Stoichiometry of reactions of α2-macroglobulin with trypsin and chymotrypsin

Björk, Ingemar; Larsson, L J; Lindblom, T; Raub, E

doi:10.1042/bj2170303

Cited by 30 publications

(43 citation statements)

References 38 publications

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“…The bait region is located approximately in the middle of the polypeptide and, in the case of human ␣ 2 M, contains a sufficient range of different amino acids to make it a suitable "bait" for a wide spectrum of proteinase specificities (41). The efficiency of inhibition is extremely high, such that there is often a stoichiometric relationship between pairs of bait regions cleaved and molecules of proteinase inhibited (42).…”

Section: Resultsmentioning

confidence: 99%

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α-Macroglobulins Are Present in Some Gram-negative Bacteria

Doan

Gettins

2008

Journal of Biological Chemistry

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␣-Macroglobulins (␣Ms) are large glycoproteins that have been identified in a wide range of vertebrate and invertebrate species and are mostly thiol ester containing proteinase inhibitors. A recent analysis of bacterial genomes (Budd, A., Blandin, S., Levashina, E. A., and Gibson, T. J. (2004) Genome Biol. 5, R38) identified many ␣-macroglobulin-like sequences that appear to have been acquired by Gram-negative bacteria from their metazoan hosts. We report the first expression and characterization of such a bacterial ␣-macroglobulin, that from Escherichia coli. This is also the first ␣-macroglobulin to be characterized that is predicted to be membrane-anchored. We found that the 183-kDa protein contains an intact thiol ester, is monomeric, and is localized to the periplasmic space. Reaction with proteinase results in limited cleavage within a bait region, rapid activation of the thiol ester, crosslinking to the attacking proteinase or other available nucleophiles, and partial protection of the proteinase against macromolecular substrates. Given these properties and the co-occurrence of the ␣M gene with one for a repair transglycosylase, this suggests a possible role for bacterial ␣Ms in cell defense following host attack. Such a role would make bacterial ␣Ms appropriate novel targets for antibiotic drugs.The ␣-macroglobulins (␣Ms) 2 are a family of large proteins (monomers of Ͼ1400 residues) present in all types of metazoans (1). Most, although not all, have been found to contain an internal thiol ester formed between the side chains of a cysteine and a glutamine residue 3 residues further C-terminal. The thiol ester is usually critical for the functioning of the ␣M. In humans the best characterized ␣M is ␣ 2 M, which is an extremely abundant tetrameric plasma glycoprotein composed of 1451 residue subunits, and which acts as a pan-proteinase inhibitor, using a unique trapping conformational change-based mechanism (2). Other human ␣Ms include pregnancy zone protein (3), CD109 (4), and CPAMD8 (5), although these have been far less well characterized. Closely related to ␣ 2 M are the complement proteins C3, C4, and C5, all three of which appear to have evolved from the same primordial gene as ␣ 2 M (6). This relationship is suggestive of a potential role for ␣ 2 M and other ␣-macroglobulins in host defense (7-9).Although no ␣M proteins have ever been reported from bacteria, a recent data base search of bacterial genomes identified ␣M-like genes in a wide range of Gram-negative bacteria from a number of different clades. These include proteobacteria, cyanobacteria, spirochetes, and thermophillic bacteria (10). The phylogenetic distribution was, however, uneven and suggestive of acquisition of the gene from the metazoan host, perhaps as a colonization factor. Most intriguingly, the ␣M gene was almost always found in tandem with a gene for a cell wall repair transglycosylase, PBP1C (11), suggesting that the ␣M-like protein, together with the transglycosylase might function in bacterial defense subsequent to breach of t...

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Section: Resultsmentioning

confidence: 99%

“…3B)). Analogously, human ␣ 2 M requires two subunits for inhibition of 1 eq proteinase to give a maximum inhibitory stoichiometry of 2 mol of proteinase per ␣ 2 M tetramer (42).…”

Section: ϫ1 Smentioning

confidence: 99%

α-Macroglobulins Are Present in Some Gram-negative Bacteria

Doan

Gettins

2008

Journal of Biological Chemistry

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“…Trypsin Inhibition Assay-Stoichiometry of trypsin trapping was determined by measuring the soybean trypsin inhibitor-resistant trypsin activity (20,21) at different trypsin:␣ 2 M ratios. ␣ 2 M (120 nM) was incubated with different molar ratios of trypsin for 5 min at 25°C.…”

