©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carboxyl-terminal Prodomain-deleted Human Leukocyte Elastase and Cathepsin G Are Efficiently Targeted to Granules and Enzymatically Activated in the Rat Basophilic/Mast Cell Line RBL (*)

Urban Gullberg (§) , Anders Lindmark , Gustav Lindgren , Ann-Maj Persson , Eva Nilsson , Inge Olsson

From the (1) Division of Hematology, Department of Medicine, University of Lund, S-221 85 Lund, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The hematopoietic neutral serine proteases leukocyte elastase and cathepsin G are synthesized as inactive precursors, but become activated by removal of an amino-terminal dipeptide and are stored in granules. Moreover, the pro forms of elastase and cathepsin G show carboxyl-terminal prodomains of 20 and 11 amino acids, respectively, which are not present in the mature enzymes. To investigate mechanisms for processing, activation, and granular targeting, we have utilized transgenic expression of myeloid serine proteases in the rat basophilic/mast cell line RBL-1 (Gullberg, U., Lindmark, A., Nilsson, E., Persson, A.-M., and Olsson, I.(1994) J. Biol. Chem. 269, 25219-25225). Leukocyte elastase was stably expressed in RBL-1 cells, and the translation products were characterized by biosynthetic labeling followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography. Processing of a main pro form of 34 kDa into mature 31- and 29-kDa forms was demonstrated. Translocation of mature forms to granule-containing fractions was shown by subcellular fractionation experiments. The processed forms were enzymatically active, judging by the occurrence of amino-terminal processing demonstrated by radiosequence analysis, the acquisition of affinity for the protease inhibitor aprotinin, and the appearance of elastase activity in transfected RBL cells. To investigate the function of the carboxyl-terminal prodomains, deletion mutants of leukocyte elastase and cathepsin G lacking the carboxyl-terminal extension were constructed and transfected into RBL cells. Our results show that as full-length proteins, the deletion mutants were converted to active enzymes and transferred to granules with kinetics similar to that of wild-type enzymes. We conclude that human leukocyte elastase and cathepsin G are converted into enzymatically active forms when expressed in RBL cells and targeted for storage in granules; the carboxyl-terminal prodomains are necessary neither for enzymatic activation nor for targeting to granules in RBL cells.


INTRODUCTION

A regulated process of proliferation and differentiation of hematopoietic cells along distinct lineages of maturation results in production of functionally competent blood cells. The neutrophil granulocyte is the dominant cell of the myeloid lineage and is of major importance in host defense. Characteristically, the neutrophil contains particular cytoplasmic granules formed in a sequential manner during its maturation and whose content is released during phagocytosis or regulated secretion. Peroxidase-positive azurophil granules are produced during the promyelocyte stage of differentiation, while the subsequent myelocyte stage gives rise to specific peroxidase-negative granules (1, 2) .

The azurophil granule contains typical lysosomal enzymes and can therefore be regarded as a specialized form of lysosome. Besides lysosomal hydrolases, the azurophil granules store bactericidal proteins that are unique for the myeloid lineage (3, 4, 5) . Among them are the neutral serine proteases leukocyte elastase and cathepsin G, both belonging to a superfamily of hematopoietic serine proteases including granzymes of cytotoxic T-lymphocytes and certain mast cell proteases (6, 7). These serine proteases are characteristically stored in granules in a processed catalytically active form, but are transiently present as inactive zymogens; activation is likely to follow the post-translational removal of an amino-terminal dipeptide in a pregranular compartment (6, 8, 9, 10, 11, 12) .

Lysosomal enzymes are often phosphorylated on mannose residues of carbohydrate side chains in order to provide binding to mannose 6-phosphate receptors, which mediate the transfer to lysosomes (13) . However, accumulating evidence indicates that alternative mechanisms, independent of mannose 6-phosphate receptors, exist for traffic to lysosomes (14, 15) . In particular, the azurophil granule proteins seem to be independent of mannose-6-phosphate for subcellular sorting, but the mechanisms by which these proteins are retrieved from the secretory pathway and targeted for storage in granules are unknown (16) . A heterogeneous pattern of oligosaccharide side chains among the different types of azurophil granule proteins of neutrophils suggests that carbohydrates are not of general importance for sorting. Promyeloperoxidase shows ``high mannose'' oligosaccharide side chains (17); procathepsin G and proelastase have complex oligosaccharide side chains (18) , while the antibacterial defensins lack carbohydrates (19) .

As for typical lysosomal enzymes, the processing of azurophil serine proteases involves late proteolytic cleavage in a pregranular or granular compartment (16) . As a consequence, prodomains are eliminated by proteolysis during the processing of the precursor into the mature enzyme. In addition to an amino-terminal dipeptide, the pro forms of leukocyte elastase and cathepsin G contain carboxyl-terminal prodomains not found in the mature enzymes (8) . Judging by comparison of the predicted sequence from cDNA (20, 21) with the amino acid sequence of the mature protein (22) , the carboxyl-terminal prodomain of leukocyte elastase comprises 20 amino acids. Cathepsin G is also subject to carboxyl-terminal processing, and although not exactly defined, cleavage may take place after Ser to remove 11 amino acids (6, 8) .

