Human β-tryptase is a ring-like tetramer with active sites facing a central pore

Pereira, Pedro José Barbosa; Bergner, Andreas; Macedo-Ribeiro, Sandra; Huber, Robert; Matschiner, Gabriele; Fritz, Hans; Sommerhoff, Christian P.; Bode, Wolfram

doi:10.1038/32703

Cited by 288 publications

(296 citation statements)

References 24 publications

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“…The MT-SP1 polypeptide fold deviates notably from that of the serine proteinase tryptase, however, which exhibits six novel surface loops through which the tryptase monomers form the intermolecular interactions that stabilize the unusual tetramer structure (31). Thus, MT-SP1 is not closely related to tryptase, and the name "matriptase" is therefore potentially confusing.…”

Section: Discussionmentioning

confidence: 98%

Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase

Friedrich¹,

Fuentes‐Prior²,

Ong³

et al. 2002

Journal of Biological Chemistry

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109

107

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The type II transmembrane multidomain serine proteinase MT-SP1/matriptase is highly expressed in many human cancer-derived cell lines and has been implicated in extracellular matrix re-modeling, tumor growth, and metastasis. We have expressed the catalytic domain of MT-SP1 and solved the crystal structures of complexes with benzamidine at 1.3 Å and bovine pancreatic trypsin inhibitor at 2.9 Å. MT-SP1 exhibits a trypsinlike serine proteinase fold, featuring a unique nine-residue 60-insertion loop that influences interactions with protein substrates. The structure discloses a trypsinlike S1 pocket, a small hydrophobic S2 subsite, and an open negatively charged S4 cavity that favors the binding of basic P3/P4 residues. A complementary charge pattern on the surface opposite the active site cleft suggests a distinct docking of the preceding low density lipoprotein receptor class A domain. The benzamidine crystals possess a freely accessible active site and are hence well suited for soaking small molecules, facilitating the improvement of inhibitors. The crystal structure of the MT-SP1 complex with bovine pancreatic trypsin inhibitor serves as a model for hepatocyte growth factor activator inhibitor 1, the physiological inhibitor of MT-SP1, and suggests determinants for the substrate specificity.The activity of proteolytic enzymes is required at multiple stages during the growth, invasion, and progression of human tumors (for a review, see Ref. 1). For example, these complex processes entail extensive re-modeling of the extracellular matrix as well as the activation of latent growth factors and pro-angiogenic proteins. Consequently, the high level expression of particular proteinases often correlates with poor patient survival for several different cancers (see, for instance, Ref. The enzyme was initially assigned as a gelatinase, because of its gelatinolytic properties and gelatinase-like molecular weight. However, isolation and sequencing of the cDNA revealed a 683-residue multidomain proteinase with a C-terminal serine proteinase domain. The enzyme was then named matriptase to emphasize its matrix degrading properties and trypsin-like specificity (5). Independently, Takeuchi and coworkers (6) cloned and characterized a type-II membranebound trypsin-like serine proteinase from a human prostatic cancer cell line, which they called membrane-type serine proteinase 1, MT-SP1. This 855-residue proteinase contained two tandem repeats of the complement component C1r/s domain (CUB, derived from complement factor/1R-urchin embryonic growth factor/bone morphogenetic protein) and four tandem repeats of the low density lipoprotein receptor (LDLR) class A domain between the N-terminal transmembrane signal anchor and the C-terminal catalytic domain (5, 6). Because the matriptase sequence reported by Lin turned out to be part of the translated MT-SP1 cDNA sequence, matriptase is likely to be a form of MT-SP1 produced by ectodomain shedding (7). Alternatively, the two cDNAs may result from alternative splicing. MT-SP1 is highly exp...

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Section: Discussionmentioning

confidence: 98%

Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase

Friedrich¹,

Fuentes‐Prior²,

Ong³

et al. 2002

Journal of Biological Chemistry

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109

107

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“…Propeptide sequence and the carboxyl-terminal 11 residues were excluded from the model. Coordinates of the crystal-derived threedimensional structure of human ␤II-tryptase (Protein Data Bank number 1AOL) (17), which is pancreasin's closest relative for which diffraction data are available, served as template for the model, which was optimized by idealizing bond geometry and removing unfavorable contacts.…”

Section: Methodsmentioning

confidence: 99%

Structure and Activity of Human Pancreasin, a Novel Tryptic Serine Peptidase Expressed Primarily by the Pancreas

