1073 (1991) 155=160 ~, 1991 ElsevierScience PublishersB.V :.31omedical Division)0304-4165/ql/SO350 030441659100083V

Bioc'himwa ct Bioph~sWu Acta.

155

ADONIS

Species-specific distribution of catheps~n E in mammalian blood ceils Satoshi ~'onezawa and Katsuhiro Nakamura Dt'partme~! of ZooloK~., ,t~,ult~. ,,f S, len, t,. /Iokkald, U~ltr'cr~:(~'. ,~;appot,, IJapan)

(R~elved 30 January 19'~0)

Key words" Cathep~,in E: Lcukt~:yt¢: Erythrt~:yt¢

The distribution of cathepsins D and E in leukocytes and erythrocyte ghosts of ~veral mammalian species, and in HL-60 and K-562 cells was examined by means of a combined application ol" electrnphoretic and immunochemical methods. Catbepsin D was found in leukocytes of all species examined, but the distribution of catbepsin E was found to be species-specific: pigs, cows and goats had no cathepsin E activity, in leukocytes or erythrncytes at all. In humans, catbepsin E occurred in erythrocytes but not in leukocytes, which contrasted with the guinea pig pattern of its presence in leukocytes and its absence in erythrocytes. No catbelx~in E-related enzymes were found in HL-60 or K-562 cells, but these human leukemic cells contained cathepsir:. D-related enzyme forms that are electrophoreticaily distinct from normal leukocyte cathepsln D. The present results are inconsistent with the view that catbepsin E may be involved as an essential factor in the biological functions o | leukocytes or erythrocytes.

Introduction Two intracellular aspartic proteinases are known to exist in mammalian blood cells. Cathepsin D, an extensively studied endopeptidase, is located in the lysosomes and is considered to function mainly in the degradation of endogenous and endocytosed proteins [1]. Cathepsin E, another aspartic proteinase, was first discovered in rabbit neutrophils, bone marrow and spleen [2]. Recent studies have demonstrated that the enzyme is distributed in a limited number of mammalian tissues and organs, including blood cells [3-8], and is localized extralysosomally as observed in rat neutrophils [8]. While the function of cathepsin E has not yet been established, these findings may be taken as the indication that not only the functions of the two cathepsins are different, but also that cathepsin E plays a part in some immune system [5-7,9]. Another possible functional aspect is that the enzyme may be involved in the self-destruction mechanism of the cell, as suggested from its occurrence in erythrocytes [7,10-12] and surface

Abbreviations: MNL, mononuclear leukocytes: PMN, polymorphonuclear leukocytes; TCA. trichloroacetic acid; DMSO, dimethyl sulfoxide:) PAGE. polyac~lamid¢ gel elcctrophorcsis. Correspondence: S. Yonezawa. Department of Zoology, Faculty of Science. Hokkaido University. S,~pporo060, Japan.

epithelial cells of the stomach [6,7]. However, since the data on cathepsin E obtained so far deal only with a few mammalian species, information on the distribution and properties of the enzyme in other species would be necessary for a better understanding of its biological function. We demonstrate here that the distribution of cathepsin E is species-specific in mammalian blood cells, while cathepsin D is distributed ubiquitously in leukocytes. Materials and Methods

A s s a y s . The activity of acid proteinases was measured by using [14C]verdohemoglobin as substrate. Verdohemoglobin. which is soluble at acidic to alkaline pHs. was prepared as described [13], and radiolabeled with [14C]KCNO (Amersham) [14], The reaction mixture consisted of 200 pl of 0.25M formate buffer (pH 3.2), 200 pl of 0.5'$ verdohemoglobin (20000 cpm) and 20 pl of enzyme solution. The reaction was allowed to proceed at 37°C for 10 rain and was stopped by adding i ml of 5(~ trichloroacetic acid (TCA), The mixture was centrifuged at 2500 rpm for 5 min and the radioactivity in the supernatant was measured. One unit of activity was defined as the amount of enzyme that produced 1 mg of TCA-soluble equivalent per rain. Alkaline phosphatase [15], ,O-glucuronidase [15] and myeloperoxidase