The aim of this work was to investigate the role of the carboxyl-terminal prodomains of leukocyte elastase and cathepsin G for enzymatic activation and granular targeting. We have recently utilized a transgenic expression model for myeloid serine proteases in a rat basophilic/mast cell line and showed that human cathepsin G is adequately processed, enzymatically activated, and targeted to granules for storage in these cells (23) . In the present work, we demonstrate that transgenic human leukocyte elastase is similarly processed and sorted to granules in this cell line. To investigate a possible role of the carboxyl-terminal prodomains in intracellular sorting or enzymatic activation, cDNA deletion mutants that lacked the prodomains were utilized. We found that the carboxyl-terminal prodomains of elastase and cathepsin G are dispensable for granular targeting in RBL cells and that deletion of these domains does not seem to interfere with folding and enzymatic activation of the native protein.


EXPERIMENTAL PROCEDURES

Materials

The eukaryotic expression vectors pRC/CMV and pCEP4 were from Invitrogen (Leek, The Netherlands). Both vectors provide cytomegalovirus promoter-driven expression of introduced cDNA and confer resistance to Geneticin and hygromycin B, respectively, which allows selection of recombinant cells. [S]Methionine/[S]cysteine (cell labeling grade) and [H]isoleucine were from Amersham International (Buckinghamshire, UK). Prior to use, [H]isoleucine was concentrated 10-fold in a vacuum centrifuge. Percoll and protein A-Sepharose CL-4B were from Pharmacia (Uppsala). Hygromycin B, protein G-agarose, aprotinin-agarose, and N-succinyl-Ala-Ala-Ala-p-nitroanilide were from Sigma. Geneticin was from Boehringer Mannheim (Mannheim, Federal Republic of Germany). Rabbit polyclonal antisera to leukocyte elastase and to cathepsin G were obtained by immunization of rabbits (24, 25) .

cDNA, Mutagenesis, and Construction of Expression Vectors

Human cDNA for leukocyte elastase was produced from total RNA from the HL-60 leukemic cell line using reverse transcription and PCR() amplification of cDNA with primers based on a published nucleotide sequence (21) using the Gene Amp® RNA PCR kit (Perkin-Elmer) according to the manufacturer's instructions. By designing the PCR primers, the Kozak consensus leader sequence for maximum translational efficiency (26) was introduced, and the flanking restriction enzyme sites HindIII and NotI were included for subsequent cloning into plasmid. PCR primers were as follows: upstream, 5`-GACTTCAGAAGCTTGCCACCATGACCCTCGGCCGCCGACTCG-3`; and downstream, 5`-GACTTCAGGCGGCCGCTCAGTGGGTCCTGCTGGCCGGGTCCGG-3` (start and stop codons in boldface). The resulting PCR product was cloned into pRC/CMV to create the expression vector pRC/CMV-elastase. Similarly, cDNA for cathepsin G (generously provided by Dr. G. Salvesen, Duke University, Durham, NC) was cloned into the expression vector pCEP4-CatG as described (23) . A carboxyl-terminal deletion mutant of leukocyte elastase was created by PCR amplification of leukocyte elastase cDNA with the downstream primer 5`-GACTTCAGGCGGCCGCTCATTGGATGATAGAGTCGATCCAG-3`, thereby introducing a stop codon (boldface) after Gln. Analogously, a deletion mutant of cathepsin G was constructed using the downstream primer 5`-GACTTCAGGCGGCCGCTCAGCTTCTCATTGTTGTCCTTATCC-3`, introducing a stop codon (boldface) after Ser. The deletion mutants of leukocyte elastase and cathepsin G were sequenced and cloned into pRC/CMV and pCEP4 to create the expression vectors pRC/CMV-elastase248-267 and pCEP4-CatG245-255, respectively. All cDNA constructs were sequenced to verify the integrity of the reading frame and the desired mutations. The deletion mutants are depicted schematically in Fig. 1.


Figure 1: Schematic view of the structures of leukocyte elastase and cathepsin G and the carboxyl-terminal deletion mutants. Amino acids are indicated by one-letter code.



Transfection Procedure

RBL-1 cells were transfected using the Bio-Rad electroporation apparatus. Transfection of RBL-1 cells by electroporation was performed with electrical settings of 960 microfarads and 300 V as described elsewhere (23) . After electroporation, Geneticin (2 mg/ml) or hygromycin B (1200 units/ml) was added to select for recombinant clones expressing the Geneticin resistance gene of pRC/CMV or the hygromycin B resistance gene of pCEP4, respectively. Individual clones growing in the presence of antibiotic were isolated, expanded into mass cultures, and screened for expression of elastase or cathepsin G by biosynthetic labeling. Clones with the most pronounced expression were chosen for further experiments.

Cell Culture

The rat basophilic/mast cell line RBL-1 was a gift from Dr. L. Hellman. Cells were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (complete medium) (GIBCO Ltd., Paisley, UK) in 5% CO at 37 °C in a fully humidified atmosphere. Exponentially growing cells were used in all experiments.

Biosynthetic Labeling

Biosynthetic labeling of newly synthesized proteins was performed as described elsewhere (23) . Unless otherwise indicated, cells were starved for 30 min, followed by pulse labeling with [S]methionine/[S]cysteine for 30 min. In chase experiments following pulse labeling, cells were resuspended in complete medium. At timed intervals, cells were withdrawn and subjected to extraction or homogenization and subsequent subcellular fractionation.