Bhagwandin

Hau

Clair

et al. 2003

Journal of Biological Chemistry

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In a search for genes encoding the serine peptidases prostasin and testisin, which are expressed mainly in prostate and testis, respectively, we identified a related, novel gene. Sequencing of cDNA allowed us to deduce the full amino acid sequence of the human gene product, which we term "pancreasin" because it is transcribed strongly in the pancreas. The idiosyncratic 6-exon organization of the gene is shared by a small group of tryptic proteases, including prostasin, testisin, and ␥-tryptase. Like the other genes, the pancreasin gene resides on chromosome 16p. Pancreasin cDNA predicts a 290-residue, N-glycosylated, serine peptidase with a typical signal peptide, a 12-residue activation peptide cleaved by tryptic hydrolysis, and a 256-amino acid catalytic domain. Unlike prostasin and other close relatives, human pancreasin and a nearly identical chimpanzee homologue lack a carboxyl-terminal membrane anchor, although this is present in 328-residue mouse pancreasin, the cDNA of which we also cloned and sequenced. In marked contrast to prostasin, which is 43% identical in the catalytic domain, human pancreasin is transcribed strongly in pancreas (and in the pancreatic ductal adenocarcinoma line, HPAC) but weakly or not at all in kidney and prostate. Antibodies raised against pancreasin detect cytoplasmic expression in HPAC cells. Recombinant, epitope-tagged pancreasin expressed in Chinese hamster ovary cells is glycosylated and secreted as an active tryptic peptidase. Pancreasin's preferences for hydrolysis of extended peptide substrates feature a strong preference for P1 Arg and differ from those of trypsin. Pancreasin is inhibited by benzamidine and leupeptin but resists several classic inhibitors of trypsin. Thus, pancreasin is a secreted, tryptic serine protease of the pancreas with novel physical and enzymatic properties. These studies provide a rationale for exploring the natural targets and roles of this enzyme.

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“…Within the mast cell granules, tryptase is found associated with heparin proteoglycan that serves to stabilize the enzyme and regulate its biological function upon release (Schwartz & Bradford, 1986). This 134 kDa tetrameric serine protease adopts a square¯at ring structure composed of four monomers, whose active sites face a central pore (Pereira et al, 1998), and consequently restrict access to potential macromolecular substrates and proteinase inhibitors. Nevertheless, tryptase has profound actions on cells, such as causing cytokine release from epithelial and endothelial cells (Cairns & Walls, 1996;Compton et al, 1998), proliferation of ®broblasts, epithelial and smooth muscle cells (Brown et al, 1995;Cairns & Walls, 1996;Ruoss et al, 1991), and ®broblast collagen synthesis (Cairns & Walls, 1997).…”

Section: Introductionmentioning

confidence: 99%

Glycosylation and the activation of proteinase‐activated receptor 2 (PAR₂) by human mast cell tryptase

Compton

Renaux

Wijesuriya

et al. 2001

British J Pharmacology

108

119

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1 Human mast cell tryptase appears to display considerable variation in activating proteinaseactivated receptor 2 (PAR 2 ). We found tryptase to be an ine cient activator of wild-type rat-PAR 2 (wt-rPAR 2 ) and therefore decided to explore the factors that may in¯uence tryptase activation of PAR 2 . 2 Using a 20 mer peptide (P20) corresponding to the cleavage/activation sequence of wt-rPAR 2 , tryptase was as e cient as trypsin in releasing the receptor-activating sequence (SLIGRL...). However, in the presence of either human-PAR 2 or wt-r PAR 2 expressing cells, tryptase could only activate PAR 2 by releasing SLIGRL from the P20 peptide, suggesting that PAR 2 expressed on the cells was protected from tryptase activation. 3 Three approaches were employed to test the hypothesis that PAR 2 receptor glycosylation restricts tryptase activation. (a) pretreatment of wt-rPAR 2 expressing cells or human embryonic kidney cells (HEK293) with vibrio cholerae neuraminidase to remove oligosaccharide sialic acid, unmasked tryptase-mediated PAR 2 activation. (b) Inhibiting receptor glycosylation in HEK293 cells with tunicamycin enabled tryptase-mediated PAR 2 activation. (c) Wt-rPAR 2 devoid of the Nterminal glycosylation sequon (PAR 2 T25 7 ), but not rPAR 2 devoid of the glycosylation sequon located on extracellular loop-2 (PAR 2 T224A), was selectively and substantially (430 fold) more sensitive to tryptase compared with the wt-rPAR 2 . 4 Immunocytochemistry using antisera that speci®cally recognized the N-terminal precleavage sequence of PAR 2 demonstrated that tryptase released the precleavage domain from PAR 2 T25 7 but not from wt-rPAR 2 . 5 Heparin : tryptase molar ratios of greater than 2 : 1 abrogated tryptase activation of PAR 2 T25 7 . 6 Our results indicate that glycosylation of PAR 2 and heparin-inhibition of PAR 2 activation by tryptase could provide novel mechanisms for regulating receptor activation by tryptase and possibly other proteases.

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Human β-tryptase is a ring-like tetramer with active sites facing a central pore

Cited by 288 publications

References 24 publications

Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase

Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase

Structure and Activity of Human Pancreasin, a Novel Tryptic Serine Peptidase Expressed Primarily by the Pancreas

Glycosylation and the activation of proteinase‐activated receptor 2 (PAR₂) by human mast cell tryptase

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Human β-tryptase is a ring-like tetramer with active sites facing a central pore

Cited by 288 publications

References 24 publications

Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase

Catalytic Domain Structures of MT-SP1/Matriptase, a Matrix-degrading Transmembrane Serine Proteinase

Structure and Activity of Human Pancreasin, a Novel Tryptic Serine Peptidase Expressed Primarily by the Pancreas

Glycosylation and the activation of proteinase‐activated receptor 2 (PAR2) by human mast cell tryptase

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About

Glycosylation and the activation of proteinase‐activated receptor 2 (PAR₂) by human mast cell tryptase