Was the Serine Protease Cathepsin G Discovered by S. G. Hedin in 1903
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Vol. 58, No 1/2011 39–44 on-line at: www.actabp.pl Regular paper Was the serine protease cathepsin G discovered by S. G. Hedin in 1903 in bovine spleen? David Palesch1, Marcin Sieńczyk2, Jozef Oleksyszyn2, Michael Reich1, Ewa Wieczerzak3, Bernhard O. Boehm1 and Timo Burster1* 1Division of Endocrinology and Diabetes, Department of Internal Medicine I, University Medical Center Ulm, Ulm, Germany; 2Wrocław University of Technology, Wrocław, Poland; 3Faculty of Chemistry, University of Gdansk, Gdańsk, Poland In the beginning of the 20th century, enzymes with pro- tive under acidic conditions, which he named b-protease teolytic activity were classified as peptidases, Erepsin, (Hedin, 1903a). and proteases. Among these, pepsin, trypsin, and auto- lytic enzymes were of the protease class. Spleen-derived After discovering these two proteases in bovine proteases were poorly characterized until Sven Gustaf spleen, Hedin performed further experiments with bo- Hedin performed several digestion experiments with vine serum and concluded that it contained a weak pro- bovine spleen. He incubated minced bovine spleen un- teolytic enzyme active in neutral medium, the activity of der acidic or neutral conditions and characterized two which could be blocked by heating at 55 °C or neutral- active proteases; the results were published in 1903. ized by antibodies (also named anti-enzymes at the time) The first protease was named α-protease and was ac- present in the serum. Hedin assumed that the α-protease tive under neutral conditions. The second was named in bovine spleen-derived leukocytes was similar to the β-protease and was active under acidic conditions. We protease found in serum. He further speculated that the replicated Hedin’s experiments according to his methods α-protease may derive from lysed leukocytes or leuko- and found, by using activity-based probes to visualize cytes that secrete proteases (Hedin, 1903b). proteases, that the historical α-protease is the present- In another experiment, Hedin mixed bovine pancreas- day serine protease cathepsin G (CatG), which is known derived trypsin with normal serum and added casein as to be important in several immune processes, including a substrate. The serum had a neutralizing effect. There- antigen processing, chemotaxis, and activation of sur- fore, the data obtained were called Reihenfolgephänomen face receptors. The β-protease, however, comprised dif- (i.e., order phenomenon), and the optimum temperature ferent proteases including CatX, B, S, and D. We suggest of neutralization was determined to be 37 °C (Hedin, that Hedin described CatG activity in bovine spleen over 1905). Hedin concluded that antibodies were necessary 100 years ago. for the anti-tryptic activity because previous antibody publications had reported anti-tryptic effects (Landstein- Keywords: proteases, cathepsin, spleen cells, Hedin er, 1900; Cathcart, 1904). Hedin defined a general prin- Received: 02 July, 2010; revised: 10 November, 2010; accepted: ciple by preincubating trypsin with serum at 37 °C before 13 December, 2010; available on-line: 07 March, 2011 adding the substrate. This successive procedure, which he established 100 years ago, is the standard protocol for biochemical inhibition of proteases. InTRoDuCTIon Moreover, Hedin stated that inside leukocytes, pro- teases were involved in “digesting processes” (Hedin, 1903a). Phagocytosis, with credit given to Metchnikoff, Proteolytic enzymes are generally broadly classified as appears intracellularly in so-called “digesting vacuoles” proteases, but in 1915, the classification of such enzymes with acidic pH (Metchnikoff, 1884; 1901). The conclu- was based on more specific substrates and included pep- sions they proposed were precise and prophetic. Antigen tidases (digest polypeptides), Erepsin (digest peptones), processing occurs inside cells and is executed by pro- and proteases (degrade proteins). Pepsin, trypsin, and teases. A gastric mucosa protease was named cathepsin autolytic enzymes were classified as proteases (Hedin, (gr. καθεφειν to digest) and was proposed to have its 1915). Towards the end of the 19th century, various sci- origin in leukocytes in the late 1920s (Willstätter, 1929). entists concentrated their research on autolytic enzymes. Lysosomes were not discovered until 1955 (De Duve et For example, in 1890, Salkowski found increased nitrog- al., 1955; Novikoff et al., 1956), and a 28 kDa protease, enous substances during long-term incubation of liver, active in alkaline pH, named cathepsin G (CatG), was muscles, and suprarenal glands at body temperature and isolated from human spleen in 1976 (Starkey & Barrett, interpreted his findings as the action of proteolytic en- 1976). zymes in these tissues (Salkowski, 1890). Thirteen years later, the Swedish scientist Sven Gustaf Hedin extended *e-mail: [email protected] Salkowski’s results by treating minced bovine spleen with Abbreviations: APC, antigen-presenting cells; Cat, cathepsin; DAP, either neutral or