Structural changes in alpha-2- and ovomacroglobulins studied by gel chromatography and electron microscopy

Nishigai, Masaaki; Osada, Toshiya; Ikai, Atsushi

doi:10.1016/0167-4838(85)90040-8

Cited by 48 publications

(29 citation statements)

References 25 publications

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“…It is easy to reconcile the two shapes as two projections differing by a 900 rotation of the prototype molecule. These images were obtained for the exposed forms of a2M (11,16), and our interpretation of the relatedness of the two shapes is in agreement with the proposal by Tapon-Bretaudiere et al (16).…”

Section: Discussionsupporting

confidence: 80%

“…They further demonstrated that these views of the complex existed only after proteolysis. The existence of a third form was assigned to the native structure, and these results were confirmed by Nishigai et al (11). In these studies, electron micrographs of native a2M consisted of only a few ordered structures in the shape of crowns with a distribution of matter resembling the petals of a flower.…”

supporting

confidence: 74%

“…When exposed to an endoprotease, a limited proteolysis of a2M occurs at a site called the "bait" region, located near the middle of the protein, resulting in a change in its structure (5)(6)(7). The structural transformation has been identified by increases in the sedimentation coefficient (8) and in the electrophoretic mobility (9) and by decreases in the radius of gyration (10) and in the Stokes' radius (11). The inactivated protease may form a covalently bound complex with a2M by reacting with an intramolecular thiol ester linkage between glutamine and cysteine residues.…”

mentioning

confidence: 99%

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Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Bretaudiere

Tapon‐Bretaudière

Stoops

1988

Proc. Natl. Acad. Sci. U.S.A.

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Well-preserved structures of native and achymotrypsin-bound a2-macroglobulin were obtained by electron microscopy. Computer processing of these images has shown that the native structure has the shape of a padlock 19 nm long. It is proposed that the native a2-macroglobulin consists of the juxtaposition of two protomers with one protomer shaped like a distorted letter "S" and with the other its reverse image, to form a binding site between the two protomers near the bottom of the complex. On cleavage of the subunits with chymotrypsin, the native structure condenses to 16.7 nm and rearranges so that the interaction between the protomers is near the middle. Two images of the a2-macroglobulin-chymotrypsin conijugate were obtained. We suggest that these images represent the end and side view of this complex. Based on the manner in which the native structure is assembled, we propose that the proteolyzed form of a2-macroglobulin is functionally asymmetric in that both protease binding sites reside on the same half of the complex. a2-Macroglobulin (a2M) is one of the major antiproteases found in the plasma of vertebrates. It has the capacity to inhibit not only endoproteases normally present in the plasma but also proteases from other origins (1). a2M, isolated from human plasma, is a large glycoprotein (Mr, 725,000) composed of four identical subunits (Mr, 180,000) (2, 3). The quaternary structure consists of two noncovalently bound protomers; each protomer is made up of two subunits covalently linked by two disulfide bonds (4). When exposed to an endoprotease, a limited proteolysis of a2M occurs at a site called the "bait" region, located near the middle of the protein, resulting in a change in its structure (5-7). The structural transformation has been identified by increases in the sedimentation coefficient (8) and in the electrophoretic mobility (9) and by decreases in the radius of gyration (10) and in the Stokes' radius (11). The inactivated protease may form a covalently bound complex with a2M by reacting with an intramolecular thiol ester linkage between glutamine and cysteine residues. Even though there are four "bait" sites available, it has been proposed that a2M has two independent and identical proteinase binding sites (12)(13)(14). By using energy transfer experiments, Pochon et al. (12) demonstrated that the two sites are 4.4 nm apart. The binding of 2 mol of proteases per mol of a2M has been observed only with smaller proteases like trypsin and chymotrypsin (Mr, 25,000); larger proteases like plasmin (Mr, 81,000) display a 1:1 molar binding ratio (15).There have been conflicting reports relating the various structures of a2M obtained by electron microscopy to the native and protease bound forms. This discrepancy seems to have resulted from the study of a2M that had undergone proteolysis during its isolation (16). Early studies reported the presence of two forms (7,(17)(18)(19): an ovate structure shaped like two crescents facing each other with a bar in the center (the "closed" form) and a ...