Subcellular Fractionation

Subcellular fractionation was performed as described elsewhere (23) . Briefly, the cell homogenate was fractionated in a Percoll density gradient, after which nine fractions were collected, with fraction 9 containing all cytosol. The distribution of lysosomes and Golgi elements in the density gradient was determined, by assaying -hexosaminidase and galactosyltransferase as described elsewhere (27, 28) , to fractions 1-3 and 5-8, respectively (data not shown).

Immunoprecipitation

For immunoprecipitation, whole cells or Percoll-containing subcellular fractions were solubilized, and biosynthetically labeled elastase or cathepsin G was immunoprecipitated and subjected to electrophoretic analysis (SDS-PAGE) followed by fluorography as described previously (18, 23) .

Adsorption to Aprotinin-Agarose

Adsorption to aprotinin-agarose was performed essentially as described by Salvesen and Enghild (8) and as previously described in detail (23) . Briefly, cells were lysed, and the lysate was allowed to react for 60 min at room temperature with 200 µl of a 50% suspension of aprotinin-agarose/10 cells. The aprotinin with bound material was washed, followed by incubation for 45 min at 4 °C in 5 ml of elution buffer (50 mM sodium acetate, pH 4.5, 300 mM NaCl) to obtain the release of bound material. Elastase or cathepsin G in the eluate or remaining in the lysate after adsorption to aprotinin-agarose (lacking affinity for aprotinin) was immunoprecipitated as described (23) .

Radiosequencing

For determination of the amino-terminal sequence of processed elastase and cathepsin G, biosynthetic labeling with [H]isoleucine followed by amino acid sequencing was performed in the following way. Cells (1 10) were starved for 30 min in 50 ml of isoleucine-free RPMI 1640 medium (Select-Amine kit, GIBCO Ltd.) in order to reduce intracellular isoleucine supplies, after which the cells were incubated in 25 ml of identical medium supplemented with [H]isoleucine (200 µCi/ml) to achieve metabolic labeling of synthesized proteins. During the subsequent chase, cells were incubated in 50 ml of complete medium. Following pulse labeling for 30 min and chase for 2 h, respectively, cell lysates were prepared, and immunoprecipitation was performed as described above. Following SDS-PAGE, the proteins were transferred to a polyvinylidene difluoride membrane by semidry electroblotting, and the membrane was subjected to autoradiography for 1 week. By guidance of the autoradiogram, radioactive bands were excised and subjected to Edman amino acid degradation, performed by the Biomedical Service Unit at University of Lund. The initial 10 degradation cycles were assayed for radioactivity using a scintillation counter.

Enzymatic Assay

The enzymatic activity of elastase was assayed using N-succinyl-Ala-Ala-Ala-p-nitroanilide as substrate (29) . A cell extract was prepared and assayed as described elsewhere (30) with minor modifications. Briefly, 1 10 cells were washed once in phosphate-buffered saline, and the cell pellet was frozen and thawed twice, after which it was resuspended in 0.9 ml of 100 mM Tris-Cl, pH 8.5, containing 1 M MgCl and 0.1% Triton X-100. After 30 s of sonication with an ultrasonicator (Kistner Lab, Stockholm, Sweden) to obtain complete homogenization of the cells, 2 ml of 5 mM Tris-Cl, pH 8.5, containing 1 M NaCl and 0.1% Triton X-100 was added, and the lysate was centrifuged at 32,000 g at 4 °C for 2 h to remove DNA. Of the remaining lysate, 500 µl was mixed with 250 µl of 200 mM Tris-Cl, pH 8.5, containing 1 M NaCl, and the mixture was heated to 40 °C, after which 250 µl of 2 mMN-succinyl-Ala-Ala-Ala-p-nitroanilide (dissolved in dimethyl fluoride) was added. The reaction mixture was incubated for 30 min, after which the reaction was stopped by the addition of 500 µl of soybean trypsin inhibitor (200 µg/ml), and the absorbance at 410 nm was read. A standard curve was obtained by replacing the cells with various amounts of porcine pancreatic elastase (Sigma). Measurements were performed within a linear range of 0.15 to 1.5 A (83-830 units/ml elastase).


RESULTS

Biosynthesis, Processing, and Granular Sorting of Transgenic Human Leukocyte Elastase in RBL Cells

Stable clones of RBL cells expressing human leukocyte elastase (RBL/elastase cells) were established by transfection with pRC/CMV-elastase. The biosynthesis of transgenic elastase was characterized by biosynthetic labeling and immunoprecipitation as described under ``Experimental Procedures.'' No labeled material was specifically immunoprecipitated from wild-type RBL cells (data not shown). Fig. 2 shows pulse labeling of RBL/elastase cells, followed by chase for the time periods indicated. After 30 min of pulse labeling, a main translation product of 34 kDa was found. In addition, two minor products with apparent masses of 37 and 32 kDa, respectively, were precipitated. The 32-kDa form was found to be an initial translation product, as it was the dominating form after 10 min of pulse labeling and was converted into the 34-kDa form following chase of the label for 20 min (data not shown); this processing from 32 to 34 kDa most probably represents glycosylation events. The nature of the 37-kDa form is not clear. After 2 h of chase, almost all of the labeled material was reduced to 31 kDa, most likely corresponding to proteolytic removal of the carboxyl-terminal prodomain (8) , and prolonged chase resulted in the appearance of an additional 29-kDa form, distinguishable after 4 h of chase. A substantial amount of the proteolytically unprocessed forms of transgenic elastase was released into the culture medium (Fig. 2), which is commonly seen with lysosomal enzymes (16) . The intracellular processed forms were stable during the 4 h of the experiment, without obvious signs of degradation. These data suggest the initial synthesis of a 34-kDa pro form of elastase that with time is carboxyl-terminally processed into a 31-kDa form and, to some extent, further into a 29-kDa form.