Section: Site-directed Mutagenesis and Expression Of Recombinant ␣ 2 mentioning

confidence: 99%

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

Bowen¹,

Gettins

1998

Journal of Biological Chemistry

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We have characterized four human ␣ 2 -macroglobulin (␣ 2 M) bait region variants (G679C, M690C, V700C, and T705C) to test the hypothesis that the bait regions are involved in the interface between noncovalently associated dimers. All four variants folded correctly as judged by many normal properties. However, the presence of a cysteine resulted in disulfide formation between otherwise noncovalently associated dimers in all four variants. The extent of disulfide cross-linking varied with the location of the cysteine and gave a mixture of species that probably contained two, one, or zero interdimer disulfides in the tetramer. This was reflected in heterogeneity of conformational change upon thiol ester cleavage by methylamine, with the presence of crosslinks correlating with blockage of conformational change. The stoichiometry of trypsin inhibition was less in all cases than for wild-type ␣ 2 M. The M690C variant also showed evidence of some species with an intramolecular disulfide between bait regions of monomers within the same dimer. Taken together, the results are consistent with a location of the four bait regions in contact with, or in very close proximity to, one another. This suggests that they form all or part of the "cavity body" seen in the low resolution x-ray structure of transformed ␣ 2 M. Inhibition of proteinases by humanresults from a series of conformational changes that are initiated by the proteinase and that result in the physical sequestration of the proteinase within the closed cage-like structure of the conformationally altered ␣ 2 M (1, 2). The initiating event is a proteolytic cleavage by the attacking proteinase near the center of the ␣ 2 M polypeptide in a region termed the bait region (3, 4). Cleavage anywhere within the bait region also results in activation of an internal thiol ester toward cleavage by nucleophiles. From studies on the kinetics of cleavage of the thiol ester by nucleophiles and of the subsequent conformational change within the ␣ 2 M, it has been shown that the conformational change occurs cooperatively after both thiol esters within one half of the ␣ 2 M tetramer have been cleaved (5, 6).Knowledge of the structural relationship between the thiol ester and the bait region and hence of the details of the activation mechanism is limited by the absence of a high resolution x-ray structure of either native or conformationally altered human ␣ 2 M. From fluorescence resonance energy transfer measurements, it was shown that the four cysteines that form the four thiol esters of the human ␣ 2 M tetramer are centrally located and are about 35 Å apart (7). This was subsequently confirmed by a low resolution (ϳ10 Å) x-ray structure of conformationally altered ␣ 2 M (8). NMR measurements on ␣ 2 M showed that the bait region of each monomer was unusually flexible (9, 10) and that it lies close (10 -17 Å) to the cysteine of the thiol ester in the transformed protein (11). However, the location of the four bait regions is still not known. Studies from this laboratory on ␣ 2 M varian...

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“…A stoichiometry of close to 2: 1 was reported for the reaction between trypsin or chymotrypsin and rY.2M by Bjork et a1. 29 ) Steiner et al reported that the equimolar rY. 2 M-thrombin complex contains a second site that is not readily cleaved by thrombin but is accessible to trypsin.…”

Section: Peg 2 -Modijication Of Sepl/mentioning

confidence: 99%

Functional Changes of Polyethylene Glycol-modified Serine Proteinase fromAspergillus sojaeand Interaction with α₂-Macroglobulin

Takoi¹,

Yamagata²,

Nakajima³

et al. 1989

Agricultural and Biological Chemistry

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Stoichiometry of reactions of α2-macroglobulin with trypsin and chymotrypsin

Cited by 30 publications

References 38 publications

α-Macroglobulins Are Present in Some Gram-negative Bacteria

α-Macroglobulins Are Present in Some Gram-negative Bacteria

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

Functional Changes of Polyethylene Glycol-modified Serine Proteinase fromAspergillus sojaeand Interaction with α₂-Macroglobulin

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Stoichiometry of reactions of α2-macroglobulin with trypsin and chymotrypsin

Cited by 30 publications

References 38 publications

α-Macroglobulins Are Present in Some Gram-negative Bacteria

α-Macroglobulins Are Present in Some Gram-negative Bacteria

Bait Region Involvement in the Dimer-Dimer Interface of Human α2-Macroglobulin and in Mediating Gross Conformational Change

Functional Changes of Polyethylene Glycol-modified Serine Proteinase fromAspergillus sojaeand Interaction with α2-Macroglobulin

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Functional Changes of Polyethylene Glycol-modified Serine Proteinase fromAspergillus sojaeand Interaction with α₂-Macroglobulin