Chimerism, point mutation, and truncation dramatically transformed mast cell delta-tryptases during primate evolution
ABSTRACT Tryptases are serine peptidases stored in mast cell granules. Rodents express 2 soluble tryptases, mast cell proteases (MCPs) 6 and 7. Human alpha- and beta-tryptases are orthologs of MCP-6. However, much of the ancestral MCP-7 ortholog was replaced by parts of other tryptases, creating chimeric delta-tryptase. Human delta-tryptase's limited activity is hypothesized to be due to truncation and processing mutations.
We sought to probe the origins and consequences of mutations in primate delta-tryptases.
Prosimian (lemur), monkey (macaque), great ape (orangutan, gorilla, and chimpanzee), and human delta-tryptase genes were identified by means of data mining and genomic sequencing. Resulting genes were analyzed phylogenetically and structurally.
The seminal conversion event generating the delta-tryptase chimera occurred early because all primates studied contain delta-tryptase genes. Truncation, resulting from a nonsense mutation of Trp206, occurred much later, after orangutans and other great apes last shared an ancestor. The Arg-3Gln propeptide mutation occurred most recently, being present in humans and chimpanzees but not in other primates. Surprisingly, the major active tryptase in monkeys is full-length delta-tryptase, not beta-tryptase, which is the main active tryptase in human subjects. Models of macaque delta-tryptase reveal that the segment truncated in human subjects contains antiparallel beta-strands coursing through the substrate-binding cleft, accounting for truncation's drastic effect on activity.
Transformations in the ancestral MCP-7-like gene during primate evolution caused dramatic variations in function. Although delta-tryptases are nearly inactive in humans, they are active and dominant in monkeys.
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ABSTRACT: The human tryptase locus on chromosome 16 contains one gene encoding only β-tryptase and another encoding either β-tryptase or the homologous α-tryptase, providing α:β gene ratios of 0∶4, 1∶3 or 2∶2 in the diploid genome, these genotypes being of potential clinical relevance in severe atopy. Using an EcoRV restriction site in α- but not β- tryptase, PCR products, spanning intron 1 to exon 5, were used to determine α/β-tryptase gene ratios using non-radioactive labels, including ethidium bromide labeling of all PCR products, and either digoxigenin-primer or DY682-primer labeling of only the final PCR cycle products. Sensitivity increased ∼60-fold with each final PCR cycle labeling technique. Ethidium bromide labeling underestimated amounts of α-tryptase, presumably because heteroduplexes of α/β-tryptase amplimers, formed during annealing, were EcoRV resistant. In contrast, both final PCR cycle labeling techniques precisely quantified these gene ratios, because only homoduplexes were labeled. Using the DY682-primer was most efficient, because PCR/EcoRV products could be analyzed directly in the gel; while digoxigenin-labeled products required transfer to a nitrocellulose membrane followed by immunoblotting. This technique for determining the α/β-tryptase genotype is sensitive, accurate, simple and safe, and should permit high-throughput screening to detect potential phenotype-genotype relations for α/β-tryptases, and for other closely related alleles.PLoS ONE 12/2014; 9(12):e114944. DOI:10.1371/journal.pone.0114944 · 3.53 Impact Factor
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ABSTRACT: The most abundant prestored enzyme of human mast cell secretory granules is the serine-protease tryptase. In humans, there are four tryptase isoforms, but only two of them, namely the alpha and beta tryptases, are known as medically important. Low levels of continuous tryptase production as an immature monomer makes up the major part of the baseline serum tryptase levels, while transient release of mature tetrameric tryptase upon mast cell degranulation accounts for the anaphylactic rise of serum tryptase levels. Serum tryptase determination contributes to the diagnosis or monitoring of mast cell disorders including mast cell activation - induced anaphylaxis, mastocytosis and a number of myeloproliferative conditions with mast cell lineage involvement. Baseline serum tryptase levels are predictive of the severity risk in some allergic conditions.Molecular Immunology 05/2014; 63(1). DOI:10.1016/j.molimm.2014.04.001 · 3.00 Impact Factor