Figure 2: Processing of transgenic leukocyte elastase. RBL/elastase cells were pulse-labeled with [S]methionine/[S]cysteine for 30 min, followed by chase for up to 4 h. At the indicated time points, 2 10 cells were withdrawn and subjected to solubilization and immunoprecipitation with anti-elastase as described under ``Experimental Procedures.'' In addition, elastase was immunoprecipitated from the incubation medium after each chase period. The immunoprecipitates were subjected to SDS-PAGE on a 5-20% gradient gel, and subsequent fluorography was performed as described under ``Experimental Procedures.'' The fluorogram was exposed for 7 days. The positions of newly synthesized 34-kDa proelastase and the 31- and 29-kDa processing forms are indicated with arrows to the right. Numbers to the left in this figure and in Figs. 3-5 are the M values of molecular weight standards.



To correlate the processing with subcellular translocation, pulse-chase experiments were performed, followed by subcellular fractionation. After 30 min of labeling, most of the newly synthesized elastase was present in fractions of intermediate density, corresponding to the endoplasmic reticulum and Golgi elements (Fig. 3), with the 34-kDa form as the dominant one. Following 90 min of chase, most of the 34-kDa pro form was processed into a 31-kDa form and transferred to dense fractions corresponding to granules. Labeled transgenic elastase still remained, after 5 h of chase, in dense fractions, and a minor portion was converted into a 29-kDa form. These data are analogous to those shown for transgenic cathepsin G in RBL cells (23) and indicate that proteolytic processing of proelastase occurs during or after transfer to a granular compartment, where the mature enzyme is stored.


Figure 3: Targeting of leukocyte elastase to granules. RBL/elastase cells were pulse-labeled for 30 min (A), followed by chase for 90 min (B) and 5 h (C). At the times indicated, 1 10 cells were homogenized, after which subcellular fractionation was performed, with subsequent collection of eight 0.8-ml subcellular fractions (fraction 9 contained all cytosol) as described under ``Experimental Procedures.'' Fractions were solubilized and subjected to immunoprecipitation with anti-elastase. Analyses of immunoprecipitates were as described in the legend to Fig. 2. The fluorograms were exposed for 7 days. The positions of 34-kDa proelastase and the 31- and 29-kDa processing forms are indicated with arrows to the right.



Processing and Granular Sorting of Carboxyl-terminal Deletion Mutants of Leukocyte Elastase and Cathepsin G in RBL Cells

By transfection of RBL cells with carboxyl-terminally deleted cDNA for leukocyte elastase or cathepsin G, cell clones were established expressing elastase (RBL/elastase248-267) and cathepsin G (RBL/CatG245-255), respectively, lacking the carboxyl-terminal prodomains. The deletion mutants are depicted schematically in Fig. 1. Fig. 4shows the initial synthesis of a 31-kDa form in RBL/elastase248-267 cells. Thus, the initially synthesized form of the carboxyl-terminal deletion mutant (31 kDa) corresponds to that of the processed full-length enzyme (31 kDa), supporting the notion that the carboxyl-terminal prodomain is removed during processing of the full-length proenzyme from 34 to 31 kDa (Fig. 2). Similar to RBL/elastase cells, the RBL/elastase248-267 cells showed, after 30 min of labeling, a small amount of elastase of lesser size (30 kDa), which disappeared while chasing the label. As for the full-length protein, this smaller (30 kDa) form may represent an early translation product that is, as the result of early glycosylation events, converted to the 31-kDa form. After 4 h of chase, a minor part was further processed into a 29-kDa form. A considerable amount of the labeled material was secreted into the medium, but after 1 h of chase, the intracellular labeled protein remained stable during the 4 h of the experiment. Most of the extracellular release occurred during the 1st h of chase, after which the secreted forms disappeared to some extent (Fig. 4). As for the full-length protein, the stability of the intracellular enzyme suggests transfer to a storage compartment, and translocation of newly synthesized elastase lacking the carboxyl-terminal prodomain was indeed demonstrated by subcellular fractionation. Fig. 5shows that elastase248-267 is targeted to dense fractions corresponding to granules, analogous to what was shown for full-length elastase in Fig. 3 . Fig. 5also demonstrates that 30 min of pulse labeling of RBL/elastase248-267 cells results in two visible translation products of 31 and 30 kDa. As seen in experiments with full-length elastase, the smaller product disappeared by chasing the label, which probably reflects glycosylation of the protein. Following 90 min of chase, the main initial translation product of 31 kDa was transferred to dense fractions, where a minor part was further processed into a 29-kDa form, distinguishable after 5 h of chase. Likewise, it was found that cathepsin G lacking the carboxyl-terminal prodomain (CatG245-255) was targeted to granule-containing fractions (data not shown). These results demonstrate that the carboxyl-terminal deletion mutants of leukocyte elastase and cathepsin G are, like full-length proenzymes, efficiently sorted for storage in a granular compartment in RBL cells.


