Decreased expression of HATL5 mRNA and protein in human carcinomas.

<p>(<b>A</b>) TMPRSS11b, encoding HATL5 in four (1–4) gene expression array studies of human carcinomas (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087675#pone.0087675.s001" target="_blank">Table S1</a>). Data are expressed as fold change in HATL5 mRNA relative to corresponding normal tissue. *<i>P</i><4·10⁻⁵, **<i>P</i><4·10⁻¹⁰, ***<i>P</i><2·10⁻²³. (<b>B</b>) IHC detection of HATL5 protein in esophageal tissue (B1, B2, B3) and cervical tissue sections (C1, C2, C3). Primary antibodies were substituted with non-immune rabbit IgG in serial section of all samples and no significant staining was observed (not shown). Strong epithelial staining (arrow heads) is detected in normal esophagus (B1) and normal cervix (C1). With carcinoma progression (Grade I) the HATL5 staining was weaker and more diffuse with only a few moderately stained carcinoma cells (arrowheads in B2 and C2) and in high grade poorly differentiated tumors (Grade III) the staining intensity was very low or below the detection limit of this assay (B3 and C3). Epi = normal epithelium, Ca = carcinoma cells. Size bars all panels; 50 µm.</p

Amino acid sequence, and predicted domain architecture of human HATL5.

<p>(<b>A</b>) Amino acid sequence of human HATL5 (UniProtKB/Swiss-Prot: Q86T26.3). Amino acid residues encoding the transmembrane domain are underlined. The region with homology to sea urchin sperm protein, enteropeptidase, agrin (SEA) domains is indicated by a solid line box and the trypsin-like serine protease domain is indicated with a dashed line box. The catalytic residues His²²⁵ (H), Asp²⁷⁰ (D), and Ser³⁶⁶ (S) are indicated with squares. Potential N-glycosylation sites are indicated with circles. The activation cleavage site is indicated with an arrow, and the two cysteine residues predicted to form a disulfide bridge linking the stem region to the serine protease domain upon activation cleavage are indicated with triangles. (<b>B</b>) Schematic representation of the predicted domain architecture of HATL5. TM = Transmembrane domain, SEA = Sea urchin <u>s</u>perm protein, <u>E</u>nteropeptidase, <u>A</u>grin domain, N indicates predicted N-glycosylation sites, the activation cleavage site is indicated with an arrow, and S-S represents the disulfide bridge linking the SEA and serine protease domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087675#pone.0087675-Somoza1" target="_blank">[31]</a>.</p

HATL5 expression and localization.

<p>(<b>A</b>) Micrographs of fluorescent confocal analysis showing cell surface expression in representative examples of HEK293 cells transiently transfected with a human full-length HATL5-V5 expression plasmid. Nonpermeabilized cells were incubated with an anti-V5 antibody (left panel) or a control non-immune antibody (right panel), followed by incubation with AlexaFluor 568-labeled secondary antibodies and Hoechst staining to visualize nuclei (blue; both panels). The cells were visualized by confocal fluorescence microscopy at 543 nm (AlexaFluor 568) and 405 nm excitation wavelengths (Hoechst). Merged images obtained at the two excitation wavelengths are shown. Size bars are 100 µm. (<b>B</b>) Expression of HATL5 (upper panel) or GAPDH (lower panel) message by RT-PCR analysis of mRNA extracted from whole mouse organs. (C) Immunohistochemical analysis of HATL5 expression in normal human tissues. HATL5 protein was detected with a rabbit-anti HATL5 antibody in esophageal musoca (C1, D1), cervical mucosa (E), oral mucosa (tongue) (F) and epiglottis (part of the supraglottic larynx) (G). Primary antibodies were substituted with non-immune rabbit IgG in serial sections of all samples and no significant staining was observed (arrowheads in C2, D2 and not shown). Strong epithelial staining (arrowheads) is detected in apical, squamous epithelial cells in normal esophagus (C1, D1), normal cervix (E), and tongue (F) with no significant staining in the mesenchymal compartment (indicated with asterisks). At high magnification, HATL5 protein is clearly localized on the cell surface of apical epithelial cells (arrow head in D1). In the epiglottis (G) staining is observed in squamous suprabasal epithelium cells in addition to apical cells. Epi = normal epithelium. Size bars all panels; 50 µm.</p

HATL5 is a 60-kDa glycoprotein.

<p>(<b>A</b>) Schematic representation of the three different recombinant HATL5 proteins generated for this study. V5-H = V5-His epitope tag (<b>B</b>) Whole cell protein lysates from HEK293, COS-7, or CHO cells expressing full-length V5-His tagged human HATL5. (<b>C</b>) Conditioned media from CHO cells expressing myc-tagged HATL5 serine protease domain or conditioned media from <i>Pichia pastoris</i> expressing cleaved, active HATL5 serine protease were analyzed. (<b>B and C</b>) Proteins were separated by SDS-PAGE and analyzed by western blotting using anti-V5, anti-myc or anti-HATL5 antibodies as indicated. Lanes with protein extracts treated with deglycosylation enzymes prior to SDS-PAGE are indicated (+), and untreated extracts (−). The black arrowheads indicate the position of the glycosylated forms of HATL5, and the open arrowheads indicate the position of the deglycosylated forms.</p

Analysis of the enzymatic activity of human HATL5.

<p>(<b>A</b>) Conditioned media samples from <i>Pichia pastoris</i> clones transfected with either the expression vector without protease insert (vector), with HATL5 serine protease domain cDNA (HATL5 #1 and #2) or matriptase serine protease domain cDNA were analyzed by western blotting using an anti-HATL5 antibody (left panel) or an anti-matriptase antibody (right panel). The positions of HATL5 (open arrow head) and matriptase serine protease domain (black arrow head) are indicated. (<b>B</b>) Samples from the conditioned media described in (A) were incubated at 37°C for 60 min with the synthetic chromogenic peptide Suc-Ala-Ala-Pro-Arg-pNA (100 µm) and the absorbance at 405 nm was recorded (<b>C</b>) 5 nM purified active recombinant HATL5 (white bars) or matriptase (black bars) serine protease domain was incubated at 37°C for 60 min with the synthetic chromogenic peptide MeOSuc-Glu-Val-Lys-Met-pNA (100 µm) in the absence or presence (inhibitor and substrate added concomitantly) of HAI-1 (60 nM), HAI-2 (40 nM), aprotinin (2 µm), leupeptin (20 µm), benzamidine (2 mM), or serpinA1 (60 nM). Enzyme activities for each enzyme are depicted relative to activity when no inhibitor was added.</p