Substrate specificity and inhibitory study of human airway trypsin-like protease

Human airway trypsin-like protease (HAT), also referred to as TMPRSS11D, is an important physiological enzyme with the main activity pronounced in an airway. In this work we have described the substrate specificity and selectivity study of the protease, performed by the combinatorial approach. Fluorogenic/chromogenic tetrapeptide library was used for this purpose. The most efficiently hydrolyzed substrates’ sequences that we selected were ABZ-Arg-Gln-Asp-Arg(Lys)-ANB-NH₂. The most active inhibitor with C-terminal Arg residue underwent detectable proteolysis action in the presence of 35 pM of HAT. Based on the selected sequences the two peptide aldehydes were synthesized and (Abz-Arg-Gln-Asp-Arg(Lys)-H) were found to be an effective HAT inhibitor, working in nanomolar range with inhibition constant 54 nM and 112 nM, respectively.     

Activation of macrophage-stimulating protein by human airway trypsin-like protease

Macrophage-stimulating protein (MSP) circulates as a proform protein and requires proteolytic processing for activation. Respiratory ciliated cells express the MSP receptor, recepteur d'origine nantais (RON), at the apical surface, which reportedly has an important role in ciliary function. Like RON, human airway trypsin-like protease (HAT) is also expressed at the apical surface of ciliated cells. Here we show that HAT cleaves proMSP at the physiological activation site, Arg483-Val484. MSP processed by HAT could induce chemotactic responses and morphological changes of peritoneal macrophages. In human respiratory epithelial cells, knock down of HAT expression reduced proMSP processing and RON autophosphorylation. We suggest that HAT is important for MSP-RON signaling in the respiratory tract.     

Development of substrate analogue inhibitors for the human airway trypsin-like protease HAT

A series of substrate analogue inhibitors of the serine protease HAT, containing a 4-amidinobenzylamide moiety as the P1 residue, was prepared. The most potent compounds possess a basic amino acid in the d-configuration as P3 residue. Whereas inhibitor 4 (K_i 13 nM) containing proline as the P2 residue completely lacks selectivity, incorporation of norvaline leads to a potent inhibitor (15, K_i 15 nM) with improved selectivity for HAT in comparison to the coagulation proteases thrombin and factor Xa or the fibrinolytic plasmin. Selected inhibitors were able to suppress influenza virus replication in a HAT-expressing MDCK cell model.     

Hepatocyte growth factor activator inhibitor type 1 inhibits protease activity and proteolytic activation of human airway trypsin-like protease

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type transmembrane serine protease inhibitor initially identified as a potent inhibitor of hepatocyte growth factor activator (HGFA), a serine protease that converts pro-HGF to the active form. HAI-1 also has inhibitory activity against serine proteases such as matriptase, hepsin and prostasin. In this study, we examined effects of HAI-1 on the protease activity and proteolytic activation of human airway trypsin-like protease (HAT), a transmembrane serine protease that is expressed mainly in bronchial epithelial cells. A soluble form of HAI-1 inhibited the protease activity of HAT in vitro. HAT was proteolytically activated in cultured mammalian cells transfected with its expression vector, and a soluble form of active HAT was released into the conditioned medium. The proteolytic activation of HAT required its own serine protease activity. Co-expression of the transmembrane full-length HAI-1 inhibited the proteolytic activation of HAT. In addition, full-length HAI-1 associated with the transmembrane full-length HAT in co-expressing cells. Like other target proteases of HAI-1, HAT converted pro-HGF to the active form in vitro. These results suggest that HAI-1 functions as a physiological regulator of HAT by inhibiting its protease activity and proteolytic activation in airway epithelium.     

Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway

Human airway trypsin-like protease (HAT) was isolated from airway secretions and localized to bronchial epithelial cells by immunohistochemistry. In the present study, we examined whether HAT could stimulate DNA synthesis and proliferation of primary human bronchial fibroblasts (HBF). HAT significantly stimulated the proliferation of HBF by 20-55%, a level similar to that of the mitogenic activity of lung mast cell tryptase (MCT). HAT also stimulated the incorporation of [3H]thymidine in HBF, and this HAT-induced DNA synthesis was abolished by leupeptin. Protease-activated receptor-2 (PAR-2) mRNA was expressed and localized to the cell surface in HBF. PAR-2 activating peptide (AP) also enhanced DNA synthesis, and both HAT and PAR-2 AP induced receptor internalization, similar to the response to trypsin. Pretreatment of HBF with anti-PAR-2 antibody significantly suppressed both HAT and PAR-2 AP-induced DNA synthesis. In addition, HAT and PAR-2 AP induced intracellular Ca2+ mobilization in HBF. The HAT-induced increase in Ca2+ was desensitized by pretreatment with trypsin or PAR-2 AP. U0126, a specific MAPK inhibitor, completely inhibited HAT-induced DNA synthesis as well as HAT-induced phosphorylation of MAPK. The effect of HAT and MCT together was additive, whereas the effect of HAT and insulin together on HBF DNA synthesis was synergistic. These results indicate that HAT stimulates fibroblast proliferation in bronchial airways through a PAR-2-dependent MEK-MAPK mediated pathway and that HAT is linked to airway processes involving fibroblasts.     

Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease

The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.     

Localization of gamma-glutamyl transpeptidase in human sweat glands: an immunohistochemical study using a monoclonal antibody

We studied the distribution of gamma-glutamyl transpeptidase (gamma-GT) by use of a monoclonal antibody (MAb) against human kidney gamma-GT in human sweat glands. In the eccrine sweat gland, the enzyme was localized along the luminal membrane and small apocrine extrusions of the superficial cells of the secretory portion. The intercellular canaliculi between basal cells were occasionally immunoreactive. In the secretory portion of the apocrine gland, luminal membrane and apocrine extrusions of various sizes and stages at the apices of the secretory cells exhibited positive reactions. Immunoreaction was also seen in the Golgi area of the cuboidal secretory cells. No positive reaction was observed in the myoepithelial cells of either gland or in the excretory duct cells.     

The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions

The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10-500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.     

Localization of a proteasomal antigen in human spermatozoa: immunohistochemical electron microscopic study

The ultrastructural localization of a proteasomal antigen in human spermatozoa was studied by means of immunolabeling with the MPC21 monoclonal antibody and secondary gold labeled antibody with 1.4 nm gold particles in combination with silver enhancement reaction using pre-embedding technique. The labeling was found in the acrosomal and postacrosomal regions, in the connecting-piece (neck) and, in some cases, in the middle-piece and also in the residual bodies. There was no significant reaction in condensed chromatin. In some abnormal forms of spermatozoa, in which the chromatin was not well condensed, the labeling in nuclei was present. The nuclear vacuoles with looser chromatin were usually strongly labeled. The nuclei of cells representing different stages of spermatogenesis, that were present in semen samples, were also labeled.     

Localization of alpha 1-microglobulin (HC protein) in normal human tissues: an immunohistochemical study using monoclonal antibodies

Summary Alpha-1-microglobulin is a low molecular weight (approximately 30 000 d) glycoprotein present in biological fluids. It is heterogeneous in charge. A monoclonal antibody was used to investigate the tissue distribution of the protein in normal human tissues and cell lines by indirect immunofluorescence and immunoperoxidase techniques. The protein was demonstrable in cells of the monocyte-macrophage lineage, in thymus and T cell dependent areas of spleen, lymph node and tonsils. It was detected in several lymphoid or nonlymphoid cell lines but not in peripheral blood lymphocytes. The microglobulin was also detectable in the cytoplasm of hepatocytes. Finally, it was observed in glandular secretions (sudoral glands and mucosal glands of the digestive tract) where it may be associated with IgA. Possible explanations for the highly divergent results previously reported with polyclonal antisera to ₁ microglobulin are discussed.     

Localization of a brain sulfotransferase, SULT4A1, in the human and rat brain: an immunohistochemical study.

Cytosolic sulfotransferases are believed to play a role in the neuromodulation of certain neurotransmitters and drugs. To date, four cytosolic sulfotransferases have been shown to be expressed in human brain. Recently, a novel human brain sulfotransferase has been identified and characterized, although its role and localization in the brain are unknown. Here we present the first immunohistochemical (IHC) localization of SULT4A1 in human brain using an affinity-purified polyclonal antibody raised against recombinant human SULT4A1. These results are supported and supplemented by the IHC localization of SULT4A1 in rat brain. In both human and rat brains, strong reactivity was found in several brain regions, including cerebral cortex, cerebellum, pituitary, and brainstem. Specific signal was entirely absent on sections for which preimmune serum from the corresponding animal, processed in the same way as the postimmune serum, was used in the primary screen. The findings from this study may assist in determining the physiological role of this SULT isoform.     