Figure 4: Processing of transgenic leukocyte elastase248-267. RBL/elastase248-267 cells were pulse-labeled with [S]methionine/[S]cysteine for 30 min, followed by chase for up to 4 h. At the indicated time points, 2 10 cells were withdrawn and subjected to solubilization, immunoprecipitation, and subsequent analyses as described in the legend to Fig. 2. The fluorogram was exposed for 7 days. The positions of 31-kDa elastase248-267 and the 29-kDa processing form are indicated with arrows to the right.




Figure 5: Targeting of leukocyte elastase248-267 to granules. RBL/elastase248-267 cells were pulse-labeled for 30 min (A), followed by chase for 90 min (B) and 5 h (C). At the times indicated, 1 10 cells were homogenized, after which subcellular fractionation, immunoprecipitation, and subsequent analyses were performed as described in the legend to Fig. 3. The fluorograms were exposed for 7 days. The positions of 31-kDa elastase248-267 and the 29-kDa processing form are indicated with arrows to the right.



Enzymatic Activation and Amino-terminal Processing of Transgenic Leukocyte Elastase and Cathepsin G

Leukocyte elastase and cathepsin G are synthesized as inactive zymogens, but are enzymatically activated in a pregranular or granular compartment. The removal of an amino-terminal dipeptide, catalyzed by the thiol protease dipeptidyl peptidase I, is a prerequisite for enzymatic activation (8, 11) . To investigate the activation of the transfected serine proteases in RBL cells, their affinity for the serine protease inhibitor aprotinin was investigated. By pulse labeling and then chasing the label, followed by adsorption to aprotinin-agarose, we demonstrated the acquisition of affinity to aprotinin for transgenic elastase, elastase248-267, and CatG245-255. Fig. 6shows similar results for all three enzyme variants, with binding of most of the labeled material to aprotinin after 2-5 h of chase. These results are consistent with results from experiments with endogenous elastase and cathepsin G (8) as well as with transgenic cathepsin G (23) , thus suggesting amino-terminal removal of the dipeptide, leading to enzymatic activation. Indeed, using a radiosequencing technique, amino-terminal processing with removal of a dipeptide was indicated for both transgenic elastase (Fig. 7A) and cathepsin G (Fig. 7B). The amino acid sequences of mature elastase and cathepsin G begin with one and two isoleucines, respectively (Fig. 1). This is in contrast to the immature forms, which contain the activation dipeptide (Ser-Glu and Gly-Glu, respectively) prior to the isoleucines. Cells were labeled with [H]isoleucine for 30 min, followed by chase for 2 h, and radiosequencing was performed as described under ``Experimental Procedures.'' After 30 min of labeling, 34-kDa proelastase and 32.5-kDa procathepsin G showed, upon sequencing, the presence of radioactivity in the third fraction, corresponding to amino acid 3 in the amino-terminal sequence, indicating the presence of the activation dipeptide followed by labeled isoleucine (Fig. 7). Following 2 h of chase, however, the processed forms of elastase and cathepsin G with masses of 31 kDa showed considerable radioactivity in fraction 1 of the sequencing chromatogram, thus indicating that cleavage of the dipeptide had occurred. The persisting radioactivity in fraction 3 following chase indicates that activation was incomplete at this point, which is concordant to results above showing incomplete acquisition of affinity for aprotinin after 2 h of chase (Fig. 6).


Figure 6: Adsorption to aprotinin-agarose. RBL/elastase, RBL/elastase248-267, and RBL/CatG245-255 cells were labeled with [S]methionine/[S]cysteine for 30 min and chased for up to 5 h. At timed intervals, aliquots of labeled cells (15 10) were withdrawn for analysis. Cell lysis and adsorption to aprotinin-agarose were performed as described under ``Experimental Procedures.'' A demonstrates the immunoprecipitates of labeled protein that did not bind to aprotinin (enzymatically nonactive). In B, the aprotinin-bound material was eluted, and immunoprecipitation with specific antiserum, SDS-PAGE, and fluorography were performed as described under ``Experimental Procedures.'' The fluorograms were exposed for 7 days. Arrows to the right indicate newly synthesized proelastase (34 kDa), elastase248-267 (31 kDa), CatG245-255 (31 kDa), and their processing forms.




Figure 7: Radiosequencing of transgenic elastase and cathepsin G. RBL/elastase (A) and RBL/CatG (B) cells were pulse-labeled with [H]isoleucine for 30 min (upper panels), followed by chase for 2 h (lower panels) as described under ``Experimental Procedures.'' Following pulse labeling and chase, 1 10 cells were withdrawn and subjected to solubiliza-tion, immunoprecipitation, SDS-PAGE, and transfer to polyvinylidene difluoride membrane as described under ``Experimental Procedures.'' Radioactive bands containing pulse-labeled 34-kDa proelastase and 32.5-kDa procathepsin G and the 31-kDa processing form of elastase and cathepsin G following 2 h of chase were excised and subjected to amino acid degradation. The amount of radioactivity in the initial 10 cycles of each sequence analysis is shown.



