title: Formation of enzymatically active, homotypic, and heterotypic tetramers of mouse mast cell tryptases. Dependence on a conserved Trp-rich domain on the surface.
abstract: Mouse mast cell protease (mMCP) 6 and mMCP-7 are homologous tryptases stored in granules as macromolecular complexes with heparin and/or chondroitin sulfate E containing serglycin proteoglycans. When pro-mMCP-7 and pseudozymogen forms of this tryptase and mMCP-6 were separately expressed in insect cells, all three recombinant proteins were secreted into the conditioned medium as properly folded, enzymatically inactive 33-kDa monomers. However, when their propeptides were removed, mMCP-6 and mMCP-7 became enzymatically active and spontaneously assumed an approximately 150-kDa tetramer structure. Heparin was not required for this structural change. When incubated at 37 degrees C, recombinant mMCP-7 progressively lost its enzymatic activity in a time-dependent manner. Its N-linked glycans helped regulate the thermal stability of mMCP-7. However, the ability of this tryptase to form the enzymatically active tetramer was more dependent on a highly conserved Trp-rich domain on its surface. Although recombinant mMCP-6 and mMCP-7 preferred to form homotypic tetramers, these tryptases readily formed heterotypic tetramers in vitro. This latter finding indicates that the tetramer structural unit is a novel way the mast cell uses to assemble varied combinations of tryptases.
authors.affiliation: Departments of Medicine, Boston, Massachusetts 02115, USA.
authors.forename: C
authors.initials: C
authors.lastname: Huang
authors.forename: G
authors.initials: G
authors.lastname: Morales
authors.forename: A
authors.initials: A
authors.lastname: Vagi
authors.forename: K
authors.initials: K
authors.lastname: Chanasyk
authors.forename: M
authors.initials: M
authors.lastname: Ferrazzi
authors.forename: C
authors.initials: C
authors.lastname: Burklow
authors.forename: W T
authors.initials: WT
authors.lastname: Qiu
authors.forename: E
authors.initials: E
authors.lastname: Feyfant
authors.forename: A
authors.initials: A
authors.lastname: Sali
authors.forename: R L
authors.initials: RL
authors.lastname: Stevens
mesh_terms: D000818:Animals; D053818:Chymases; D002942:Circular Dichroism; D004765:Enteropeptidase / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005990:Glycerol / Q000494:pharmacology; D006493:Heparin / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008407:Mast Cells / Q000201:enzymology*; D051379:Mice; D011485:Protein Binding; D011489:Protein Denaturation; D011498:Protein Precursors / Q000378:metabolism; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D053802:Tryptases
chemical_list: D011498:Protein Precursors; D011994:Recombinant Proteins; C509190:Tpsb2 protein, mouse; D006493:Heparin; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509222:Tpsab1 protein, mouse; D053802:Tryptases; D004765:Enteropeptidase; D005990:Glycerol