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. 2008 Dec 5;283(49):34178-87.
doi: 10.1074/jbc.M807553200. Epub 2008 Oct 14.

Alternate mRNA Splicing in Multiple Human Tryptase Genes Is Predicted to Regulate Tetramer Formation

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Free PMC article

Alternate mRNA Splicing in Multiple Human Tryptase Genes Is Predicted to Regulate Tetramer Formation

Nicole E Jackson et al. J Biol Chem. .
Free PMC article

Abstract

Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (alphaII, betaI, betaIII, and deltaI), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1' or S2' binding pocket and results in the complete loss of the "47 loop," reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates.

Figures

FIGURE 1.
FIGURE 1.
A, cDNA and putative amino acid sequence of αII and αIISV tryptase. The top line represents the translated amino acid sequence; the middle represents the nucleotide sequence ofαII tryptase (uppercase); and the bottom line represents the nucleotide sequence of αIISV tryptase (lowercase). The nucleotides deleted in the splice variant are denoted by a hyphen. Nucleotide numbering begins from the translation initiation codon (Met). B, partial cDNA and amino acid sequences of δI and δISV tryptase. C, partial cDNA and amino acid sequences of βI and βISV tryptase. D, partial cDNA and amino acid sequences of βIII and βIIISV tryptase.
FIGURE 2.
FIGURE 2.
mRNA splicing pattern. A, in αIISV tryptase, recognition of a novel 3′ splice site within exon 4 results in shortening of exon 4. B, detail of intron 3/exon 4 splice site junctions for αII and αIISV tryptase. C, nucleotide sequence alignment of intron 3/exon 4 splice site junctions for αII and αIISV tryptase.
FIGURE 3.
FIGURE 3.
Amino acid sequences of αIISV, βISV, and δISV tryptase compared with their full-length counterparts and to βII tryptase. A dash indicates the presence of an identical amino acid. Numbering begins at the first residue of the mature enzyme, which is indicated by a down arrow. The location of the 47 loop is indicated by a thick bar. His, Asp, and Ser of the catalytic triad are marked with a number sign.
FIGURE 4.
FIGURE 4.
A, three-dimensional surface electrostatic models of wild-type (or full-length) αII tryptase (left panel) and αIISV tryptase (right panel). B, surface representation of βII tryptase as reported by Pereira et al. (21) showing the tryptase tetramer ring structure. The 9 amino acids that are deleted in the splice variants are colored orange, and due to the orientation of the individual monomers they are visible in the A and C monomers only. Monomers A–D are depicted as dark blue, light blue, green and yellow, respectively.
FIGURE 5.
FIGURE 5.
Splice variant tryptases are transcribed in various tissues. The relative abundance of splice variant tryptase transcripts in a range of human tissues was determined using RT-quantitative PCR. The data represents the mean (± S.D.) from a single experiment. All samples were tested in triplicate and have been tested in at least two independent experiments.
FIGURE 6.
FIGURE 6.
A, affinity-purified anti-αIISV tryptase antibody recognizes rβISV tryptase but not rβIorrβII tryptase. B, anti-tryptase antibody AA1 recognizes rβISV, rβI, and rβII tryptases. Sizes are as indicated in kDa. rβISV and rβI tryptases were expressed in E. coli.
FIGURE 7.
FIGURE 7.
Splice variant tryptase protein is expressed in human aorta (adventitia), spleen, and breast tumor tissues. Sections were stained immunohistochemically using anti-αIISV tryptase antibody (A, C, and E), the monoclonal anti-tryptase antibody AA1 (B, D, and F), and isotype control sections are shown in the inset. A and B, C and D, and E and F are serial sections.
FIGURE 8.
FIGURE 8.
βISV tryptase expressed in yeast and activated by EK cleavage of the propeptide cleaves the substrate tosyl-GPR-pNA, whereas unactivated βISV tryptase does not. EK activated, but not unactivated, βI tryptase also cleaves tosyl-GPR-pNA. The EK control had a similar level of activity as the unactivated proteins.
FIGURE 9.
FIGURE 9.
βISV tryptase cleaves human fibronectin. Purified and activated βISV tryptase (lane 3) and βI tryptase (lane 4) were incubated with human fibronectin (1 mg) in the presence of heparin for 18 h at room temperature. Samples were separated on a nonreduced 7.5% Tris-Tricine SDS-polyacrylamide gel and silver-stained. Arrow indicates a fibronectin digestion band. Controls are human fibronectin alone (lane 1) and fibronectin with EK (lane 2). Sizes are as indicated in kDa.

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