To estimate the relative enzymatic activity in RBL cells expressing transgenic elastase, an enzymatic assay was employed as described under ``Experimental Procedures.'' As might be predicted from the results of experiments with adsorption to aprotinin (Fig. 6), RBL/elastase248-267 cells contained elastase activity comparable to that of RBL/elastase cells. The elastase activity in RBL/elastase248-267 cells was 561 ± 95 units, and the corresponding value for RBL/elastase cells was 490 ± 108 units, while wild-type RBL cells contained 75 ± 27 units (per 5 10 cells, ± S.E., n = 3). These results demonstrate that transgenic elastase is enzymatically activated and that a deletion of the carboxyl-terminal prodomain obviously does not interfere with the conversion of the zymogen form into catalytically competent enzyme. A similar enzymatic assay for cathepsin G (31) could not be employed due to strong interference of endogenous proteases from the RBL cells.


DISCUSSION

The aim of this work was to elucidate processing and sorting mechanisms for the granule serine protease family of myeloid origin (3, 6) . Certain mast cell proteases belong to this family of enzymes, and therefore, the rat basophilic/mast cell line RBL should also be equipped with processing and sorting mechanisms for the hematopoietic serine proteases. Indeed, granzyme A and cathepsin G cDNAs transfected into this cell line both produced functional enzymes (23, 32) . For this reason, RBL cells were employed in both our previous (23) and present studies. Our data show that transgenic human leukocyte elastase is converted into active enzyme and targeted to granules in RBL cells. Moreover, this study demonstrates that both leukocyte elastase and cathepsin G lacking the carboxyl-terminal prodomain are still targeted to dense granule-containing fractions and converted into catalytically active forms. Thus, the carboxyl-terminal extension of these enzymes is obviously unnecessary for sorting and enzymatic activation in RBL cells.

Generally speaking, lysosomal enzymes are synthesized as larger precursors that undergo limited proteolysis during and after intracellular sorting, involving cleavage of both amino-terminal and carboxyl-terminal prodomains (16) . In some cases, either amino- or carboxyl-terminal prodomains have proved important for folding, intracellular targeting, and enzymatic activation of the protein (33) . However, the biological role of prodomain processing remains obscure for most lysosomal enzymes. Our results show that processing and sorting of human leukocyte elastase transfected into RBL cells follow a scheme that corresponds to previous findings concerning the processing of endogenous neutrophil elastase (6, 8, 18, 34) and are similar to results from studies on endogenous and transgenic cathepsin G (23) . The mature form of leukocyte elastase had an apparent mass of 31 kDa and was found in dense granule fractions isolated by Percoll density gradient separation. This form appeared in granule fractions after 90 min of chase, suggesting that transport into granules requires <2 h. The 31-kDa product was stable for at least 5 h, although some slow processing into a 29-kDa form did occur. Several lines of evidence strongly indicate that the protein was converted into enzymatically active forms; the acquisition of affinity for the serine protease inhibitor aprotinin was demonstrated, and it was confirmed by radiosequencing for both elastase and cathepsin G that amino-terminal processing occurred with removal of the activation dipeptide. Moreover, we also demonstrated the presence of elastase activity in extracts of transfected RBL cells. A fraction of newly synthesized leukocyte elastase failed to be retained in the cells and was secreted as precursor forms, which is a common feature in lysosomal enzyme processing (16) .

In the promyelocyte, leukocyte elastase and cathepsin G are stored in azurophil granules, which can be regarded as specialized forms of lysosomes, and they seem to be targeted to granules in a mannose 6-phosphate-independent manner (16) . In yeast, some enzymes contain prodomains that are necessary and sufficient for mannose 6-phosphate-independent targeting into the vacuole, the yeast analogue of the mammalian lysosome (35, 36) . Also in mammalian cells, mechanisms for lysosomal delivery that are independent of mannose 6-phosphate exist (14, 15) . A transient mannose 6-phosphate-independent membrane association has been demonstrated for procathepsins D, C, and L (37-40). Among these, the membrane association of cathepsin D involves interaction with prosaposin that binds to cathepsin D immediately after synthesis in the endoplasmic reticulum (41) . The recent demonstration that the prodomain of cathepsin L, which shows a close resemblance to vacuolar sorting sequences of enzymes in yeast, binds to an intracellular transmembrane proenzyme receptor (42, 43) makes a functional role of prodomains in lysosomal mannose 6-phosphate-independent targeting likely. And thus, the carboxyl-terminal prodomains of leukocyte elastase and cathepsin G may be necessary for granular sorting (8) . Consequently, carboxyl-terminal deletion mutants were constructed to investigate this hypothesis. Our results show that deletion of the prodomain does not interfere with sorting, as the deletion mutants were transferred to granules with the same kinetics as full-length proteins. Targeting mechanisms in RBL cells may, however, be different from those operating in the promyelocytes, as the granules of RBL cells are not identical to azurophil granules of promyelocytes.

