Structural Requirements and Mechanism for Heparin-induced Activation of a Recombinant Mouse Mast Cell Tryptase, Mouse Mast Cell Protease-6

Jenny Hallgren,Dorothe Spillmann,Gunnar Pejler

doi:10.1074/jbc.m105531200

Abstract

Mast cell tryptase is stored as an active tetramer in complex with heparin in mast cell secretory granules. Previously, we demonstrated the dependence on heparin for the activation/tetramer formation of a recombinant tryptase. Here we have investigated the structural requirements for this activation process. The ability of heparin-related saccharides to activate a recombinant murine tryptase, mouse mast cell protease-6 (mMCP-6), was strongly dependent on anionic charge density and size. The dose-response curve for heparin-induced mMCP-6 activation displayed a bell-shaped appearance, indicating that heparin acts by binding to more than one tryptase monomer simultaneously. The minimal heparin oligosaccharide required for binding to mMCP-6 was 8-10 saccharide units. Gel filtration analyses showed that such short oligosaccharides were unable to generate tryptase tetramers, but instead gave rise to active mMCP-6 monomers. The active monomers were inhibited by bovine pancreatic trypsin inhibitor, whereas the tetramers were resistant. Furthermore, monomeric (but not tetrameric) mMCP-6 degraded fibronectin. Our results suggest a model for tryptase tetramer formation that involves bridging of tryptase monomers by heparin or other highly sulfated polysaccharides of sufficient chain length. Moreover, our results raise the possibility that some of the reported activities of tryptase may be related to active tryptase monomers that may be formed according to the mechanism described here.

Highlights

Mast cell tryptase is stored as an active tetramer in complex with heparin in mast cell secretory granules
We showed that heparin is required for the assembly of monomeric inactive tryptase to active tetramers and that tetramerization only occurs at acidic pH [10]
Structural Requirements for the Activation of mouse mast cell protease-6 (mMCP-6) by Heparin—In a previous study, we showed that the activation and tetramer formation of a recombinant tryptase, mMCP-6, is dependent on heparin and acidic pH [10]

Summary

EXPERIMENTAL PROCEDURES

Active mMCP-6 was obtained after incubating the protein with heparin at pH 6 as described [10] (see below). MMCP-6 (100 –200 ng) was incubated with different saccharides (445 pM to 44.5 ␮M) in PBS (total volume of 130 –260 ␮l), pH 6, for 30 min. In one set of experiments, the standard PBS buffer (0.14 M NaCl) was replaced by phosphate buffers containing 0 – 0.5 M NaCl. Subsequently, mMCP-6 activity was recorded using a Titertek Multiscan spectrophotometer (Flow Laboratories) after addition of 20 ␮l of a 2 mM solution of S-2288 in H2O. 3 ␮g of enterokinase-digested His6-EK-mMCP-6 was incubated with [3H]heparin (intact; 0.48 –360 pmol, 180 –136,000 cpm) or [3H]heparin oligosaccharides of various sizes (80 pmol; 4500 cpm) in a total volume of 400 ␮l of PBS, pH 6, for 30 min. After an incubation time of 30 min, residual tryptase activity was determined as described above

RESULTS

Mechanism for Tryptase Activation

DISCUSSION