| 1. |
- Fu, Zhirong, et al.
(författare)
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Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity
- 2021
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 16:5
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Tidskriftsartikel (refereegranskat)abstract
- Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.
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| 2. |
- Fu, Zhirong, et al.
(författare)
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Extended cleavage specificity of sheep mast cell protease-2 : A classical chymase with preference to aromatic P1 substrate residues
- 2019
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Ingår i: Developmental and Comparative Immunology. - : ELSEVIER SCI LTD. - 0145-305X .- 1879-0089. ; 92, s. 160-169
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Tidskriftsartikel (refereegranskat)abstract
- Serine proteases constitute the major protein content of mammalian mast cell granules and the selectivity for substrates by these proteases is of major importance for the role of mast cells in immunity. In order to address this subject, we present here the extended cleavage specificity of sheep mast cell protease-2 (MCP2), a chymotrypsin-type serine protease. Comparison of the extended specificity results to a panel of mammalian mast cell chymases show, in almost all aspects, the same cleavage characteristics. This includes preference for aromatic residues (Phe, Tyr, Trp) in the P1 position of substrates and a preference for aliphatic residues in most other substrate positions around the cleavage site. MCP2 also cleaved, albeit relatively low efficiency, after Leu in the P1 position. In contrast to the human, mouse, hamster and opossum chymases that show a relatively strong preference for negatively charged amino acids in the P2'position, the sheep MCP2, however, lacked that preference. Therefore, together with the rat chymase (rMCP1), sheep MCP2 can be grouped to a small subfamily of mammalian chymases that show fairly unspecific preference in the P2'position. In summary, the results here support the view of a strong evolutionary conservation of a potent chymotrypsin-type protease as a key feature of mammalian mast cells.
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| 3. |
- Fu, Zhirong, et al.
(författare)
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Highly Selective Cleavage of Cytokines and Chemokines by the Human Mast Cell Chymase and Neutrophil Cathepsin G
- 2017
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Ingår i: Journal of Immunology. - : AMER ASSOC IMMUNOLOGISTS. - 0022-1767 .- 1550-6606. ; 198:4, s. 1474-1483
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Tidskriftsartikel (refereegranskat)abstract
- Human mast cell chymase (HC) and human neutrophil cathepsin G (hCG) show relatively similar cleavage specificities: they both have chymotryptic activity but can also cleave efficiently after leucine. Their relatively broad specificity suggests that they may cleave almost any substrate if present in high enough concentrations or for a sufficiently long time. A number of potential substrates have been identified for these enzymes and, recently, these enzymes have also been implicated in regulating cytokine activity by cleaving numerous cytokines and chemokines. To obtain a better understanding of their selectivity for various potential in vivo substrates, we analyzed the cleavage of a panel of 51 active recombinant cytokines and chemokines. Surprisingly, our results showed a high selectivity of HC; only 4 of 51 of these proteins were substantially cleaved. hCG cleaved a few additional proteins, although this occurred after adding almost equimolar amounts of enzyme to target. The explanation for this wide difference in activity against peptides or other linear substrates compared with native proteins is most likely related to the reduced accessibility of the enzymes to potential cleavage sites in folded proteins. In this article, we present evidence that sites not exposed on the surface of the protein are not cleaved by the enzyme. Interestingly, both enzymes readily cleaved IL-18 and IL-33, two IL-1-related alarmins, as well as the cytokine IL-15, which is important for T cell and NK cell homeostasis. Cleavage of the alarmins by HC and hCG suggests a function in regulating excessive inflammation.
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| 4. |
- Fu, Zhirong, et al.
(författare)
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Mast Cells and Basophils in the Defense against Ectoparasites : Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:23
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Tidskriftsartikel (refereegranskat)abstract
- Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.
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| 5. |
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| 6. |
- Iwamura, Masatsugu, et al.
