵VGFYESDVMGR⁷¹⁵ is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (¹⁸O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that trypsin's modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.
... Purified human fibrinogen was from Hyphen Biomed (plasminogen-free) or from Sigma-Aldrich (premium quality essentially plasminogen-free). Native α 2 M was purified from normal blood plasma as described in (Wyatt et al. 2015). The collection of human plasma was approved by the joint UOW & ISLHD Health and Medical Human Research Ethics Committee (HE02/080) or the Central Adelaide Local Health Network Research Ethics Committee (HREC/18/CALHN/421). ...
Fibrinogen, a major constituent of blood plasma, is highly susceptible to reaction with biological oxidants. It has been proposed that fibrinogen plays a role in antioxidant defence, but oxidation of fibrinogen is also known to disrupt normal blood clotting and is implicated in the pathology of atherosclerosis. In the present study, we show that the biological oxidant hypochlorite promotes the formation of soluble high molecular weight fibrinogen assemblies ≥ 40 x 10⁶ Da, that do not accumulate when fibrinogen is induced to aggregate by other stresses such as heating or hydroxyl-mediated damage in vitro. Hypochlorite-modified fibrinogen is stable at 37ºC as assessed by precipitation assays, and has reduced susceptibility to iron-induced (hydroxyl-mediated) precipitation compared to native fibrinogen. In contrast to hypochlorite-modified albumin, which is known to be immunostimulatory, hypochlorite-modified fibrinogen does not induce RAW 264.7 (macrophage-like) cells or EOC 13.31 (microglia-like) cells to produce reactive oxygen species or induce cell death. Furthermore, depletion of fibrinogen from human blood plasma increases the immunostimulatory property of blood plasma after it is supplemented with hypochlorite in situ. We propose that reaction of hypochlorite with fibrinogen in blood plasma potentially reduces the accumulation of other hypochlorite-modified species such as immunostimulatory hypochlorite-modified albumin. The latter represent a novel role for fibrinogen in blood plasma antioxidant defence.
... That would explain changes in antigen/antibody interaction and, as a consequence, the increase in '()#* . Moreover, it has been established that A2M structure is acutely sensitive to freezing and lyophilization, which is a crucial factor with lyophilized A2M standards [54]. . CC-BY-ND 4.0 International license perpetuity. ...
One of the main functions of alpha-2-macroglobulin (A2M) in human blood serum is the binding of all classes of protease. It is known that trypsin, after such interaction, possesses modified pro-teolytic activity. Trypsin first hydrolyzes two bonds in A2M's 'bait region', and the peptide 705 VGFYESDVMGR 715 is released from A2M. In this work, specifics of the A2M-trypsin interaction were used to determine A2M concentration directly in human blood serum using MALDI mass-spectrometry. Following exogenous addition of trypsin to human blood serum in vitro, the concentration of the VGFYESDVMGR peptide was measured, using its isotopically-labeled analogue (18 O), and A2M concentration was calculated. The optimized mass spectrometric approach was verified using a standard method for A2M concentration determination (ELISA) and the relevant statistical analysis methods. It was also shown that tryp-sin's modified proteolytic activity in the presence of serum A2M can be used to analyze other serum proteins, including potential biomarkers of pathological processes. Thus, this work describes a promising approach to serum biomarker analysis that can be technically extended in several useful directions.
... Studies using stored pooled human plasma have suggested that two identical α 2 -macroglobulin subunits of size 182 kDa are disulfide-bonded to form dimers, which interact non-covalently to yield a tetrameric structure 4,5 . In biologic fluids, α 2 -macroglobulin tetramers appear to be predominant, but may undergo structural changes during manipulation and preservation 6 . Thus, the exact circulating molecular forms of α 2 -macroglobulin associated with health and diseases have yet to be elucidated. ...
α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosis and cardiac hypertrophy. However, its circulating molecular form and exact concentrations in human health/diseases are not known. Blue native-polyacrylamide gel electrophoresis of human serum was used to confirm the native conformation of α2-macroglobulin. We created an enzyme-linked immunosorbent assay suitable for quantifying its circulating molecular form and undertook a cross-sectional study to measure its serum levels in 248 patients with diabetes mellitus and 59 healthy volunteers. The predominant circulating molecular form of α2-macroglobulin was the tetramer, whereas its dimer was detectable in patients with high serum levels of α2-macroglobulin. The serum α2-macroglobulin concentration was not associated with glycated hemoglobin or any other glycemic variable as evaluated from 48-h continuous glucose monitoring, but showed close correlation with left ventricular posterior wall thickness, carotid artery intima-media thickness, urinary albumin:creatinine ratio (ACR) and brachial–ankle pulse wave velocity (baPWV). Multivariate analysis revealed only the ACR and baPWV to be independent variables influencing serum levels of α2-macroglobulin. Thus, an increased ACR and baPWV are associated with higher serum concentrations of α2-macroglobulin, and the latter may contribute to the mechanism by which albuminuria increases the risk of developing cardiovascular diseases.
