(A) Exon–intron structure of the mouse matriptase-3 gene and domain architecture of the deduced protein. The locations of the five potential N-glycosylation sites are indicated by arrowheads. The exons encoding the catalytic residues His⁶³² (H), Asp⁶⁸⁰ (D) and Ser⁷⁷⁶ (S) are shown. (B) Nucleotide sequence of the mouse matriptase-3 cDNA, as predicted by bioinformatic analysis and verified by direct molecular cloning, and the deduced amino acid sequence of the encoded protein. Amino acid residues encoding the predicted transmembrane domain are underlined, the activation cleavage site is boxed, the residues constituting the catalytic triad are highlighted in boldface and enlarged font, the potential N-glycosylation sites are encircled, and the predicted polyadenylation signal sequence in the 3′-untranslated region is underlined.

(A) RT–PCR analysis demonstrating matriptase-3 mRNA in brain, testis, skin and eye of adult mice. RNA was isolated from mouse liver (lane 1), brain (lane 2), thymus (lane 3), heart (lane 4), lung (lane 5), spleen (lane 6), testis (lane 7), ovary (lane 8), kidney (lane 9), skin (lane 10) and eye (lane 11), reverse transcribed, amplified by PCR with a mouse matriptase-3-specific primer pair (Mat3, upper panel) or a ribosomal protein S15 specific primer pair (S15, lower panel), and the amplification products were analysed by agarose gel electrophoresis. (B) Analysis of mouse matriptase-3 expression by dot-blot hybridization confirms the expression of matriptase-3 mRNA in brain, testis and eye, reveal high expression of matriptase-3 mRNA in the epididymis and salivary gland, and lower expression in the heart, skeletal muscle, thymus, ovary, prostate and uterus. The relative ratio of matriptase-3 expression to ubiquitin (UQ) expression is shown. Au, arbitrary units. (C) RT–PCR analysis of RNA isolated from spinal cord (lane 1), pons/medulla (lane 2), midbrain (lane 3), thalamus/hypothalamus (lane 4), cerebellum (lane 5), olfactory bulb (lane 6), frontal cortex (lane 7), posterior cortex (lane 8), hippocampus (lane 9), and striatum (lane 10) shows expression of matriptase-3 mRNA in all parts of the mouse brain. (D) RT–PCR analysis of RNA isolated from whole mouse skin (lane 1), primary mouse fibroblasts (lane 2) and primary mouse keratinocytes (lane 3) reveals the expression of matriptase-3 mRNA in whole mouse skin, but not in cultured keratinocytes or fibroblasts.

(A) N-glycosylation of mouse matriptase-3. Western-blot analysis with HA antibodies of whole cell lysates of COS7 cells transfected with empty expression vector (lanes 1 and 2) or a full-length, C-terminally HA-tagged mouse matriptase-3 cDNA expression plasmid (Mat3–HA; lanes 3 and 4). The protein extracts in lanes 2 and 4 were treated with endo H before SDS/PAGE, and the protein extracts in lanes 1 and 3 were treated with deglycosylation buffer (50 mM sodium citrate, pH 5.5) without endo H. The upper arrow indicates the position of the N-glycosylated form of mouse matriptase-3 (90 kDa), and the lower arrow indicates the position of the N-deglycosylated mouse matriptase-3 protein (80 kDa). The positions of molecular-mass standards (kDa) are indicated on the left. (B) The abnormal migration of mouse matriptase-3 in SDS/polyacrylamide gels is not caused by proteolytic processing. Whole cell lysates of COS7 cells transfected with empty expression vector (lane 1), a full-length, N-terminally HA-tagged mouse matriptase-3 cDNA expression plasmid (HA–Mat3; lane 2), or a full-length, C-terminally HA-tagged mouse matriptase-3 cDNA expression plasmid (Mat3–HA) were analysed by Western blotting using HA antibodies. The arrow indicates the position of N- and C-terminally tagged mouse matriptase-3 (90 kDa). The positions of molecular-mass standards (kDa) are indicated on the left. (C) Mouse matriptase-3 is expressed on the plasma membrane. COS7 cells transfected with a full-length, C-terminally HA-tagged mouse matriptase-3 cDNA (Mat3–HA; top panel) or with a full-length, C-terminally HA-tagged mouse DESC1 cDNA (DESC1–HA; middle panel) were treated with Sulfo-NHS-Biotin to biotinylate cell-surface proteins. Whole cell lysates were prepared and either analysed directly by Western blotting using HA antibodies (lysate, lane 1) or separated into non-biotinylated proteins (wash, lane 2) and biotinylated proteins (avidin-bound, lane 3) by avidin–agarose batch affinity chromatography. Bottom panel: lysates from mouse matriptase-3 cDNA expression plasmid-transfected cells were analysed by Western blotting against the cytoplasmic protein GAPDH. The absence of biotinylated GAPDH shows that the biotinylation agent did not penetrate the plasma membrane.

(A) Purified recombinant mouse matriptase-3 (10 nM) or thrombin (10 nM) were incubated with 100 μM of Suc-Ala-Ala-Pro-Arg-pNA (AAPR), Suc-Ala-Ala-Pro-Lys-pNA (AAPK), Suc-Ala-Ala-Pro-Val-pNA (AAPV), Suc-Ala-Ala-Pro-Phe-pNA (AAPF), H-Glu-Gly-Arg-pNA (EGR), Z-Gly-Gly-Arg-pNA (GGR), and Z-Phe-Val-Arg-pNA (FVR) at 35 °C, and the changes in A at 405 nm with time were monitored. (B) Purified recombinant mouse matriptase-3 (10 nM) was incubated with the synthetic peptide Suc-Ala-Ala-Pro-Arg-pNA in the absence or presence of PMSF (1 mM), egg white trypsin inhibitor (TI; 100 nM), EDTA (2.5 mM), aprotinin (3 μM), leupeptin (20 μM), E-64 (30 μM), human recombinant PAI-1 (50 nM) or soluble mouse recombinant HAI-1 (50 nM). The conversion rate in each reaction is presented as the change in A₄₀₅ with time (OD/hour, absorbance units/h).