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Section: Discussionsupporting

confidence: 80%

supporting

confidence: 74%

mentioning

confidence: 99%

See 1 more Smart Citation

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Bretaudiere

Tapon‐Bretaudière

Stoops

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…3C and Table 1). For example, the Stokes radius of p250 (10.4 nm) was calculated to be larger than that of ␣ 2 -macroglobulin (8.8 nm), a 720-kDa tetrameric protein (49). To test whether the exceptionally large Stokes radii reflected association with multiple detergent micelles, cell lysates were prepared and analyzed by size exclusion chromatography using detergents with different micelle sizes: n-octylglucoside (average micelle size of 8 kDa (50)), dodecyl-␤-D-maltoside (average micelle size of 50 -76 kDa (50)) and Triton X-100 (average micelle size of 81 kDa (51)).…”

Section: P250 and P160 Are Disulfide-bonded Species Composed Ofmentioning

confidence: 99%

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation

Watt

Spruce

et al. 2016

Journal of Biological Chemistry

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The formation of functional amyloid must be carefully regulated to prevent the accumulation of potentially toxic products. Premelanosome protein (PMEL) forms non-toxic functional amyloid fibrils that assemble into sheets upon which melanins ultimately are deposited within the melanosomes of pigment cells. PMEL is synthesized in the endoplasmic reticulum but forms amyloid only within post-Golgi melanosome precursors; thus, PMEL must traverse the secretory pathway in a non-amyloid form. Here, we identified two pre-amyloid PMEL intermediates that likely regulate the timing of fibril formation. Analyses by non-reducing SDS-PAGE, size exclusion chromatography, and sedimentation velocity revealed two native high M r disulfide-bonded species that contain Golgi-modified forms of PMEL. These species correspond to disulfide bond-containing dimeric and monomeric PMEL isoforms that contain no other proteins as judged by two-dimensional PAGE of metabolically labeled/immunoprecipitated PMEL and by mass spectrometry of affinity-purified complexes. Metabolic pulse-chase analyses, small molecule inhibitor treatments, and evaluation of site-directed mutants suggest that the PMEL dimer forms around the time of endoplasmic reticulum exit and is resolved by disulfide bond rearrangement into a monomeric form within the late Golgi or a post-Golgi compartment. Mutagenesis of individual cysteine residues within the non-amyloid cysteine-rich Kringlelike domain stabilizes the disulfide-bonded dimer and impairs fibril formation as determined by electron microscopy. Our data show that the Kringle-like domain facilitates the resolution of disulfide-bonded PMEL dimers and promotes PMEL functional amyloid formation, thereby suggesting that PMEL dimers must be resolved to monomers to generate functional amyloid fibrils.

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“…accumulated knowledge on the trap mechanism, we know that (1) one molecule of tetrameric human c~2M can trap up to two molecules of small proteinases such as chymotrypsin or trypsin, (2) when two mol of chymotrypsin are trapped per mol of c~2M under molar abundance of the former, all four bait regions of tetrameric ~2M are cleaved. The structural changes of CZzM and its homologues that takes place after bait region cleavage can be detected by a variety of physical and biochemical methods including electron microscopy [5,6], X-ray solution scattering [7], circular dichroism [8], electrophoresis [9], gel chromatography [10], fluorescence spectroscopy and sedimentation velocity [11]. We have little information, however, on the kinetic aspects of the reaction such as (1) how fast the bait regions are cleaved after the encounter of ~2 M with proteinases, (2) how many bait regions must be cleaved before the proteinases are trapped, (3) how closely connected in time are the bait region cleavage and the structural change?…”

Section: Introductionmentioning

confidence: 99%

Direct kinetics of bait region cleavage of α‐2‐macroglobulin by a rapid quenching method

Shibuya

Ikai

1993

FEBS Letters

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The rate of bait region cleavage of human c~-2-macroglobulin by chymotrypsin was determined by a rapid quenching method under conditions where the bimolecular encounter between the two reactants was not rate-limiting. ~2M was first mixed with a 30 molar excess of chymotrypsin in a sequential stopped-flow apparatus and after programmed time intervals the activity of chymotrypsin was quenched with 1 N HCI. The fraction of uncleaved subunits was quantitated by SDS-PAGE under reducing conditions. The result indicated that the bait region cleavage proceeded following a two-exponential decay curve with respective rate constants of k~ = 40 s -l and k 2 = 2 s ~.c~-2-Macroglobulin; Bait region cleavage; Rapid quenching method

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Structural changes in alpha-2- and ovomacroglobulins studied by gel chromatography and electron microscopy

Cited by 48 publications

References 25 publications

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

Structure of native alpha 2-macroglobulin and its transformation to the protease bound form.

The Kringle-like Domain Facilitates Post-endoplasmic Reticulum Changes to Premelanosome Protein (PMEL) Oligomerization and Disulfide Bond Configuration and Promotes Amyloid Formation

Direct kinetics of bait region cleavage of α‐2‐macroglobulin by a rapid quenching method

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