A novel heparin-dependent processing pathway for human tryptase. Autocatalysis followed by activation with dipeptidyl peptidase I.

Abstract

Tryptase is the major protein constituent of human mast cells, where it is stored within the secretory granules as a fully active tetramer. Two tryptase genes ( ␣ and ␤ ) are expressed by human mast cells at the level of mRNA and protein, each with a 30 amino acid leader sequence. Recombinant precursor forms of human ␣ -and ␤ -tryptase were produced in a baculovirus system, purified, and used to study their processing. Monomeric ␤ -protryptase first is shown to be intermolecularly autoprocessed to monomeric ␤ -pro'tryptase at acid pH in the presence of heparin by cleavage between Arg Ϫ 3 and Val Ϫ 2 in the leader peptide. The precursor of ␣ -tryptase has an Arg Ϫ 3 to Gln Ϫ 3 mutation that precludes autoprocessing. This may explain why ␣ -tryptase is not stored in secretory granules, but instead is constitutively secreted by mast cells and is the predominant form of tryptase found in blood in both healthy subjects and those with systemic mastocytosis under nonacute conditions. Second, the NH 2 -terminal activation dipeptide on ␤ -pro'tryptase is removed by dipeptidyl peptidase I at acid pH in the absence of heparin to yield an inactive monomeric form of tryptase. Conversion of the catalytic portion of ␤ -tryptase to the active homotetramer at acid pH requires heparin. Thus, ␤ -tryptase homotetramers probably account for active enzyme detected in vivo. Also, processing of tryptase to an active form should occur optimally only in cells that coexpress heparin proteoglycan, restricting this pathway to a mast cell lineage. ( J. Clin. Invest. 1996. 97: 988-995.)

Human Tryptases α and β/II Are Functionally Distinct Due, in Part, to a Single Amino Acid Difference in One of the Surface Loops That Forms the Substrate-binding Cleft

“…Although Mirza et al (31) were able to obtain enzymatically active tryptase ␣ in transiently transfected COS cells, Sakai et al (32) concluded that this human MC protease probably is not on recombinant mouse tryptases have revealed that this family of serine proteases must spontaneously form a tetramer to become enzymatically active. It is therefore assumed that the percentage of pseudozymogen in each preparation which is converted by EK into active enzyme is the same.…”

“…The latter investigators noted that the propeptide of tryptase ␣ differs from that of mouse, rat, and other human tryptase zymogens at residue Ϫ3. Based on their inability to obtain active enzyme in an insect cell expression system, Sakai et al (32) concluded that all tryptases probably undergo an unusual multistep activation process in MCs that is exquisitely heparin-dependent. To explain their in vitro findings, these investigators proposed that a conformation-dependent change occurs when a tryptase zymogen initially binds to heparin, which in turn causes a partial autocatalytic event resulting in the removal of the first 8 residues of the propeptide.…”

“…It was then proposed that dipeptidyl peptidase I removes the remaining 2 residues of the zymogen in the granule to create the mature enzyme. Unexplained in the Sakai et al (32) mechanism of tryptase activation is how a heparin-bound tryptase zymogen can partially activate itself yet still fail to cleave varied low molecular weight substrates.…”

Dipeptidyl Peptidase I Is Essential for Activation of Mast Cell Chymases, but Not Tryptases, in Mice

“…Recombinant human ␤-protryptase can be activated in vitro by two sequential steps of propeptide processing (7). First, ␤-protryptase is autoprocessed to the inactive intermediate proЈ-tryptase, which has a residual pro-dipeptide.…”

“…Available evidence suggests that activation of tryptases is more complicated than that of chymases and other granule-associated serine proteases. In vitro studies indicate that, after removal of a signal peptide by signal peptidase, human ␤-tryptase requires two additional, sequential cleavages of its propeptide for activation (7). The first such cleavage is thought to be by tryptase itself, which leaves a two-amino acid activation dipeptide to be removed by DPPI.…”