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Protease inhibitors of the alpha-macroglobulin family (αM) have a unique mechanism that allows them to trap proteases that is dependent not on the protease's class, but rather on its cleavage specificity. Proteases trigger a conformational change in the αM protein by cleaving within a "bait region," resulting in the sequestering of the protease inside the αM molecule. This nonspecific inhibitory mechanism appears to have arisen early in the αM family, and the broad protease-trapping capacity that it allows may play a role in pathogen defense.Human α 2 -macroglobulin (A2M) is a tetrameric αM whose bait region is permissive to cleavage by most proteases, making it a broad-spectrum protease inhibitor. Recent work has demonstrated that the inhibitory capacity of A2M derives directly from its bait region sequence: modifying the bait region sequence to introduce or remove protease cleavage sites will modify A2M's inhibition of the relevant proteases accordingly. Thus, changing the amino acid sequence of the bait region presents an effective avenue for protein engineering of new protease inhibitors if the substrate specificity of the target protease is known. The design of new A2M-based protease inhibitors with tailored inhibitory capacities has potential applications in basic research and the clinic. In this chapter, we describe the general approach and considerations for the bait region engineering of A2M., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Abstract: An alpha 2-macroglobulin (α2-M) gene was cloned from the haemocytes of tiger shrimp Penaeus monodon by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the α2-M cDNA consists of 4876bp with an open reading frame (ORF) of 4494bp, a 52bp 5′-untranslated region, and a 327bp 3′-untranslated region containing a poly A signal. The open reading frame encodes a protein of 1498 amino acids with 18 residues signal sequence. The predicted molecular mass of the mature protein (1480 amino acids) is 167.7kDa with an estimated pI of 5.30. The P. monodon α2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate α2-Ms. Sequence comparison showed that α2-M deduced amino acid sequence of P. monodon has an overall similarity of 85, 52 and 49% to that of kuruma shrimp Marsupenaeus japonicus, American horseshoe crab Limulus polyphemus and mud crab Scylla serrata, respectively. Alignment of the deduced amino acid sequence to other species α2-M showed that the overall structure is evolutionarily conserved and phylogentic analysis revealed that P. monodon α2-M is closely related to other arthropod α2-M, and displays the highest similarity to M. japonicus α2-M. The α2-M was mainly expressed in haemocytes, but not in eyestalk, gill, muscle, hepatopancreas, and intestine. Quantitative real-time RT-PCR analysis showed that α2-M mRNA transcript in haemocytes of P. monodon increased significantly in 12, 24 and 48h post-peptidoglycan (PG) injection, but returned to the original values in 72h post-PG injection. [Copyright &y& Elsevier]

Abstract: Molecular approaches were used to clone thioester-containing α2-macroglobulin (α2-M) genes in the haemocytes of mud crab Scylla serrata. The full length sequence of α2-M was determined by RT-PCR, cloning and sequencing of overlapping PCR and rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the α2-M cDNA clone consists of 5491bp with an open reading frame (ORF) of 4986bp encoding a protein of 1662 amino acids with 22 residues signal sequence. The calculated molecular mass of the mature protein is 184.2kDa with an estimated pI of 8.41. The S. serrata α2-M sequence contains putative functional domains including a GCGEQNM thioester region, a bait region, and a receptor-binding domain which are present in other invertebrate and vertebrate α2-Ms. Sequence comparison showed that α2-M deduced amino acid sequence of S. serrata has an overall similarity of 68% and 48% to that of kuruma shrimp Marsupenaeus japonicus and American horseshoe crab Limulus polyphemus, respectively. Phylogentic analysis revealed that S. serrata α2-M is closely related to other arthropod α2-M, and displays the highest similarity to M. japonicus α2-M. The α2-M was mainly expressed in haemocytes. Quantitative real-time RT-PCR analysis showed that α2-M mRNA transcript in haemocytes of S. serrata increased significantly in 24h- and 48h-post lipopolysaccharide (LPS) injection. [Copyright &y& Elsevier]

Abstract: The plasma protein α2-macroglobulin (α2M) was purified by gel filtration and anion-exchange chromatography from grass carp plasma. The α2M consists of two different subunits of molecular weight 95kDa and 80kDa, respectively. The characteristics of grass carp α2M are similar to mammalian α2M, in that grass carp α2M exists in two forms: a fast-form and a slow-form. The former is complexed with protease. The sequence of grass carp α2M-conserved region and a region containing the bait region was determined by sequence analysis using polymerase chain reaction (PCR). The deduced amino acid sequence of the conserved region is similar to the α2M sequence of common carp, however, the bait region amino acid sequence is dramatically distinct from that of common carp. This may partially explain the differential ability of α2M of different species to inhibit different proteases. The α2M was able to inhibit Aeromonas hydrophila extracellular protease (AhECPase) and thus may play a role in resistance to infection by this pathogen. [Copyright &y& Elsevier]

The sequence of a α2-macroglobulin (α2M) from the soft tick Ornithodoros moubata (TAM) was determined by cloning and sequencing of overlapping polymerase chain reaction (PCR) and rapid amplification of cDNA ends PCR products. The TAM cDNA sequence is 4944 bp long and contains one open reading frame coding for a protein precursor composed of 1494 amino-acid residues, including a 24-residue signal sequence. The mature protein is cleaved into two subunits similarly to the C3 and C4 components of complement and fish α2Ms. Phylogeny analysis revealed that TAM is closely related to Limulus α2M and displays the highest similarity to the partial sequence of α2M from hard tick Ixodes scapularis. The comparison of conserved cysteine residues between TAM and human and Limulus α2Ms made it possible to predict the pattern of disulfide bridges and explain the atypical molecular arrangement of TAM. Four variants of the TAM bait region differing only in a short central segment were found; our data indicate that TAM exists as a single-copy gene in the tick genome and its bait region variants likely arise by alternative splicing. TAM is produced by tick hemocytes and it is also significantly expressed in salivary glands. TAM mRNA levels were shown to be up-regulated upon blood meal. [Copyright &y& Elsevier]

Alpha 2-macroglobulin (α2M), a plasma glycoprotein produced in the liver, inhibits a variety of proteinases and thus considered to play important homeostatic roles in the body. This broad inhibitory spectrum has been explained by the trapping theory by which a proteinase recognizes a region of 25–30 amino acid peptide in α2M called bait region and cleaves it, leading to the conformational change of α2M, and to the subsequent entrapment and inhibition of the proteinase. We constructed α2M cDNAs with mutated DNA sequences in the bait region, and obtained recombinant CHO cell lines producing either wild type α2M, or mutant α2Ms, i.e., α2M/K692 and α2M/K696, each with substitution of Arg with Lys at codons 692 and 696, respectively. We tested if lysyl endopeptidase is not inhibited by wild type α2M, but could be inhibited by these engineered mutant α2Ms. Thus, recombinant α2M/K696 protein successfully inhibited lysyl endopeptidase activity, while recombinant α2M/K692 protein was not sensitive to lysyl endopeptidase, suggesting that not all bait region peptide bonds can equally be accessible and susceptible to proteinases. The present results not only provided the trapping theory with additional supportive evidence, but the first experimental evidence for the value of engineered α2M-derived proteinase inhibitor with an artificial proteinase inhibitory spectrum of potential industrial and/or therapeutic usefulness. [ABSTRACT FROM AUTHOR]