(A) The predicted amino acid sequences of the serine protease domains of mouse (MmMAT3), rat (RnMAT3), human (HsMAT3), chimpanzee (PtMAT3), dog (CfMAT3), chicken (GgMAT3), tiger pufferfish (TnMAT3), spotted green pufferfish (FrMAt3), Danio zebrafish (DrMAT3) matriptase-3, mouse matriptase/MT-SP1 (MmMAT1) and mouse matriptase-2 (MmMAT2) were aligned with the Clustal W program. Amino acids essential for serine protease activity are highlighted. The activation cleavage site is boxed and highlighted in boldface, disulphide bridges between the eight conserved Cys residues (boxed) are indicated by solid lines, catalytic residues within their canonical motifs are highlighted in grey, and residues forming the substrate-binding site are indicated by asterisks. (B) Phylogenetic tree of the 19 known murine members of the TTSP family. Serase-1, -2 and -3 represent individual serine protease subunits of polyserase-1 [21]. The amino acid sequences of the catalytic domains were aligned with the Clustal W program using default settings. The phylogenetic tree was constructed with MEGA2.1 software using the neighbour-joining method and was tested for validity by 1000 bootstrap repetitions.

(A) RT–PCR analysis of human tissue samples reveals matriptase-3 mRNA expression in brain, lung, ovary, testis, trachea and salivary gland. RNA from human adipose tissue (lane 1), bladder (lane 2), brain (lane 3), cervix (lane 4), colon (lane 5), oesophagus (lane 6), heart (lane 7), kidney (lane 8), liver (lane 9), lung (lane 10), ovary (lane 11), placenta (lane 12), prostate (lane 13), skeletal muscle (lane 14), small intestine (lane 15), spleen (lane 16), testis (lane 17), thymus (lane 18), thyroid (lane 19), trachea (lane 20) and salivary gland (lane 21) was reversetranscribed, amplified by PCR with a human matriptase-3-specific primer pair (Mat3, upper panel) or a human ribosomal protein S15 specific primer pair (S15, lower panel), and the amplification products were analysed by agarose gel electrophoresis. (B) Northern-blot analysis of matriptase-3 expression in human tissues. Polyadenylated RNA (2 μg/lane) from tissue samples of human brain (lane 1), liver (lane 2), placenta (lane 3), small intestine (lane 4), colon (lane 5), thymus (lane 6), spleen (lane 7), prostate (lane 8), testis (lane 9) and ovary (lane 10) was separated by agarose gel electrophoresis, immobilized to a membrane and probed with a ³²P-labelled human matriptase-3 cDNA probe that contained nt 464–980 of the predicted open reading frame. The positions of RNA markers (kb) are indicated on the left.

(A) Top: C-terminally V5-tagged mouse matriptase-3 serine protease domain was purified to homogeneity from insect cell conditioned medium and analysed by Western blotting using anti-V5 antibodies before (lane 1) and after (lane 2) incubation for 60 min at 37 °C in 50 mM Tris/HCl (pH 8.0), 1 mM CaCl₂ and 0.1% Tween 20. The position of the activation site-cleaved mouse matriptase-3 serine protease domain is indicated. The positions of molecular-mass standards (kDa) are indicated on the left. Bottom: amino acid sequence of the N-terminus of the recombinant matriptase-3 zymogen (first 30 amino acids are shown). Amino acids forming the predicted activation cleavage site are highlighted in boldface, and the P₁–P₁′ bond is indicated with an arrowhead. (B) Activated mouse matriptase-3 serine protease domain cleaves and forms SDS-stable complexes with α₂M. Purified inactive (lanes 1–3) or activated (lanes 4–6) mouse matriptase-3 serine protease domain was pretreated with PMSF (lanes 3 and 6) or solvent (lanes 1, 2, 4 and 5) and incubated with α₂M (lanes 2, 3, 5 and 6) or buffer (lanes 1 and 4). Complex formation was analysed by Western blotting using anti-V5 antibodies under reducing conditions. The positions of mouse matriptase-3–α₂M complexes (Mat3:α₂M) and the uncomplexed activated mouse matriptase-3 serine protease domain (Mat3) are indicated. The position of molecular-mass standards (kDa) are shown on the left. (C) Matriptase-3 shows proteolytic activity against denatured collagens, casein and BSA. Gelatin (80 μg; lanes 1–3), denatured casein (lanes 4–6) or denatured BSA (lanes 7–9) were incubated with the buffer (lanes 1, 4 and 7), the purified activated mouse matriptase-3 serine protease domain (lanes 2, 5 and 8) or trypsin (lanes 3, 6 and 9). All reactions were analysed on SDS/PAGE followed by a Coomassie Brilliant Blue staining to visualize substrate cleavage.

The active mouse matriptase-3 catalytic domain was incubated for 30 min at room temperature with buffer (lane 1), 30 nM α₂M (lane 2), 100 nM human PAI-1 (lane 3), PAI-1_A P₁ mutant (lane 4), PAI-1_R insertion loop mutant (lane 5), PCI (lane 6), PCI with heparin (lane 7), α1-PI (lane 8), α2-AP (lane 8), AT III (lane 10), HC II (lane 11) or HC II with heparin (lane 12). The positions of the activated mouse matriptase-3 serine protease domain (Mat3) and the complexes of the protease with serpins (Mat3:serpins) are indicated. The positions of molecular-mass standards (kDa) are indicated on the left.