(författare)
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Alteration of the hormonal bioactivity of parathyroid hormone-related protein (PTHrP) as a result of limited proteolysis by prostate-specific antigen
- 1996
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Ingår i: Urology. - 0090-4295. ; 48:2, s. 317-325
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Tidskriftsartikel (refereegranskat)abstract
- Objectives. To discover whether the proteolytic activity of prostate- specific antigen (PSA) affects the structure and function of parathyroid hormone-related protein (PTHrP), as both are abundant components of human seminal plasma. Methods. The ability of PTHrP to act as a substrate was studied by incubating a synthetic polypeptide, consisting of 34 amino acid residues of the amino-terminal domain of PTHrP, with purified PSA. The incubate was then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, high-pressure liquid chromatography separation, amino- terminal peptide sequencing, and mass spectrometry. The physiologic effect of the proteolytic activity of PSA on PTHrP was studied by measuring any alteration in PTHrP (1-34)-induced elevation of cyclic adenosine monophosphate (cAMP) production by UMR-106 rat osteosarcoma cells in culture. All cell culture experiments were performed with PSA and PTHrP (1-34) at physiologic concentrations. Results. Our data show that PSA proteolytically cleaves PTHrP (1-34) after either residue 22 or 23, generating three peptide fragments. Both cleavages occur carboxy terminally of a phenylalanine residue. The cAMP production in rat osteosarcoma cells, induced by the amino- terminal portion of PTHrP (1-34), as a result of its structural similarity with parathyroid hormone (PTH), was abated by PSA in a dose- and time- dependent fashion. In contrast, heat-inactivated PSA had no effect on cAMP production. Conclusions. Our study demonstrates that PTHrP is a substrate for PSA. The cleavage of the amino-terminal portion of PTHrP completely disrupts its ability to interact with the PTH/PTHrP receptor and thus inhibits its PTH-like activity. The proteolytic processing of PTHrP by PSA may play an important role in the post-translational/post-secretional regulation of prostatic PTHrP activities, which are believed to include regulation of prostate growth and differentiation.
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| 7. |
- Malm, Johan, et al.
(författare)
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Enzymatic action of prostate-specific antigen (PSA or hK3) : Substrate specificity and regulation by Zn2+, a tight-binding inhibitor
- 2000
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Ingår i: Prostate. - 0270-4137. ; 45:2, s. 132-139
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND. In semen, prostate-specific antigen (PSA or hK3) digests the gel proteins semenogelin I and II, resulting in liquefaction and the release of motile spermatozoa. We characterized the substrate specificity and zinc-mediated inhibition of PSA. METHODS. The proteolysis of human semenogelin I (SgI) and II (SgII) by PSA was characterized by purification of generated SgI and SgII fragments, N-terminal sequencing, and mass spectrometry. Zn2+-inhibition of PSA was studied using a chromogenic substrate. RESULTS. Eighteen cleavage sites in SgI and 16 in SgII were identified. Cleavages were identified mainly as the C-terminal of certain tyrosine and glutamine residues, but also the C-terminal of histidine, aspartic acid, leucine, serine, and asparagine residues. No cleavages were identified at any arginine, lysine, phenylalanine, tryptophan, or methionine residues, indicating that the substrate specificity of PSA is distinct from that of trypsin, chymotrypsin, tissue kallkrein (hK1), and kallikrein 2 (hK2). Zn2+ ions have a dramatic effect on PSA activity; the data indicate that Zn2+ is a tight-binding inhibitor of PSA activity. CONCLUSIONS. The data will enable the optimized design of PSA activity assays, which may prove instrumental to uncovering the role of PSA in cancer and reproduction. The inhibition data indicate that Zn2+ could regulate PSA activity, which may prove important in the development of efficient inhibitors of PSA activity. (C) 2000 Wiley-Liss, Inc.
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| 8. |
- Malm, Johan, et al.
(författare)
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Isolation and characterization of the major gel proteins in human semen, semenogelin I and semenogelin II
- 1996
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 238:1, s. 48-53
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Tidskriftsartikel (refereegranskat)abstract
- Semenogelin I and semenogelin II constitute the major gel-forming proteins in human semen. The gel proteins were rapidly solubilized and separated from spermatozoa in ejaculates collected at pH 9.7 in buffer containing 4 mol/l urea and dithiothreitol. This protected the semenogelins from proteolytic degradation by prostate-specific antigen, and allowed their isolation by affinity chromatograph:g on heparin-sepharose. Semenogelins I and II were almost selectively retained and eluted partially separated in 0.25 mol/l NaCl. Further purification was achieved by chromatography on Superose. Approximately 10-20 mg semenogelin I and 2-5 mg semenogelin II were recovered from each sample with a purity exceeding 95% as judged by SDS/PAGE. The molecular mass of semenogelin I (49,958 Da) and the major form of semenogelin II (63,539 Da) measured by mass spectrometry was consistent with the reported cDNA data. The occurrence of a second, larger form of semenogelin II was due to asparagine-linked glycosylation. The amino-termini of the purified proteins were blocked, but digestion with pyroglutamate aminopeptidase enabled the identification of amino-terminal sequences consistent with the reported cDNA data. The amino acid compositions of the purified proteins were also consistent with those derived from cDNA data. The absorption coefficients (280 nm, 1%, 1 cm) for semenoaelins I and II. were 5.5 and 5.4, respectively, and the isoelectric point was above pH 9.5 for both proteins.