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.     

Melanocyte subpopulation turnover during the human hair cycle: an immunohistochemical study

The human hair cycle is characterized by successive phases of growth and involution that imply tissue regression and regeneration. As a consequence, the hair melanin unit has to be renewed in a cyclic manner. Actually, the behavior of human hair follicle melanocytes throughout the hair cycle has been poorly studied. Thus, the origin of melanocytes present in the bulb after human hair regeneration is still not clarified, and neither are the events that control the melanin biosynthesis activity in the human hair bulb. In this study, we showed at the cellular level that in human pigmented hair follicles, the expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) was detectable during the anagen phases III/IV through VI, only in those melanocytes which were located in the bulb. During the catagen phase, the two evaluated melanogenic enzymes were detectable no more, although melanocytes were still present in the preceding bulbar area. The epithelial column of catagen follicles and the capsule of telogen follicles also contained inactive melanocytes as evidenced by pMel-17 labeling. At the induction of a new anagen hair follicle, some melanocytes were committed to cell division, but only when located in the nascent bulb close to the dermal papilla. Our results emphasize the close relationship between melanogenesis and the hair cycle and suggest that in humans, melanogenesis is restricted to anagen hair follicles not because of the regulation of tyrosinase activity, but because of melanogenic enzyme expression, e.g., tyrosinase and TRP-1. Furthermore, the fact that in the newly developing anagen hair follicles, cell-division commitment and tyrosinase and TRP-1 expression were observed in melanocytes only when located in the nascent bulb suggests a highly regio-specific melanocyte stimulation in early the anagen phase.     

Localization of proinsulin and insulin in human insulinoma: preliminary immunohistochemical results

We have carried out an immunohistochemical investigation of 15 human insulinomas applying monoclonal antibodies specifically recognizing proinsulin and insulin. Our results demonstrate that the epitopes unique to proinsulin and insulin can be detected with the respective monoclonal antibodies using the protein A-gold technique after routine formaldehyde fixation and paraffin embedding of the tissues. The immunostaining pattern for proinsulin and insulin in the insulinomas was different from the observed in B cells of pancreatic islets present in the adjacent normal pancreas. Furthermore, the pattern of immunostaining was found to vary from tumor to tumor. These findings strongly suggest the possibility of a disturbed proinsulin to insulin conversion in human insulinomas.     

Age-related decrease in rod bipolar cell density of the human retina: an immunohistochemical study

During normal ageing, the rods (and other neurones) undergo a significant decrease in density in the human retina from the fourth decade of life onward. Since the rods synapse with the rod bipolar cells in the outer plexiform layer, a decline in rod density (mainly due to death) may ultimately cause an associated decline of the neurones which, like the rod bipolar cells, are connected to them. The rod bipolar cells are selectively stained with antibodies to protein kinase C-α. This study examined if rod bipolar cell density changes with ageing of the retina, utilizing donor human eyes (age: 6–91 years). The retinas were fixed and their temporal parts from the macula to the mid-periphery sectioned and processed for protein kinase C-α immunohistochemistry. The density of the immunopositive rod bipolar cells was estimated in the mid-peripheral retina (eccentricity: 3–5 mm) along the horizontal temporal axis. The results show that while there is little change in the density of the rod bipolar cells from 6 to 35 years (2.2%), the decline during the period from 35 to 62 years is about 21% and between seventh and tenth decades, it is approximately 27%.     