156 [161 were assayed by slight modifications of the respective methods. Protein was determined by the method of Lowry et al. [17 I. Isolation of human leukocytes and segregation of subcellular granules. Human blood samples were freshly collected, anticoagulated with heparin and mixed with 0.23 vol. of 3% polyvinylpyrrolidone in 0.9% NaCI. After 45 rain at room lemperature, leukocyte-rich supernatants were siphoned off, layered on Ficoll-Paque (Pharmacia) and centrifuged at 450 x g for 45 min according to B~Syum [181. The cells at the interface were collected, washed and used as a fraction of mononuclear leukocytes (MNL). The cells precipitated through Ficoll-Paque were treated twice with hypotonic saline to lyse residual erythrocytes, and used as a fraction of polymorphonuclear leukocytes (neutrophils; PMN). The purity of both fractions was more than 90% as examined under a light microscope. The isolation of subcellular granules of human neutrophils was carried out essentially as described previously 181. Briefly, cells were disrupted by the nitrogen cavitation method [15] and the cytoplasmic granules and plasma membrane fragments were segregated by density centrifugation on discontinuous Percoll gradients (65 and 30%). After centrifugation at 48000 × g for 15 min, fractions of 23 drops were collected, treated with Triton X-100, frozen-thawed three times and recentrifuged at 144000 x g for 90 min to remove Percoll. Isolation of PMN, M N L and erythrocyte ghosts from other mammals. Blood samples from rats, guinea pigs and pigs were freshly collected, anticoagulated, and treated in the same way as for the preparation of h u m a n MNL and PMN. Each cell fraction was washed in Dulbecco's PBS and frozen at - 7 0 " C until use. Bovine and goat leukocytes were prepared according to Doussiere and Vignais [191, followed by purification of M N L and PMN with FicolI-Paque as above. The purities of the fractions obtained from different animals were lower (70-80%) than those of the human fractions. In some experiments, treatment of leukocytes with FicolI-Paque was omitted, because no difference was found in the distribution pattern of acid proteinases between PMN and MNL fractions of each animal (see Results). Erythrocyte ghosts were prepared by the method of Dodge et al. [20]. Preparation of crude extracts. Each of the isolated ce u fractions was sonicated in 20 mM Tris/1 m M EDTA/0.1% Triton X-100 (pH 7.5) (5.107 cells/ml of buffer) and centrifuged at 15000 × g for 20 rain. The resulting supernatant was used for quantitative and electrophoretic analyses. Erythrocyte ghosts were suspended in 5 mM Tris/1 mM EDTA/0.1% Triton X-100 (pH 7.5) and frozen-thawed three times. Cell lines. Cells from human HL-60 and K-562 lines were separately cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (Whittaker, MA) in a

humidified atmosphere with 5% CO 2. K-562 cells and HL-60 cells were induced to differentiate along the erythroid pathway with 0.1 mM hemin [21] and along the granular leukocytic pathway with 1.23% DMSO [22], respectively. Electrophoretic experiments. Polyacrylamide gel electrophoresis (PAGE) of native acid proteinases was carried out as described previously [8 i. For identification of proteinases, samples were preteated with polyclonal antibody specific to rat cathepsin D or rat cathepsin E, as described previously [3.4,8]. Results

Acid proteinases in human blood cells and in HL-60 and K-562 cells In extension of our previous works [8], we first attempted to examine the subcellular localization of human neutrophil cathepsin E. Three particulate fractions (P1, lowest, azurophil granule-rich; P2, middle, specific granule-rich; P3, highest, plasma membranerich) were successfully segregated by centrifugation on discontinuous Percoll gradients of posmuclear cavitate of human neutrophils (Fig. 1). However, the distribution pattern of acid proteinase activity was found to be markedly different from that of rat neutrophiis [8]; an extremely low activity in h u m a n P3 and no detectable activity in h u m a n S fraction (Fig. ID). Upon electrophoresis, only one somewhat broad band of activity that moved at the same speed as that observed with crude extracts of isolated human PMN (Fig. 2) and M N L (not shown) was detected in all of PI, P2 and P3 fractions. The enzyme cross-reacted with anti-rat cathepsin D antibody, but not with anti-rat cathepsin E antibody (Fig. 2), thus being identified as cathepsin D. H u m a n erythrocyte ghost acid proteinase, cathepsin E [10-12], migrated faster than leukocyte cathepsin D and crossreacted with anti-rat cathepsin E antibody (Fig. 2). Fig. 2 also shows that cells of HL-60 and K-562 lines have unique acid proteinase species that are immunochemically cathepsin D-related, but migrate faster than normal leukocyte cathepsin D. No cathepsin E-related acid proteinase was found in the promyelocytic leukemia cells, the erythroleukemia cells, DMSO-treated neutrophil-like HL-60 cells or hemin-treated K-562 erythroid cells. Acid proteinases in blood cells of several mammalian species The finding that the distribution of acid proteinases in blood cells was different between rats and h u m a n s prompted us to examine their distributional patterns in blood cells from other mammals. As a result, no cathepsin E activity was found in leukocytes or even in erythrocyte ghosts of pigs, cows or goats (Fig. 3). Guinea pigs contained cathepsin E in leukocytes but not in

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Fig. l. Distribution of protein (A). myeloperoxidasc and #-glucuronidusc (B). alkaline phosphatasc (C) and acid protcinase (D) after centrifugation of po.~muclear cavitate of human neutrophils on discontinuous Percoll gradients. The positions of three particulate fractions (Pl, P2 and P3) and ,,~oluble fraction (S) are indicated in A.

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Fig. 2. Upper panel: Zymogran~ of acid proteinas¢ of four different human neutrophil samples (lanes 2-5), HL-60 cells (lane 6) and K-562 cells (lane 7). For comparison, the pattern of rat neutrophil acid proteinase is shown in lane I. Lower panel: Immunochemical identification of acid proteinases of human neatr,~phils (lanes 1-3), HL-60 cells (lanes 4-6), K-562 cells (lanes 7-9) and human crythrocyte ghosts (lanes 10-11). Each sample was subjected to electrophoresis before (lanes 1, 4, 7 and 10) and after treatment with anti-rat cathepsin D antibody (lanes 2, 5 and 8) or anti-rat cathepsin E antibody (lanes 3, 6. 9 and I I). In lanes 2 and 5, the presence of antigen-antibody complex can be recognized at the origin.

158 TABLE I

TABLE II

Distribution of

Species-specific distribution of cathepsin E in mammalian blood cells.

The distribution of cathepsins D and E in leukocytes and erythrocyte ghosts of several mammalian species, and in HL-60 and K-562 cells was examined by...
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