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| 9. |
- Malm, Johan, et al.
(författare)
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The pentraxin serum amyloid P component is found in the male genital tract and attached to spermatozoa
- 2008
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Ingår i: International Journal of Andrology. - : Wiley. - 0105-6263 .- 1365-2605. ; 31, s. 508-517
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Tidskriftsartikel (refereegranskat)abstract
- Serum amyloid P component (SAP) belongs to the pentraxin family of proteins, members of which are characterized by radial pentameric structure and calcium-dependent ligand binding. SAP is present in all types of amyloidosis and has been shown to bind to several ligands, but the physiological function of this protein has not been fully elucidated. The present study identified and characterized SAP in human semen and immunolocalized it to the male reproductive tract. SAP was also detected in seminal plasma by immunoblotting and purification by affinity chromatography followed by mass spectrometry. According to electroimmunoassay, the concentration of SAP in semen is approximately 2 mg/L, and flow cytometry revealed SAP attached to the surface of spermatozoa. Moreover, immunohistochemistry showed positive staining of spermatozoa, subsets of epithelial cells, and the stroma of accessory male genital glands and testis. Presence of mRNA supports local production of SAP, as shown with reverse transcription polymerase chain reaction. We identified SAP in a new setting - the human male reproductive system. SAP was detected on ejaculated spermatozoa, in seminal plasma and in tissue sections from the male reproductive tract. Further functional studies are needed to explain the role of SAP in human reproduction.
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| 10. |
- Ramos-Ramirez, Patricia, et al.
(författare)
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House Dust Mite and Cat Dander Extract Induce Asthma-Like Histopathology with an Increase of Mucosal Mast Cells in a Guinea Pig Model
- 2023
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Ingår i: Journal of Immunology Research. - : Hindawi Publishing Corporation. - 2314-8861 .- 2314-7156. ; 2023
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Tidskriftsartikel (refereegranskat)abstract
- Background. Asthma is a chronic inflammatory disease with structural changes in the lungs defined as airway remodelling. Mast cell responses are important in asthma as they, upon activation, release mediators inducing bronchoconstriction, inflammatory cell recruitment, and often remodelling of the airways. As guinea pigs exhibit anatomical, physiological, and pharmacological features resembling human airways, including mast cell distribution and mediator release, we evaluated the effect of extracts from two common allergens, house dust mite (HDM) and cat dander (CDE), on histopathological changes and the composition of tryptase- and chymase-positive mast cells in the guinea pig lungs. Methods. Guinea pigs were exposed intranasally to HDM or CDE for 4, 8, and 12 weeks, and airway histology was examined at each time point. Hematoxylin and eosin, Picro-Sirius Red, and Periodic Acid-Schiff staining were performed to evaluate airway inflammation, collagen deposition, and mucus-producing cells. In addition, Astra blue and immunostaining against tryptase and chymase were used to visualize mast cells. Results. Repetitive administration of HDM or CDE led to the accumulation of inflammatory cells into the proximal and distal airways as well as increased airway smooth muscle mass. HDM exposure caused subepithelial collagen deposition and mucus cell hyperplasia at all three time points, whereas CDE exposure only caused these effects at 8 and 12 weeks. Both HDM and CDE induced a substantial increase in mast cells after 8 and 12 weeks of challenges. This increase was primarily due to mast cells expressing tryptase, but not chymase, thus indicating mucosal mast cells. Conclusions. We here show that exposure to HDM and CDE elicits asthma-like histopathology in guinea pigs with infiltration of inflammatory cells, airway remodelling, and accumulation of primarily mucosal mast cells. The results together encourage the use of HDM and CDE allergens for the stimulation of a clinically relevant asthma model in guinea pigs.
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