Development of the basal lamina in xenografted human carcinomas: an ultrastructural and immunohistochemical study

Summary The development of the basal lamina (BL), the key structure of the basement membrane (BM), was investigated in three xenografted human carcinomas of the sigmoid colon (CA 1), the lung (L 261), and the hypopharynx (H-Stg 1) following heterotransplantation to athymic mice. The study involved the use of electron microscopy and indirect immunofluorescence techniques employing highly specific antibodies against the intrinsic BL components, heparan sulfate proteoglycan, laminin and type-IV collagen. Following transplantation, the extracellular matrix material of the transplanted tumors decomposed and was phagocytozed by invading macrophages within 1 to 2 days. During this stage, no specific binding of the applied antibodies to BL components could be detected within the xenografts. Following the ingrowth of host-derived connective tissue between days 2 to 6, small fluorescence-positive granules appeared within the cytoplasm and around those tumor cells that were located close to the invaded strands of connective tissue. Ultrastructurally, typical secretory granules were detectable in the cytoplasm of many xenografted carcinoma cells. Thereafter, a tannic acid-positive, patchy material appeared in the extracellular space of CA 1 and L 261 and aggregated to form small fragments of a discontinuous BL. In the H-Stg 1 xenografts, this material assembled to form continuous mono-, bi- and multilayered structures. Large amounts of excess BL material remained accumulated in the L 261 and H-Stg 1 xenografts until the end of the observation period (day 24). These findings reveal that discontinuities of the BL occur independent of the active invasion processes of tumor cells, since xenografted human carcinomas neither grow invasively nor metastasize in nude mice. Moreover, they confirm that these discontinuities are not caused by a quantitatively insufficient production of BL material, but rather arise from qualitative imbalances of the composition of the synthesized BL material.     

Protein phosphatase 2Calpha expression in normal human tissues: an immunohistochemical study

The recent development of fluorescent proteins has rapidly and radically altered the way cell biology is performed by allowing simple, non-invasive imaging of cellular processes in real time. The special properties of the nervous system, such as synaptic morphology, axonal/dendritic maturation, and neuronal migration are especially amenable to investigation using fluorescent proteins. This review focuses on the various genetic and viral vectors used to express fluorescent proteins in vivo and in slice culture, and the strengths and limitations associated with them.     

Crystallographic and kinetic evidence of allostery in a trypsin-like protease

Protein allostery is based on the existence of multiple conformations in equilibrium linked to distinct functional properties. Although evidence of allosteric transitions is relatively easy to identify by functional studies, structural detection of a pre-existing equilibrium between alternative conformations remains challenging even for textbook examples of allosteric proteins. Kinetic studies show that the trypsin-like protease thrombin exists in equilibrium between two conformations where the active site is either collapsed (E*) or accessible to substrate (E). However, structural demonstration that the two conformations exist in the same enzyme construct free of ligands has remained elusive. Here we report the crystal structure of the thrombin mutant N143P in the E form, which complements the recently reported structure in the E* form, and both the E and E* forms of the thrombin mutant Y225P. The side chain of W215 moves 10.9 ? between the two forms, causing a displacement of 6.6 ? of the entire 215-217 segment into the active site that in turn opens or closes access to the primary specificity pocket. Rapid kinetic measurements of p-aminobenzamidine binding to the active site confirm the existence of the E*-E equilibrium in solution for wild-type and the mutants N143P and Y225P. These findings provide unequivocal proof of the allosteric nature of thrombin and lend strong support to the recent proposal that the E*-E equilibrium is a key property of the trypsin fold.     

Degradation of DNA topoisomerase I by a novel trypsin-like serine protease in proliferating human T lymphocytes

DNA topoisomerase I (Topo I) contributes to various important biological functions, and its activity is therefore likely regulated in response to different physiological conditions. Increases in both the synthesis and degradation of Topo I were previously shown to accompany phytohemagglutinin stimulation of proliferation in human peripheral T lymphocytes. The mechanism of this degradation of Topo I has now been investigated with both in vivo and in vitro assays. The activity of a nuclear protease that specifically degrades Topo I was induced in proliferating T lymphocytes. The full-length Topo I protein (100 kDa) was sequentially degraded to 97- and 82-kDa fragments both in vivo and in vitro. The initial site of proteolytic cleavage was mapped to the NH(2)-terminal region of the enzyme. The degradation of Topo I in vitro was inhibited by aprotinin or soybean trypsin inhibitor, suggesting that the enzyme responsible is a trypsin-like serine protease. Furthermore, Topo I degradation by this protease was Mg(2+)-dependent. The Topo I-specific protease activity induced during T lymphocytes proliferation was not detected in Jurkat (human T cell leukemia) cells and various other tested human cancer cell lines, possibly explaining why the abundance of Topo I is increased in tumor cells.