acidic medium and analyzing the prod- a-aminoalkylphosphonate diphenyl ester; DDT, dithiothreitol; LHVS, ucts of autodigestion. Hedin published his data in 1903, morpholinurea-leucine-homophenylalanine-vinyl phenyl sulfone; PBMC, peripheral blood mononuclear cells; PMSF, phenylmethyl- showing that bovine spleen cells harbor at least two dif- P sulfonyl fluoride; Suc-VPF, Suc-Val-Pro-Phe (OPh)2; ZRLR, Z-Arg-Leu- ferent proteolytic enzymes, one active under neutral con- Arg-α-aza-glycyl-Ile-Val-OMe ditions, which he named α-protease, and the other ac- 40 D. Palesch and others 2011 The function of cathepsins in the endocytic com- Active site labeling and Western blotting. PBMC partment (endosome and lysosome) within antigen-pre- and spleen cells were lysed (10 mM Tris, pH 7.5, 150 senting cells (APC) is to digest antigens into antigenic mM NaCl, and 0.5% NP-40) and adjusted to equal to- peptides capable of binding to major histocompatibility tal protein concentration (quantified by Bradford assay), complex (MHC) class II molecules. These MHC II anti- and 20 µg of the cell lysate or filtrates were incubated genic peptide complexes migrate to the cell surface, ac- with PBS (pH 7.4) in the presence of DAP (DAP22c, tivate respective T cells, and cause an immune response. 2 µM) (Oleksyszyn & Powers, 1991; Reich et al., 2009a). Cysteine (CatB, C, F, H, S, X, V, L) and asparagine en- Alternatively, 5 µg of cell lysate or filtrates were incubat- doprotease (Watts et al., 2005; Turk & Turk, 2009), serine ed with reaction buffer (1 mM EDTA, 50 mM citrate, (CatA (Reich et al., 2010) and CatG (Burster et al., 2010)), pH 5.0, and 50 mM DTT) in the presence of DCG- and apartyl (CatD and CatE) proteases are involved in 04 (10 µM; probe kindly donated by M. Bogyo, Stan- antigen processing of different APC (for review, see ford University, Palo Alto, CA, USA) (Greenbaum et al., Colbert et al., 2009). The serine proteases CatG, neu- 2000) for 1 h at room temp. Some of the samples were trophil elastase, and proteinase 3 are highly expressed in treated with CatG inhibitor I (CatG inhibitor, 10 µM, granulocytes and can be secreted into the blood by these Calbiochem, Merck, Darmstadt, Germany), Suc-Val-Pro- P cells under inflammatory conditions (Meier et al., 1985). Phe (OPh)2 (Suc-VPF, 10 µM, (Oleksyszyn and Powers, CatG is important in the processing of antigens in pri- 1991)), Z-Arg-Leu-Arg-α-aza-glycyl-Ile-Val-OMe (10 µM, mary APC (Reich et al., 2009a), has anti-bacterial capacity ZRLR, (Wieczerzak et al., 2007)), phenylmethylsulfonyl (Ohlsson et al., 1977), regulates chemotaxis (Nufer et al., fluoride (PMSF, 2 mM, Sigma-Aldrich, Steinheim, Ger- 1999), activates the protease-activated receptor 4 (Sam- many), or morpholinurea-leucine-homophenylalanine- brano et al., 2000), and is inhibited by thrombospondin 1 vinyl phenyl sulfone (LHVS, 20 nM, H. Kalbacher, Uni- present in the serum or by serpins found in cells (Born- versity of Tübingen, Germany) and pre-incubated for stein et al., 1991; Heutinck et al., 2010). 15 min at 37 °C. Purified CatG from human sputum The spleen is the body’s largest filter of blood and was purchased from Sigma-Aldrich and 50 ng was used functions to promote the innate and adaptive immune in all experiments. Samples were resolved by 12 % so- response. The red pulp of the spleen is responsible for dium dodecyl sulfate polyacrylamide gel electrophoresis clean-up of older erythrocytes and effective iron recy- (SDS/PAGE), blotted, and visualized using streptavidin- cling. The white pulp is formed by sheathed lymphoid horseradish peroxidase (HRP, Vectastain, Burlingame, tissue composed of T and B cell compartments that fa- CA, USA). For Western blotting, 20 µg of protein from cilitate T cell interactions with dendritic cells (DC) and crude cell extracts was loaded on SDS/PAGE, and the mediate B cell expansion, respectively. APC from the immune blot was performed using anti-CatD antibody bloodstream enter the white pulp through the marginal (Calbiochem, Schwalbach, Germany) or anti-CatX (R&D zone and initiate a potent adaptive immune response. Systems, Wiesbaden, Germany). Anti-β-actin antibody We replicated Hedin’s 1903 experiment by analyzing and secondary HRP-conjugated antibodies were obtained protease activity after treatment of bovine spleen with from Sigma-Aldrich (Taufkirchen, Germany). neutral or acidic medium. Activity-based probes and Determination of CatG activity. Kinetic measure- Western blotting were used to determine the presence of ment of CatG activity was accomplished by adding 15 cysteine, aspartyl, or serine proteases in the collected fil- mg of the indicated samples to the colorimetric sub- trates. We found that CatG was abundant in the fraction strate Suc-Val-Pro-Phe-pNA (200 mM) in buffer (0.5 M which Hedin described as filtrate B (FB; Fig. 1). Thus, MgCl2 in PBS, pH 7.4). The colorimetric substrate was we provide evidence, using several biochemical meth- prepared as followed: Boc-Phe-OH (Iris-biotech, Mark- ods, that Hedin’s α-protease, first described in 1903, was tredwitz, Germany) was reacted with p-nitro aniline to most likely CatG.