The timing for cleavage of the carboxyl-terminal prodomains of elastase and cathepsin G was reported to parallel the removal of an amino-terminal activation dipeptide (8) . However, other data suggesting that the carboxyl-terminal processing may precede cleavage of the amino-terminal dipeptide (23) are further supported by our results from radiosequencing indicating that following 2 h of chase, when the carboxyl-terminal processing was accomplished judging by reduction in apparent mass, the removal of the amino-terminal dipeptide was still incomplete. These data suggest that amino-terminal processing and activation of the protein need to take place on protein lacking the carboxyl-terminal prodomain. The present demonstration that the carboxyl-terminal deletion mutants of elastase and cathepsin G were transformed into active enzymes, as judged by the acquisition of affinity for aprotinin and the occurrence of elastase activity in transfected cells, strongly argues against an important function of the carboxyl-terminal prodomains in enzyme folding and prevention of premature activation of the zymogen forms. However, the question of whether or not previous removal of the carboxyl-terminal prodomain is a prerequisite for the cleavage of the amino-terminal dipeptide to occur is still not resolved. Among the myeloid serine proteases, azurocidin has been reported to have a carboxyl-terminal prodomain, comprising three amino acids, that is removed during processing (44) . For other members of the hematopoietic serine protease superfamily, the information is incomplete, and it is not known whether all members are carboxyl-terminally processed. Whatever the case, our present data indicate that the carboxyl-terminal prodomain seems to be dispensable for folding, activation, and granular targeting of the enzymes. Besides neutral serine proteases, the azurophil granules contain several lysosomal hydrolases and thus expose the content to a highly proteolytic environment. Similarly, the granules of RBL cells, used in this experimental model, store both serine proteases and lysosomal enzymes (45, 46) . Thus, the carboxyl-terminal processing of leukocyte elastase and cathepsin G might merely be the result of exposure of protease-sensitive regions, but the enzyme(s) responsible for this action are not identified.

In conclusion, this investigation has demonstrated that human leukocyte elastase, following transfection into rat RBL cells, is targeted for storage in granules and converted into enzymatically active forms. Furthermore, the carboxyl-terminal prodomains of both leukocyte elastase and cathepsin G were found to be dispensable for enzymatic activation as well as granular targeting of the enzymes in RBL cells.


FOOTNOTES

*
This work was supported by the Greta and Johan Kock Foundation, the Alfred sterlund Foundation, the Swedish Cancer Society, Funds of Lunds Sjukv, the Thelma Zoega Foundation, the John Persson Foundation, and the Medical Faculty of Lund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Research Dept. 2., E-block, University Hospital, S-221 85 Lund, Sweden. Fax: 46-46-18-44-93.

The abbreviations used are: PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Björn Hultberg for help with -hexosaminidase assays.


REFERENCES
  1. Bainton, D. F.(1992) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I., Goldstein, I. M., and Snyderman, R., eds) pp. 303-324, Raven Press, New York
  2. Borregaard, N., Lollike, K., Kjeldsen, L., Sengel, H., Bastholm, L., Nielsen, M. H., and Bainton, D. F.(1993) Eur. J. Haematol. 51, 187-198 [Medline] [Order article via Infotrieve]
  3. Lehrer, R. I., and Ganz, T.(1990) Blood 76, 2169-2181 [Medline] [Order article via Infotrieve]
  4. Elsbach, P., and Weiss, J.(1992) in Inflammation: Basic Principles and Clinical Correlates (Gallin, J. I., Goldstein, I. M., and Snyderman, R., eds) pp. 603-636, Raven Press, New York
  5. Gabay, J. E., and Almeida, R. P.(1993) Curr. Opin. Immunol. 5, 97-102 [Medline] [Order article via Infotrieve]
  6. Salvesen, G., and Enghild, J. J.(1991) Biomed. Biochim. Acta 50, 665-671 [Medline] [Order article via Infotrieve]
  7. Hudig, D., Ewoldt, G. R., and Woodard, S. L.(1993) Curr. Opin. Immunol. 5, 90-96 [Medline] [Order article via Infotrieve]
  8. Salvesen, G., and Enghild, J. J.(1990) Biochemistry 29, 5304-5308 [Medline] [Order article via Infotrieve]
  9. Brown, G. R., McGuire, M. J., and Thiele, D. L.(1993) J. Immunol. 150, 4733-4742 [Abstract/Free Full Text]
  10. Caputo, A., Garner, R. S., Winkler, U., Hudig, D., and Bleackley, R. C. (1993) J. Biol. Chem. 268, 17672-17675 [Abstract/Free Full Text]
  11. McGuire, M. J., Lipsky, P. E., and Thiele, D. L.(1993) J. Biol. Chem. 268, 2458-2467 [Abstract/Free Full Text]
  12. Urata, H., Karnik, S. S., Graham, R. M., and Husain, A.(1993) J. Biol. Chem. 268, 24318-24322 [Abstract/Free Full Text]
  13. Kornfeld, S., and Mellmann, I.(1989) Annu. Rev. Cell Biol. 5, 483-525 [CrossRef]
  14. Rijnboutt, S., Kal, A. J., Geuze, H. J., Aerts, H., and Strous, G. J. (1991) J. Biol. Chem. 266, 23586-23592 [Abstract/Free Full Text]
  15. Glickman, J. N., and Kornfeld, S.(1993) J. Cell Biol. 123, 99-108 [Abstract]
  16. Hasilik, A.(1992) Experientia (Basel) 48, 130-151 [Medline] [Order article via Infotrieve]
  17. Nauseef, W. M., Olsson, I., and Arnljots, K.(1988) Eur. J. Haematol. 40, 97-110 [Medline] [Order article via Infotrieve]
  18. Lindmark, A., Persson, A.-M., and Olsson, I.(1990) Blood 76, 2374-2380 [Abstract]
  19. Valore, E. V., and Ganz, T.(1992) Blood 79, 1538-1544 [Abstract]
  20. Farley, D., Salvesen, G., and Travis, J.(1988) Biol. Chem. Hoppe-Seyler 369, (suppl.) 3-7 [Medline] [Order article via Infotrieve]
  21. Takahashi, H., Nukiwa, T., Yoshimura, K., Quick, C. D., States, D. J., Holmes, M. D., Whang-Peng, J., Knutsen, T., and Crystal, R. G.(1988) J. Biol. Chem. 263, 14739-14747 [Abstract/Free Full Text]
  22. Sinah, S., Watorek, W., Karr, S., Giles, J., Bode, W., and Travis, J. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2228-2232 [Abstract]
  23. Gullberg, U., Lindmark, A., Nilsson, E., Persson, A.-M., and Olsson, I. (1994) J. Biol. Chem. 269, 25219-25225 [Abstract/Free Full Text]
  24. Ohlsson, K., and Olsson, I.(1974) Eur. J. Biochem. 42, 519-527 [Medline] [Order article via Infotrieve]
  25. Olsson, I., and Venge, P.(1974) Blood 44, 235-246 [Medline] [Order article via Infotrieve]
  26. Kozak, M.(1987) Nucleic Acids Res. 15, 8125-8148 [Abstract]
  27. Hultberg, B., Lindsten, J., and Sjöblad, S.(1976) Biochem. J. 155, 599-605 [Medline] [Order article via Infotrieve]
  28. Bretz, R., and Stäubli, W.(1977) Eur. J. Biochem. 77, 181-192 [Medline] [Order article via Infotrieve]
  29. Bieth, J., Spiess, B., and Wermuth, C. G.(1974) Biochem. Med. 11, 350-357 [Medline] [Order article via Infotrieve]
  30. Barrett, A. J.(1981) Methods Enzymol. 80, 581-588 [Medline] [Order article via Infotrieve]
  31. Barrett, A. J.(1981) Methods Enzymol. 80, 561-565 [Medline] [Order article via Infotrieve]
  32. Nakajima, H., and Henkart, P. A.(1994) J. Immunol. 152, 1057-1063 [Abstract/Free Full Text]
  33. Baker, D., Shiau, A. K., and Agard, D. A.(1993) Curr. Opin. Cell Biol. 5, 966-970 [Medline] [Order article via Infotrieve]
  34. Lindmark, A., Gullberg, U., and Olsson, I.(1994) J. Leukocyte Biol. 55, 50-57 [Abstract]
  35. von Figura, K.(1991) Curr. Opin. Cell Biol. 3, 642-646 [Medline] [Order article via Infotrieve]
  36. Stack, J. H., and Emr, S. D.(1993) Curr. Opin. Cell Biol. 5, 641-646 [Medline] [Order article via Infotrieve]
  37. Burge, V., Mainferme, F., and Wattiaux, R.(1991) Biochem. J. 275, 797-800 [Medline] [Order article via Infotrieve]
  38. McIntyre, G. F., and Erickson, A. H.(1991) J. Biol. Chem. 266, 15438-15445 [Abstract/Free Full Text]
  39. Rijnboutt, S., Aerts, H. M. F. G., Geuze, H. J., Tager, J. M., and Strous, G. J.(1991) J. Biol. Chem. 266, 4862-4868 [Abstract/Free Full Text]
  40. Grässel, S., and Hasilik, A.(1992) Biochem. Biophys. Res. Commun. 182, 276-282 [Medline] [Order article via Infotrieve]
  41. Zhu, Y., and Conner, G. E.(1994) J. Biol. Chem. 269, 3846-3851 [Abstract/Free Full Text]
  42. McIntyre, G. F., and Erickson, A. H.(1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10588-10592 [Abstract]
  43. McIntyre, G. F., Godbold, G. D., and Erickson, A. H.(1994) J. Biol. Chem. 269, 567-572 [Abstract/Free Full Text]
  44. Morgan, J. G., Sukiennicki, T., Pereira, H. A., Spitznagel, J. K., Guerra, M. E., and Larrick, J. W.(1991) J. Immunol. 147, 3210-3214 [Abstract/Free Full Text]
  45. Metzger, H., Alcaraz, G., Hohman, R., Kinet, J. P., Pribluda, V., and Quarto, R.(1986) Annu. Rev. Immunol. 4, 419-470 [CrossRef][Medline] [Order article via Infotrieve]
  46. Kido, H., Izumi, K., Otsuka, H., Fukusen, N., Kato, Y., and Katunuma, N.(1986) J. Immunol. 136, 1061-1065 [Abstract/Free Full Text]

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